KR102682305B1 - Method for isolating liver derived exosome using liver-vein closed circulation system and pharmaceutical composition for treating diabetes related disease comprising liver derived exosome as an active ingredient - Google Patents

Abstract

The present invention relates to a method for extracting liver-derived exosomes using a hepatic vascular occlusive circuit system and a pharmaceutical composition for treating diabetes-related diseases containing exosomes derived from liver or liver epithelial cells as an active ingredient. It was confirmed that exosomes loaded with TM4SF5, which is specifically derived from cells, can promote glucose absorption into the blood in muscle cells, adipocytes, muscle tissue, or adipose tissue, and the hepatic vascular occlusive circuit system of the present invention and Through this liver-derived exosome extraction method, TM4SF5-loaded exosomes specifically derived from the liver can be efficiently extracted, and the characteristics and secretion characteristics of the exosomes are secreted according to the level or presence of nutrients in the extracellular fluid. From this point of view, a composition containing the extracted exosomes as an active ingredient can be usefully used as a treatment for diabetes-related diseases.

Description

Method for extracting liver-derived exosomes using the liver-vein closed circulation system and pharmaceutical composition for treating diabetes-related diseases containing liver-derived exosomes as an active ingredient {Method for isolating liver derived exosome using liver-vein closed circulation system and pharmaceutical composition for treating diabetes related disease comprising liver derived exosome as an active ingredient}

The present invention relates to a method for extracting liver-derived exosomes using a hepatic vascular occlusive circuit system and a pharmaceutical composition for treating diabetes-related diseases containing liver-derived exosomes as an active ingredient.

Extracellular vesicles include exosomes, ectosomes, microvesicles, and apoptotic bodies released or secreted from cells. Exosomes are 30-200nm long. It is a biological nanoparticle that has the size of and is produced from intracellular multivesicular bodies (MVB). Exosomes are released from cells into the extracellular space and contain specific proteins, mRNA, or microRNA derived from cells and transport them to adjacent cells or distant cells to transmit signals. Do it. While playing a role in signaling between cells, exosomes play a wide range of roles, ranging from normal physiological signaling in various organs to signaling in pathological conditions.

Therefore, although much research is being conducted on exosomes, exosome research is experiencing difficulties due to the following technical limitations. The first limitation is that it is difficult to fractionate and analyze various sizes, and the second is that in vivo exosome collection technology has not been developed. The first limitation, although in its infancy, is being studied with techniques using chromatography and field-flow fractionation, while the second limitation has not yet made much progress. Most exosome research studies exosomes obtained from cultured cell lines or body fluids/blood, but the amount of exosomes obtained from cells cultured in vitro is very limited and difficult to represent the various environments in the body. In addition, exosomes collected from body fluids and blood can be obtained very easily, but because exosomes from various organs in the body are mixed, it is difficult to specifically study the properties and functions of exosomes in specific organs. If we can determine the characteristics of exosomes secreted from various organs in various situations, we will be able to easily diagnose changes in exosomes obtained from body fluids.

On the other hand, Transmembrane 4 L six family member 5 (TM4SF5) is a tetraspanin that passes through the cell membrane four times. The present inventors have shown that it can damage liver epithelial cells and liver epithelial cells through the action of various cytokines and chemokines following chronic damage to liver epithelial cells. It was confirmed that expression was increased in phagocytes, and therefore, the interaction between liver epithelial cells and macrophages depends on the expression of TM4SF5, which can lead to liver fibrosis or cirrhosis beyond steatohepatitis by remodeling the inflammatory response and immune environment of the liver. It has been confirmed that it exists.

Prior literature related to TM4SF5 includes Republic of Korea Patent No. 10-2112760, which discloses that overexpression of TM4SF5 causes fatty liver and steatohepatitis, and a kit for diagnosing liver fibrosis, cirrhosis, or alcoholic liver damage containing an antibody against TM4SF5 protein. There is Korean Patent No. 10-1368871, which discloses, but the action of exosomes loaded with TM4SF5 to promote glucose absorption in the blood by muscle and fat cells or tissues has not been disclosed.

Accordingly, the present inventors confirmed that exosomes secreted from liver epithelial cells expressing TM4SF5 can access other organs, especially muscle and fat tissue, and positively help muscle and fat cells absorb glucose, and the exosomes In order to obtain exosomes with high efficiency, the present invention was completed by constructing a “liver vein-closed circulation system”, a system for obtaining exosomes only from living liver tissue of mice.

The present invention provides a hepatic vascular occlusion circuit system for extracting hepatic epithelial cell or liver-derived exosomes to obtain exosomes containing TM4SF5 protein, a treatment substance for diabetes-related diseases, with high efficiency, and a liver-derived exosome extraction method using the same. The purpose is to provide a pharmaceutical composition for treating diabetes-related diseases containing the exosome as an active ingredient, and analyzing its characteristics.

In order to achieve the above purpose,

The present invention relates to a liver vascular occlusion circuit system for extracting liver-derived exosomes,

A peristaltic pump for circulating the hepatic vascular occlusive circuit;

Exosome circulating media and flushing buffer circulated along the hepatic vascular occlusive circuit by the peristaltic pump;

A three-way connector including a switch to control circulation of the exosome circulation media and flushing buffer;

a first tubing connecting the inlet of the peristaltic pump with the three-way connector and connecting the outlet of the peristaltic pump with a third tubing;

A second tubing connecting the exosome circulation media and the flushing buffer with the three-way connector, respectively;

A first catheter inserted into the subject's portal vein;

a third tubing connecting the first catheter and the first tubing;

A first connector connecting the first catheter and the third tubing;

a second catheter inserted into the subject's inferior vena cava;

A fourth tubing connecting the second catheter and the exosome circulation media;

A second connector connecting the second catheter and the fourth tubing; and

A fifth tubing connecting the exosome circulation media and fluffing buffer with an oxygen tank;

It provides a hepatic vascular occlusive circuit system including.

In addition, the present invention relates to a method of extracting liver-derived exosomes using the liver vascular occlusion circuit system,

inserting the first catheter into the subject's portal vein;

Operating the peristaltic pump;

Connecting the first catheter and a first connector connected to the pump;

inserting the second catheter into the inferior vena cava of the subject;

Connecting the second catheter to a fourth tubing and connecting the other end of the fourth tubing to the exosome circulating media tube;

stopping the peristaltic pump and turning the switch on the three-way connector to supply the exosome circulating media to the pump;

Connecting the exosome circulation media and flushing buffer with an oxygen tank;

Reactivating the peristaltic pump to circulate the exosome circulation media;

Centrifuging the circulated exosome circulation media to separate the supernatant;

Concentrating the separated supernatant; and

It provides a liver-derived exosome extraction method comprising the step of isolating exosomes from the concentrated supernatant.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes-related diseases, containing exosomes containing TM4SF5 (Transmembrane 4 L6 family member 5) protein.

The present inventors confirmed that exosomes loaded with TM4SF5, which is specifically derived from the liver, can promote glucose absorption into the blood in muscle cells, adipocytes, muscle tissue, or adipose tissue, and the hepatic vascular occlusion of the present invention TM4SF5-loaded exosomes specifically derived from the liver can be efficiently extracted through a circuit system and a method of extracting exosomes derived from liver or liver epithelial cells using the same, and a composition containing the extracted exosomes as an active ingredient is effective in treating diabetes. It can be useful as a treatment for related diseases.

Figure 1 is a schematic diagram of the liver vein-closed circulation system of the present invention.
Figure 2a shows the expression of APEX2-tagged TM4SF5 (TM4SF5-APEX2) in the multivesicular body (MVB) of the cells after transfection of TM4SF5-APEX2 plasmid into liver epithelial cells SNU449, followed by DAB staining. This is a diagram confirmed through a transmission electron microscope (TEM).
Figure 2b is a diagram confirming TM4SF5-APEX2 in exosomes isolated from the cells of Figure 2a through cryo-electron microscopy (cryo-EM).
Figure 3 is a diagram showing liver-derived exosomes in the sample obtained through a transmission electron microscope after obtaining a sample from a normal C57BL/6 mouse using the liver vascular occlusion circuit system.
Figure 4a is a diagram confirming the size of liver-derived exosomes using nanoparticle tracking analysis (NTA).
Figure 4b compares the sizes of liver-derived exosomes isolated from normal mice, Tm4sf5 knock-out (KO) mice (Tm4sf5 ^-/- ), and mice overexpressing liver epithelial cell-specific Tm4sf5 (Alb-Tm4sf5-FLAG TG). It's a graph.
Figure 4c is a diagram confirming the size of mouse plasma-derived exosomes using nanoparticle tracking analysis.
Figure 5a shows the level of Tm4sf5 expression in primary hepatic epithelial cells isolated from Tm4sf5 knockout (KO) mice (Tm4sf5 ^-/- ) and hepatic epithelial cell-specific Tm4sf5 overexpressing mice (Alb-Tm4sf5-FLAG). am.
Figure 5b is a diagram confirming the expression level of exosome markers Alix, TSG101, and Flottlin in the sample after obtaining exosome samples from Alb-Tm4sf5-FLAG mice using the hepatic vascular occlusion circuit system.
Figure 5c shows the expression level of TM4SF5 (-Strep) and exosome markers CD54 and ALIX after injecting control vector (EV, empty vector) and TM4SF5 expression vector (TM4SF5) into liver epithelial SNU449 cells to induce expression. It's a degree.
Figure 5d shows the expression of TM4SF5 by injecting a control vector and a TM4SF5 expression vector into liver epithelial SNU449 cells, and simultaneously injecting control siRNA (NS, non-specific) or siTM4SF5 (siRNA targeting #4 sequence of TM4SF5). This is a diagram confirming the expression levels of TM4SF5, GLUT1, and ALIX after control.
Figure 5e is a diagram confirming the expression levels of Tm4sf5, Alix, TSG101, CD63, CD81, and CD9 in the liver epithelial cells of Figure 5a and the exosome sample of Figure 5b.
Figure 5f is a diagram confirming the expression levels of TM4SF5, CD63, ALIX, and CD54 after liver epithelial Huh7 cells were maintained in a glucose-free cell culture medium for 24 hours and then treated with glucose at a given concentration for 2 hours.
Figure 5g shows the expression of TM4SF5, CD63, and ALIX in cell exudate or liver epithelial cell-derived exosomes after hepatic epithelial Huh7 cells were maintained in a glucose-free cell culture medium for 24 hours and then treated with glucose at a concentration of 25mM for a given time. This is a degree that confirms the degree.
Figure 5h shows liver epithelial SNU449 cells transfected with CD151-strep or TM4SF5-strep expression vector together with HA-GLUT1 expression vector, and then maintained in cell culture medium without glucose for 24 hours with (-) serum (10%). After treatment with 25mM of glucose and 10mM of amino acid or arginine for 24 hours (+), the cell exudate was obtained and the Strep-tagged protein was precipitated with avidin-sephrose beads. Western analysis showed that HA-GLUT1 was co-precipitated in the precipitate. This is a diagram confirmed through blotting.
Figure 5i is a diagram confirming the degree of binding between GLUT1 and TM4SF5 under the same experimental conditions as Figure 5h after expressing TM4SF5-Strep WT or several mutants in liver epithelial SNU449 cells.
Figure 5j shows that after controlling the expression of TM4SF5 by injecting control shRNA or ShTM4SF5 into liver epithelial Hep3B cells, the expression was maintained in cell culture medium without glucose for 4 hours, and then (0) glucose was added to 25mM for a given time (10 or 20 minutes) After treatment, the degree of phosphorylation of S6K1 (ribosomal protein S6 kinase beta-1) and AMPKα (catalytic subunit of AMP-activated protein kinase, AMPK) and the expression level of TM4SF5 were confirmed.
Figure 5k shows that liver epithelial Huh7 cells were maintained in cell culture medium with or without glucose (No Starv.) for 24 hours, and then treated with glucose at a concentration of 25mM for a given time, and then AMPK, ACCα (acetyl CoA carboxylase α), and mTOR. This is a diagram confirming the degree of phosphorylation of (mechanistic target of rapamycin) and S6K1 and the expression of exosomal markers.
Figure 5l shows that after controlling the expression of TM4SF5 by injecting control shRNA or ShTM4SF5 into hepatic epithelial Huh7 cells, the cells were grown normally in RPMI culture medium containing glucose (RPMI) or glucose in the cell culture medium was excluded for 24 hours. This figure shows the average size of the isolated exosomes after treatment (Glu+) or untreated (Glu-) with 25 mM for 24 hours using nanoparticle tracking analysis.
Figure 5m shows that after controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial cells Huh7 cells, cells were grown normally in RPMI culture medium (RPMI), or glucose in the cell culture medium was excluded for 24 hours, and then grown again at 25 This figure shows the average size of isolated exosomes analyzed by turnable resistive pulse sensing (TRPS) without treatment (glu-) or after treatment (Glu+) with mM for 24 hours.
Figure 5n is a diagram showing the size and concentration of exosomes derived from Huh7 cells after undergoing size-exclusion chromatography under the conditions of Figure 5m, and confirming the size and concentration using the single angle dynamic light scattering (DLS) method.
Figure 5o shows exosomes were isolated after controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial Huh7 cells. This is a diagram showing the analysis of proteins present in isolated exosomes using a MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) device. Figure 5o a shows PANDER (Protein Analysis Through Evolutionary Relationships, http://pantherdb. org) analysis (PANDER-GO-slim). Figure 5o b and c show the same analysis of the proteins of exosomes obtained from cells injected with shTM4SF5 (targeting #4 or #12 sequences, respectively). The proteins listed in red in Figure 5o a are proteins that exist only in exosomes obtained from cells expressing TM4SF5.
Figure 5p shows the precipitates (precipitates) of Strep-linked proteins after expression by injecting the control Strep-vector or Strep-linked TM4SF1, TM4SF4, TM4SF5, TM4SF18, TM4SF20, CD9, and CD151 together with HA-GLUT4, respectively, into the HEK293FT cell line. This is a diagram confirming the degree of binding to each other (left) or the level of expression of HA-GLUT4 and Strep (right) by confirming the presence of HA-GLUT4 in the genus by Western blot. The band (*) visible under EV injection conditions is a non-specific factor.
Figure 6a shows livers derived from three normal mice (#1 to #3) fed a normal diet and two normal mice (#4, #5) starved by restricting food for 24 hours using a hepatic vascular occlusive circuit system. This is a diagram showing the size of the exosomes after isolating them.
Figure 6b shows exosomes obtained by flowing media containing glucose for 1 hour when perfusing exosome circulating media in the same mouse using a hepatic vascular occlusion system, and then flowing media without glucose. This is a diagram in which the size of exosomes obtained during sending was confirmed based on the presence or absence of glucose.
Figure 6c shows control Strep-vector or Strep-TM4SF5 was injected together with HA-GLUT1, HA-GLUT2, HA-GLUT3, HA-GLUT4, or HA-GLUT9 into the HEK293FT cell line to express it, and then glucose was excluded from the cell culture medium. Or (-) after treatment with 25 mM for 24 hours (+), the presence of HA-GLUTs in the precipitates (precipitates) of Strep-TM4SF5 was confirmed by Western blot to show the degree of binding to each other (top) or HA-GLUTs and Strep -This is a diagram confirming the expression level of TM4SF5 (bottom).
Figure 6d is a diagram confirming the distribution location of TM4SF5-FLAG (red) and HA-GLUT1 (green) through fluorescence immunostaining after TM4SF5-FLAG and HA-GLUT1 were simultaneously injected and expressed in the SNU449 cell line. The presence of areas where colors overlap each other indicates the area where they combine.
Figure 6e shows the results of measuring glucose uptake into the cells after controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial Huh7 cells or treating them with 100 μM fasentin (a GLUT1/4 specific inhibitor). This is a diagram showing .
Figure 6f shows AML12 cells, which are normal liver epithelial cells in mice, were expressed by injecting a control vector or TM4SF5-HA, and then using a Seahorse This diagram shows the results of a glycolytic stress test by measuring ECAR (extracellular acidification rate) while sequentially treating deoxy-D-glucose.
Figure 6g shows ECAR by injecting control vector or TM4SF5-HA into AML12 cells and expressing them, followed by expression by injecting control vector (EV) or TM4SF5-HA, followed by treatment with oligomycin 1 μM and treatment with increasing concentrations of glucose. This diagram shows the results of measuring and confirming glucose sensitivity.
Figure 6h shows exosomes isolated after injecting control vector, control siRNA, Glut1, Glut2, or Glut4 siRNA into AML12 cells, and exosomes isolated from normal mouse brown adipose tissue (BAT) cells differentiated for 7 days. This diagram shows the results of measuring ECAR while sequentially adding glucose, oligomycin, and 2-DG to BAT cells after reaction.
Figure 6i After injecting control vector, control siRNA, Glut1, Glut2, or Glut4 siRNA into AML12 cells, exosomes were isolated and reacted with normal mouse brown adipose tissue (BAT) cells differentiated for 7 days. After treatment, BAT cells were treated with 1.0 μM of oligomycin and 0.5 mM of glucose. The concentration of glucose was increased to 1.0, 2.0, and 5.0 every 30 minutes, and ECAR was measured to confirm glucose sensitivity.
Figure 6j shows exosomes isolated from AML12 cells expressing control vector (EV) or Tm4sf5-Strep with DMSO or TSAHC [4'-(p-toluenesulfonylamido)-4-hydroxychalcone, TM4SF5 specific inhibitor] in brown adipose tissue. This is a diagram confirming the binding of Strep-TM4SF5 and GLUT4 by checking the expression level of Strep and Glut4 present in the precipitate of Strep-TM4SF5 in BAT cell extract after treatment with (BAT) cells.
Figure 6k shows Glucose Uptake-Glo?? after exosomes isolated from AML12 cells expressing control vector (EV) or Tm4sf5-Strep were treated with brown adipose tissue cells. This diagram shows the results of measuring glucose absorption using Assay (J1343, Promega, WI).
Figure 7a shows changes in glucose concentrations of 2.0, 4.0, 1.0, and 0.5 g/kg in hepatic epithelial cell-specific Tm4sf5-overexpressing mice and Tm4sf5 ^-/- mice (single-cohort study with repeated weekly measurements of about 10 mice each). This diagram shows the results of repeatedly examining blood glucose levels after injections once a week. After the initial four rounds of analysis, AAV8-Tbg-EV or AAV8-Tbg-Tm4sf5-HA (adeno-associated virus subtype 8 encoding the HA-tagged Tm4sf5 gene linked to the liver epithelial cell-specific thyroxine binding globulin (Tbg) promoter) (AAV8) This diagram shows the results of examining blood glucose levels after IV injection of the virus, waiting for 5 weeks, and then injecting 2.0 and 4.0 g/kg of glucose once a week for 2 weeks when the animal became 16 weeks old. .
Figure 7b shows the blood glucose levels of Tm4sf5 ^-/- mice and control normal mice when liver-derived exosomes isolated from liver epithelial cell-specific Tm4sf5 overexpressing mice (Alb-Tm4sf5 TG) were not treated with glucose (2 g/kg). This diagram shows the results of the investigation by time after intraperitoneal injection.
Figure 7c shows the blood glucose levels of Tm4sf5 ^-/- mice and control normal mice when treated with liver-derived exosomes isolated from liver epithelial cell-specific Tm4sf5 overexpressing mice (Alb-Tm4sf5 TG) using glucose (2 g/kg). This diagram shows the results of the investigation by time after intraperitoneal injection.
Figure 8 shows the extent to which exosomes obtained from Alb-Tm4sf5 TG mice or Tm4sf5 ^-/- KO mice migrate to various organs of normal mice 24 hours after IV injection using the hepatic vascular occlusion circuit system by performing surgery on each organ. This was confirmed using in vivo animal imaging equipment.
Figure 9a shows exosomes isolated from the AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector, separated from BAT, treated with differentiated adipocytes, and treated with BAT using Seahorse equipment with various drugs added. This diagram shows the results of a glycolysis stress test of adipocytes by exosome treatment by measuring the acidification of the extracellular solution of adipocytes.
Figure 9b shows that exosomes isolated from the AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector were treated with adipocytes isolated from brown adipose tissue and differentiated, and various concentrations of glucose were added using Seahorse equipment. This diagram shows the results of measuring the degree of activation of the glycolysis function by measuring the acidification of the extracellular solution due to glycolysis during addition.
Figure 9c shows exosomes (AML12-sEV ^HA-Tm4sf5 ) isolated from AML12 cells expressing HA-Tm4sf5 at a concentration of 4 x 10 ⁸ particles/condition and incubated in brown adipose tissue cells differentiated from normal mice for 24 hours. This diagram shows the results observed with a confocal fluorescence microscope after processing and staining Glut1 (left) or Glut4 (right) in red and HA-Tm4sf5 in green.
Figure 9d shows H&E staining and HA using brown adipose tissue cells isolated from normal WT mice or KO mice 3 weeks after injection of the control virus AAV8-EV or AAV8-Tbg-Tm4sf5-HA expressing the Tm4sf5-HA gene. This diagram shows the results of observing the degree of staining by immunostaining -Tm4sf5.
Figure 9e is a diagram showing the results of confirming the expression of signaling factors related to improved thermogenesis, mTOR expression and phosphorylation, and UCP1 (uncoupling protein-1) expression in the brown adipose tissue cells obtained in Figure 9d.
Figure 10a shows the results of treating C2C12 muscle cells with exosomes isolated from the AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector and analyzing the degree of glucose uptake into the muscle cells.
Figure 10b shows TM4SF5-exosomes (Huh7-Exo-from cells in RPMI) isolated from human liver epithelial cells Huh7, which originally express TM4SF5, were cultured in normal RPMI-1640 culture medium, and cultured in glucose-excluded culture medium. TM4SF5-exosomes (Huh7-Exo-from cells in glu-media), isolated after culturing in a culture medium containing 25 mM glucose, and TM4SF5-exosomes (Huh7-Exo-from cells in glu+ media), respectively. This diagram shows the results of confirming the sensitivity of glycolysis to various glucose concentrations after treating A204 muscle cells for 45 minutes.

Hereinafter, the present invention will be described in detail.

The present invention relates to a liver vascular occlusion circuit system for extracting liver-derived exosomes,

A peristaltic pump for circulating the hepatic vascular occlusive circuit;

Exosome circulating media and flushing buffer circulated along the hepatic vascular occlusive circuit by the peristaltic pump;

A three-way connector including a switch to control circulation of the exosome circulation media and flushing buffer;

a first tubing connecting the inlet of the peristaltic pump with the three-way connector and connecting the outlet of the peristaltic pump with a third tubing;

A second tubing connecting the exosome circulation media and the flushing buffer with the three-way connector, respectively;

A first catheter inserted into the subject's portal vein;

a third tubing connecting the first catheter and the first tubing;

A first connector connecting the first catheter and the third tubing;

a second catheter inserted into the subject's inferior vena cava;

A fourth tubing connecting the second catheter and the exosome circulation media;

A second connector connecting the second catheter and the fourth tubing; and

A fifth tubing connecting the exosome circulation media and fluffing buffer with an oxygen tank;

It provides a hepatic vascular occlusive circuit system including.

The exosomes refer to small membrane vesicles with a lipid bilayer derived from endosomes that are released by all cells in the body. Exosomes may refer to small vesicles secreted outside the cell with proteins, DNA, RNA, etc. in order to transmit signals between cells, and may include, for example, proteins, lipids, mRNA, microRNA (miRNA), and genomic DNA. . In addition, exosomes are known to play a variety of roles, such as binding to other cells and tissues and delivering membrane components, proteins, and RNA.

In a specific embodiment of the present invention, exosomes may be derived from any one or more selected from the group consisting of liver tissue, liver tissue/extracellular fluid, liver epithelial cells, and hepatocytes, and are preferably mouse. It may be derived from any one or more selected from the group consisting of liver tissue, liver epithelial cells, and hepatocytes, but is not limited thereto.

The flushing buffer may be contained in a constant temperature water bath. The constant temperature water tank can maintain a temperature of 40°C to 45°C, and preferably, the constant temperature water tank can maintain a temperature of 42°C.

The exosome circulation media and the flushing buffer may be connected to an oxygen tank.

In addition, the present invention relates to a method of extracting liver-derived exosomes using the liver vascular occlusion circuit system,

inserting the first catheter into the subject's portal vein;

Operating the peristaltic pump;

Connecting the first catheter and a first connector connected to the pump;

inserting the second catheter into the inferior vena cava of the subject;

Connecting the second catheter to a fourth tubing and connecting the other end of the fourth tubing to the exosome circulating media tube;

stopping the peristaltic pump and turning the switch on the three-way connector to supply the exosome circulating media to the pump;

Connecting the exosome circulation media and flushing buffer with an oxygen tank;

Reactivating the peristaltic pump to circulate the exosome circulation media;

Centrifuging the circulated exosome circulation media to separate the supernatant;

Concentrating the separated supernatant; and

It provides a liver-derived exosome extraction method comprising the step of isolating exosomes from the concentrated supernatant.

The step of circulating the exosome circulation media may be performed for 30 to 90 minutes, preferably for 45 to 75 minutes, and more preferably for 50 to 70 minutes. It is not limited to this.

In the step of centrifuging the circulated exosome circulation media to separate the supernatant, the centrifuge may be operated at a centrifugal force of 2,000 x g for 5 to 20 minutes, and a centrifugal force of 10,000 x g for 30 to 60 minutes. Typically, the centrifuge can be operated for 7 to 15 minutes at a centrifugal force of 2,000 x g, and for 40 to 50 minutes at a centrifugal force of 10,000 The centrifuge can be operated for 45 minutes, but is not limited to this.

In the step of concentrating the separated supernatant, the supernatant is obtained by centrifuging at 4°C for 10 minutes at 2000 do. The filtered supernatant was centrifuged at 10,000 It can be concentrated to 200 to 700 μl, and more preferably, 400 to 600 μl can be obtained and concentrated to about 5Х10 ⁹ particles/ml, but is not limited thereto.

The step of separating exosomes may be performed using size exclusion chromatography, but is not limited thereto.

The liver-derived exosomes may have an average size distribution area narrowed to 200 nm or less, and may also refer to exosomes with a wide distribution area including a size smaller than 200 nm.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes-related diseases, containing exosomes containing TM4SF5 (Transmembrane 4 L6 family member 5) protein.

The exosome may be derived from any one or more selected from the group consisting of liver tissue, liver extracellular fluid, liver epithelial cells (hepatocytes), and hepatocytes, and the exosome is capable of controlling blood sugar levels. Preferably, the exosome is It can regulate homeostasis by promoting glucose uptake in the blood by muscle cells, adipocytes, muscle tissue, or adipose tissue.

The exosomes allow the loaded TM4SF5 to bind to Glut1, require the activity of Glut1, promote the uptake of glucose in the blood into liver epithelial cells, activate glycolysis in liver epithelial cells, or regulate glucose homeostasis in the blood. (homeostasis) can be controlled.

The exosomes allow the loaded TM4SF5 to bind to Glut4, require the activity of Glut4, promote the absorption of glucose in the blood into muscle cells/tissues or fat cells/tissues, or promote glycolysis in muscle or fat tissues and cells. It can activate or regulate glucose homeostasis in the blood.

The liver-derived exosomes may have an average size distribution area narrowed to 200 nm or less, and may also refer to exosomes with a wide distribution area including a size smaller than 200 nm.

The diabetes-related disease may be any one or more diseases selected from the group consisting of fatty liver, steatohepatitis, diabetes, hyperlipidemia, hypertension, obesity, microvascular damage, nerve damage, ketoacidosis, arteriosclerosis, cardiovascular disease, and cerebrovascular disease, , Preferably, the diabetes-related disease is diabetes, but is not limited thereto.

In a specific example of the present invention, to confirm whether TM4SF5 protein is loaded in exosomes present in human liver epithelial cells, TM4SF5-APEX2 plasmid is transfected into liver epithelial cells SNU449, and then 48 hours. After DAB staining and transmission electron microscopy, the expression and location of TM4SF5 protein (TM4SF5-APEX2 protein) labeled with APEX2 (ascorbate peroxidase 2) was confirmed. As a result, black was found within the multivesicular body (MVB) of the liver epithelial cells. It was confirmed that the color-stained TM4SF5-APEX2 protein was surrounded by a membrane (see Figure 2a), and as a result of separating exosomes from the liver epithelial cells and confirming the presence of TM4SF5 protein through cryo-electron microscopy, the exosomes TM4SF5-APEX2 protein stained black was confirmed within (see Figure 2b).

In addition, in order to confirm whether exosomes are present in the sample obtained through the hepatic vascular occlusion system, the liver of a normal mouse (C57BL/6) was used and the liver occlusion was performed for 1 hour through the hepatic vascular occlusion system. As a result of obtaining a sample by circulating it and confirming the presence of exosomes in the obtained sample through a transmission electron microscope, it was confirmed that exosomes were present in the sample, and the exosomes were generally confirmed to have a diameter of 200 nm or less ( 3).

In addition, in order to confirm the difference in physical properties between general exosomes obtained from plasma (serum) and liver-derived exosomes obtained by circulating in the liver vascular occlusion circuit for 1 hour, the sizes of liver-derived exosomes and plasma-derived exosomes were measured. As a result of the measurement comparison, it was confirmed that the size of exosomes derived from the liver of Tm4sf5 KO mice was generally larger than that of normal mice (see Figure 4a). In addition, it was confirmed that the higher the expression of Tm4sf5 in liver epithelial cells, the smaller the size of liver epithelial cell-derived exosomes (see Figure 4b), and the average size of exosomes obtained from the plasma of normal mice was relatively higher than that of the liver-derived exosomes. It was confirmed to be smaller compared to the moth (see Figure 4c).

In addition, Tm4sf5 KO mice (Tm4sf5 ^-/- ) and transgenic mice (Alb-Tm4sf5-FLAG) in which the Tm4sf5 gene is linked to the promoter of the albumin gene present in liver epithelial cells, causing the Tm4sf5 gene to be overexpressed only in the liver epithelial cells of the animal. As a result of performing a Western blot using primary hepatocytes isolated from After circulating the hepatic vascular occlusive circuit for 1 hour and separating the samples, Western blotting was performed to identify proteins expressed in exosomes contained in the separated samples. As a result, Alix, a well-known marker for exosomes, was used. , Expression of TSG101 and Flottlin proteins was confirmed (see Figure 5b), and expression was induced by injecting control vector (EV, empty vector) and TM4SF5 expression vector (TM4SF5) into hepatic epithelial SNU449 cells, and then TM4SF5 (-Strep) ) and the expression level of exosome markers CD54 and ALIX, the N138A/N155Q mutation, which is unable to glycosylate TM4SF5, was not present in exosomes. On the other hand, when TM4SF5 was present, it was confirmed that the presence of ALIX was significantly reduced (see Figure 5c). The control vector and TM4SF5 expression vector were injected into liver epithelial SNU449 cells, and the expression of TM4SF5 was controlled by simultaneously injecting control siRNA (NS, non-specific) or siTM4SF5 (siRNA targeting #4 sequence of TM4SF5). , As a result of checking the expression levels of TM4SF5, GLUT1, and ALIX, when the presence of TM4SF5 in exosomes increased, the expression levels of GLUT1 and ALIX decreased, and when the presence of TM4SF5 was decreased (by siTM4SF5), GLUT1 and ALIX were present. The amount did not decrease (see Figure 5d).

As a result of confirming several proteins expressed in the primary liver epithelial cells and exosomes isolated through the liver vascular occlusive circuit system, it was confirmed that Tm4sf5(-FLAG), TSG101, and ALIX were expressed in the primary liver epithelial cells, and the liver blood vessels It was confirmed that Tm4sf5, TSG101, ALIX, CD63, and CD81 were expressed in exosomes isolated through a closed-circuit system (see Figure 5e).

The control vector and TM4SF5 expression vector were injected into liver epithelial SNU449 cells, and the expression of TM4SF5 was controlled by simultaneously injecting control siRNA (NS, non-specific) or siTM4SF5 (siRNA targeting #4 sequence of TM4SF5). , As a result of checking the expression levels of TM4SF5, GLUT1, and ALIX, the amount of TM4SF5 and CD63 loaded into exosomes gradually increased over time after glucose treatment, but ALIX showed a weak increase and then a tendency to decrease. (See Figure 5f).

Liver epithelial cells Huh7 cells were maintained in a cell culture medium without glucose for 24 hours and then treated with 25 mM glucose for a given period of time. As a result of checking the expression levels of TM4SF5, CD63, and ALIX, TM4SF5 and CD63 were observed after treatment with glucose. Although the amount loaded into exosomes tended to gradually increase over time, ALIX showed a tendency to increase more weakly (see Figure 5g).

Liver epithelial Huh7 cells were maintained in cell culture medium without glucose for 24 hours and then treated with glucose at a concentration of 25mM for a given period of time. As a result of checking the expression levels of TM4SF5, CD63, and ALIX, when glucose was excluded from the outside of the cells, TM4SF5 In contrast to the fact that the binding to GLUT1 was successful, it was confirmed that the binding was inhibited when glucose was supplied (i.e., when TM4SF5-loaded exosomes were secreted) (see Figure 5h).

After transfecting HA-GLUT1 with a CD151-strep or TM4SF5-strep expression vector into hepatic epithelial SNU449 cells, the expression level of HA-GLUT1 was confirmed. As a result, WT, Pro153Ala, and Val156Ala mutant TM4SF5 were affected by treatment with extracellular glucose. It was confirmed that the binding affinity to GLUT1 was lost, but the binding affinity of the Thr157Ala mutant TM4SF5 was maintained (see Figure 5i).

After expressing TM4SF5-Strep WT or several mutants in hepatic epithelial cells SNU449 cells, the expression level of GLUT1 was checked. As a result, in case of TM4SF5 expression, in case of expression repression, S6K1 (ribosomal protein S6 kinase beta-1) ) phosphorylation increased, and phosphorylation of AMPKα (catalytic subunit of AMP-activated protein kinase, AMPK) decreased, thereby ensuring a high-energy state (see Figure 5j).

After controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into liver epithelial Hep3B cells, S6K1 (ribosomal protein S6 kinase beta-1), ACCα (acetyl CoA carboxylase α), mTOR, AKT and AMPKα (catalytic subunit of The degree of phosphorylation of AMP-activated protein kinase (AMPK) and the expression of exosome biomarkers were confirmed. Compared to the case where glucose was not treated, the phosphorylation of AMPK and ACC gradually decreased over time, indicating a high energy state, and the phosphorylation of mTOR (mechanistic target of rapamycin), S6K1, and AKT. increased (see Figure 5k).

After controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial Huh7 cells, the cells were grown normally in RPMI culture medium (RPMI), or glucose in the cell culture medium was excluded for 24 hours, and then grown again at 25 mM for 24 hours. As a result of nanoparticle tracking analysis of the average size of exosomes isolated after treatment (Glu-) or treatment (Glu+) for a period of time, when TM4SF5 was expressed, exosomes were larger when treated again compared to when glucose was excluded. The average size of the moths was reduced, but this phenomenon was not observed when the expression of TM4SF5 was suppressed (see Figure 5l).

After controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial Huh7 cells, the cells were grown normally in RPMI culture medium (RPMI), or glucose in the cell culture medium was excluded for 24 hours, and then grown again at 25 mM for 24 hours. As a result of analyzing exosomes isolated for a period of time without treatment (glu-) or after treatment (Glu+), it was confirmed that the size of exosomes decreased with glucose treatment (Figure 5m). reference).

As a result of analyzing the size and concentration of Huh7 cell-derived exosomes that underwent size-exclusion chromatography under the same conditions as in Figure 5M using single angle dynamic light scattering (DLS), it was found that TM4SF5-loaded exosomes secreted from liver epithelial cells were outside the cell. When glucose was excluded and treated with high concentration, it was secreted, and it was confirmed that the average size was much smaller (see Figure 5n).

After controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial Huh7 cells, exosomes were isolated. As a result of analyzing the proteins present in the isolated exosomes using a MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) device, TM4SF5 was present in the exosome (a) isolated from cells expressing TM4SF5. Compared to exosomes obtained from non-expressing cells (b and c), it was confirmed that proteins related to biological adhesion (listed in red) were additionally present (see Figure 5o).

HEK293FT cell line was injected with a control Strep-vector or Strep-linked TM4SF1, TM4SF4, TM4SF5, TM4SF18, TM4SF20, CD9, and CD151, respectively, together with HA-GLUT4, and then combined with HA-GLUT4 and Strep-linked proteins. As a result of confirming the extent, it was confirmed that TM4SF5 binds well to GLUT4, unlike TM4SF18 and CD151, with TM4SF1, TM4SF4, TM4SF20, and CD9 (see Figure 5p).

In addition, in order to determine whether liver-derived exosomes change depending on the blood concentration of glucose, physical changes in exosomes according to the blood glucose concentration were measured using a hepatic vascular occlusion system. As a result, three normal mice under normal diet conditions were found. The average size of liver-derived exosomes isolated from 179.433 ± 11.272 nm was 179.433 ± 11.272 nm, while the average size of liver-derived exosomes isolated from 2 normal mice under feed-restricted conditions was 276.9 ± 3.818 nm, which is equivalent to the average size of 276.9 ± 3.818 nm under normal diet conditions. It was confirmed that the size distribution of exosomes was very wide compared to mouse exosomes (see Figure 6a), and when perfusion of exosome circulation media in the same normal mouse, media containing glucose was flowed for 1 hour. As a result of checking the size of the obtained exosomes and the exosomes obtained by flowing media that did not contain glucose according to the presence or absence of glucose, the size distribution of the exosomes when converted from the state in which glucose was present to the state in which it was not present was found to be It was confirmed that it was widening (see Figure 6b).

After injecting the control Strep-vector or Strep-TM4SF5 together with HA-GLUT1, HA-GLUT2, HA-GLUT3, HA-GLUT4, and HA-GLUT9 into the HEK293FT cell line to express them, glucose was excluded from the cell culture medium or (-) After treatment with 25 mM for 24 hours (+), the binding of HA-linked protein(s) and Strep-TM4SF5 was confirmed, and it was confirmed that TM4SF5 bound to GLUT1, GLUT4, and GLUT9 (see Figure 6c).

After TM4SF5-FLAG and HA-GLUT1 were simultaneously injected and expressed in the SNU449 cell line, the distribution location of TM4SF5-FLAG and HA-GLUT1 was confirmed through fluorescence immunostaining, and the results showed that TM4SF5 and GLUT1 coexist in the liver epithelial cell membrane. It was confirmed that binding was possible by doing this (see Figure 6d).

After controlling the expression of TM4SF5 by injecting control shRNA or shTM4SF5 into hepatic epithelial Huh7 cells or treating them with 100 μM facentin, glucose uptake into the cells was measured, and results showed that TM4SF5 expression was suppressed or GLUT1/4 inhibitor ( It was confirmed that glucose absorption was reduced when treated with fasentin (see Figure 6e).

After injecting and expressing control vector or TM4SF5-HA into AML12 cells, which are normal liver epithelial cells of mice, a glycolytic stress test was performed. As a result, the degree of ECAR was much higher in cells expressing TM4SF5, confirming that glycolysis was activated. (See Figure 6f).

After expressing by injecting control vector or TM4SF5-HA into AML12 cells, and then expressing by injecting control vector (EV) or TM4SF5-HA, glucose sensitivity was confirmed. As a result, ECAR was observed even when treated with low glucose in cells expressing TM4SF5. The degree of was much higher, confirming that glycolysis can be activated more sensitively to blood sugar (see Figure 6g).

After injecting control vector, control siRNA, Glut1, Glut2, or Glut4 siRNA into AML12 cells, exosomes were isolated and reacted with normal mouse brown adipose tissue (BAT) cells differentiated for 7 days, followed by glucose, oligomycin, As a result of measuring ECAR while sequentially adding and 2-DG, exosomes secreted when TM4SF5 and Glut1/Glut4 are expressed in liver epithelial cells cause activation of glycolysis in target cells (brown adipose tissue cells). It has been confirmed that this can be done. (See Figure 6h).

After injecting control vector, control siRNA, Glut1, Glut2, or Glut4 siRNA into AML12 cells, exosomes were isolated, reacted with normal mouse brown adipose tissue (BAT) cells differentiated for 7 days, and incubated with 1.0 μM of oligomycin. While treating with 0.5mM of glucose, the concentration of glucose was increased to 1.0, 2.0, and 5.0 every 30 minutes, and glucose sensitivity was confirmed by measuring ECAR. As a result, the target cell glycolysis of exosomes derived from liver epithelial cells expressing TM4SF5 was confirmed. It was confirmed that Glut4 in liver epithelial cells is not required for the activation function (see Figure 6i).

After processing exosomes isolated from AML12 cells expressing control vector (EV) or Tm4sf5-Strep into brown adipose tissue (BTA) cells, the degree of binding of Strep and Glut4 was confirmed. As a result, hepatic epithelial cell-derived Tm4sf5-loaded exosomes It was confirmed that the microbes attached to BAT target cells allowed Tm4sf5-Strep and Glut4 to bind (see Figure 6j).

Exosomes isolated from AML12 cells expressing control vector (EV) or Tm4sf5-Strep were treated with brown adipose tissue (BAT) cells, and glucose uptake was measured. As a result, hepatic epithelial cell-derived exosomes expressing Tm4sf5 When treated, it was confirmed that glucose absorption increased, especially depending on concentration (see Figure 6k).

In addition, 8-week-old liver epithelial cell-specific Tm4sf5-overexpressing mice and Tm4sf5 ^-/- mice were injected with glucose once a week at concentrations of 2.0, 4.0, 1.0, and 0.5 g/kg, and the blood glucose levels were examined. As a result, WT and alb-TG mice were confirmed to have lower blood glucose levels than Tm4sf5-/- KO mice (left of the dotted line). Meanwhile, as a result of checking the blood glucose level 5 weeks after IV injection of AAV8-Tbg-EV and AAV8-Tbg-Tmsf5-HA, there was a difference in the blood glucose level between WT mice and Tm4sf5-/- KO mice. It was confirmed that it did not appear (right side of the dotted line) (see Figure 7a).

In addition, to confirm the effect of liver-derived exosomes loaded with TM4SF5 on glucose uptake and blood sugar level control in various cells or tissues, the Tm4sf5 gene was overexpressed in the liver using the albumin promoter (Alb-Tm4sf5). ), exosomes obtained through the hepatic vascular occlusion circuit system were separated through size exclusion chromatography, and liver-derived exosomes loaded with TM4SF5 were obtained from normal mice (WT) or Tm4sf5 KO mice (Tm4sf5 ^-/- ). After administration, the results were confirmed. In general, Tm4sf5 ^-/- mice exhibit a phenotype in which their blood glucose processing ability is lower than that of normal mice for a certain period of time after glucose injection (see Figure 7b), whereas exosomes derived from the liver of Alb-Tm4sf5 mice are injected into normal mice. When injected into Tm4sf5 ^-/- mice, the level of glucose in the blood was lowered, confirming that the glucose processing ability was significantly improved compared to the control group (see Figure 7c).

To confirm the extent to which liver-derived exosomes obtained by the hepatic vascular occlusion circuit system migrate to tissues and organs, liver-derived exosomes were injected into normal mice and observed using in vivo animal imaging equipment. As a result, Alb-Tm4sf5 There was no significant difference between exosomes derived from the liver of TG mice or exosomes derived from the liver of Tm4sf5 ^-/- KO mice, but exosomes derived from the liver of Tm4sf5 ^-/- KO mice reached the lungs and leg bones more during 24-hour treatment. It was confirmed that exosomes derived from the liver of Alb-Tm4sf5 TG mice loaded with TM4SF5 reached and were absorbed further into the brown adipose tissue (BAT) tissue on the back of Tm4sf5 ^-/- (see Figure 8).

In addition, exosomes isolated from the rodent hepatic epithelial AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector were treated with adipocytes isolated from mouse BAT and differentiated, and various drugs were administered using Seahorse equipment. By measuring the acidification of the extracellular solution of BAT adipocytes in the added state and performing a glycolysis stress test on adipocytes by exosome treatment, Tm4sf5 was present when treated with exosomes loaded with Tm4sf5. It was confirmed that the glycolysis capacity of adipocytes increased significantly compared to the control group treated with exosomes (see Figure 9a). When exosomes loaded with Tm4sf5 were treated together with an inhibitor of Glut1/4 (fasentin), the extracellular acidification rate (ECAR) was confirmed to be low, confirming that the treatment effect of exosomes loaded with Tm4sf5 exists in adipocytes. It was confirmed that it depends on the presence (and therefore binding to TM4SF5) and activation of Glut4.

In addition, exosomes isolated from the rodent hepatic epithelial AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector were treated with adipocytes isolated from mouse BAT and differentiated, and then mixed at various concentrations using Seahorse equipment. As a result of measuring the degree of activation of the glycolysis function by measuring the acidification of the extracellular solution by glycolysis while additionally adding glucose, it was found that exosomes isolated from AML12 cells expressing TM4SF5 could be processed into BAT adipocytes. In this case, compared to exosomes isolated from cells that do not express TM4SF5, it was confirmed that the function of adipocyte glycolysis was improved under various glucose addition conditions (see Figure 9b).

Exosomes (AML12-sEV ^HA -Tm4sf5) isolated from AML12 cells expressing HA-Tm4sf5 were isolated from normal mice at a concentration of 4 x 10 ⁸ particles/condition and treated with differentiated brown adipose tissue cells for 24 hours. After staining Glut1 (left) or Glut4 (right) in red and HA-Tm4sf5 in green, the results were observed under a confocal fluorescence microscope. As a result, AML12-sEV ^HA -Tm4sf5 was found to be stained around Glut1 or cell surface areas in BAT cells. It was confirmed that it coexisted with Glut4 and was stained yellow (see Figure 9c).

Three weeks after injection of the control virus AAV8-EV or AAV8-Tbg-Tm4sf5-HA expressing the Tm4sf5-HA gene, the degree of HA-Tm4sf5 staining was observed using brown adipose tissue cells isolated from normal WT mice or KO mice. As a result, it was confirmed that the BAT of animals injected with the Tm4sf5-HA expressing AAV8 virus had smaller and less fat droplets than the BAT of animals injected with AAV8-EV (see Figure 9d).

As a result of identifying signaling factors related to improved thermogenesis in brown adipose tissue cells, the expression level of UCP1 protein did not change in brown adipose tissue cells of each animal to be unrelated to different thermogenesis, but compared to injection of AAV8-EV virus into KO mice. It was confirmed that mTOR phosphorylation increased in animals injected with the AAV8 virus expressing Tm4sf5-HA (see Figure 9e).

In addition, to confirm the effect of liver-derived exosomes on glucose metabolism in muscle cells, exosomes isolated from the AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector were treated with C2C12 muscle cells and incubated with the muscles. As a result of analyzing the degree of glucose uptake into cells, it was confirmed that when exosomes isolated from the AML12 cell line or AML12 cell line expressing only the control vector were treated, the amount of glucose uptake increased compared to the case where exosomes were not treated. In particular, it was confirmed that when exosomes loaded with Tm4sf5 were treated, the degree of glucose absorption was higher compared to when exosomes not loaded with Tm4sf5 were treated (see Figure 10a).

In addition, TM4SF5-exosomes (Huh7-Exo-from cells in RPMI) were isolated and obtained from human liver epithelial cells Huh7, which originally express TM4SF5, while cultured in normal RPMI-1640 culture medium, and then cultured in glucose-excluded culture medium. The isolated TM4SF5-exosomes (Huh7-Exo-from cells in glu-media) and the isolated TM4SF5-exosomes (Huh7-Exo-from cells in glu+ media) were cultured in a culture medium containing 25 mM glucose, respectively, at A204. After treating muscle cells for 45 minutes, the sensitivity of glycolysis to various glucose concentrations was confirmed. TM4SF5- isolated from Huh7 cells cultured in RPMI media without glucose control or cultured in culture medium from which glucose was removed. It was confirmed that the sensitivity of glycolysis to glucose was higher when A204 muscle cells were treated with TM4SF5-exosomes isolated from a culture medium containing 25 mM glucose than exosomes (see FIG. 10b).

Hereinafter, the present invention will be described in detail through the following examples and experimental examples. However, the following examples and experimental examples only illustrate the present invention, and the present invention is not limited thereto.

<Example 1> Production of liver-vein closed circulation system

In order to obtain exosomes specifically derived from animal liver tissue, a liver-vein closed circulation system was created (Figure 1).

Specifically, the inlet part of the peristaltic pump was connected to a 3-way connector with a switch with tubing 1 (Customized 1x1, ID - 1 mm, OD 2 mm - EMS Tech), and the remaining two connections of the connector were connected to the oxygen tank. It was connected to the flushing buffer and the exosome circulation media using tubing 2 (Masterflex Platinum L/S 96410-13), and the flushing buffer was stored in a 42°C constant temperature water bath (Benchmark (B2000-4- E) It was kept immersed in water. Tubing 3 (Masterflex Platinum L/S 96410-14) was connected to the peristaltic pump outlet of tubing 1, and a Luer lock connector (Sungho Sigma, C20-140- 660 (PVLM20)) was connected, and tubing 4 (Silicon tubing ID-3 mm, OD 6 mm - Daihan) was connected to a luer lock connector on one side and to exosome circulation media on the other side. At this time, all tubing was positioned on the same plane.

<Example 2> Isolation of exosomes using a hepatic vascular occlusion system

Anesthetize the experimental mouse by placing it in an airtight glass container (~200 ml size) containing a cotton ball soaked with 2 ml 30% isoflurane, then remove the plunger from the 10 ml syringe and inject 2 ml A cotton ball soaked in 30% isoflurane was placed and the mouth and nose of the mouse were inserted into the entrance. At this time, the area where the mouse's mouth and nose come in contact with the anesthetic solution was prevented. The anesthetized mouse was placed on a heating pad (JD-OT-03/06) set at 42°C, the front and hind legs were fixed with tape, and the abdomen was disinfected with ethanol. Afterwards, an incision was made 3 to 4 cm from 1 cm above the genitals with surgical scissors, the organs were pushed to the right and taken out, and the liver was attached to the diaphragm using the side part of a straight forcep to reveal the portal vein. Separate the portal vein and the surrounding fat using a microdissecting forcep (3 3/4" Octagoanl grip, (BRI surgical-10-2105)) at a point 1 to 1.5 cm away from the liver, and place a surgical suture (Iri, Black) into the separated area. Silk No. 3) was passed, and a loose knot was made. Then, a catheter (24G, Becton Dickinson, Insyte Autoguard 381812) was inserted into the portal vein, and the needle was released by pressing the retract button on the catheter. Upon removal, it was confirmed that the blood was flowing back. The position of the catheter was fixed and the knot was tightened, and the tip of the catheter was placed 1 to 2 mm away from the entrance into the liver. /MC4 pump head) was operated to adjust the flow rate to about 1 ml/min so that the blood flowing back from the catheter and the flushing buffer formed on the luer lock connector connected to the pump met and the connection was tightened to prevent air bubbles from entering. The liver was placed back in its original position, and the chest was incised to expose the heart and inferior vena cava. The vein where the catheter would be inserted was clearly visible and connected to the inferior vena cava. A catheter was inserted into the inferior vena cava using a wide opening and fixed with a suture. At this time, the blood coming out of the liver was mixed with flushing buffer [1x HBSS - 10x HBSS (Sigma Aldrich, H4641) diluted 1:10, 4.2 mM NaHCO. ₃ (Sigma Aldrich-S5761), 0.5 mM EGTA (Sigma Aldrich-E4378), pH 7.2, 0.2 μm sterilized filter] was checked and the tube was supported and maintained for 30 minutes while the blood was removed from the liver. Body temperature was maintained using Seonglim Electronic Medical Device (HH-2500), and the open chest and abdomen were covered with sterile gauze soaked in saline (Jungwae Pharmaceutical, Crinkle) for 30 minutes, and then placed on the catheter connected to the inferior vena cava. Tubing 4 was connected and the other end was connected to the tube containing exosome circulation media. Stop the pump for a moment, turn the switch, and transfer the exosome circulating media [RPMI 1640 (Hyclone, SH30027.01), 10% 100 kDa Ultrafiltered FBS (Gendepot, F0600-050), FBS to a 100 kDa membrane centrifugal concentrator (Sartorius, VS2041). ) was used to filter, 1 Х Penicillin/Streptomycin (Gendepot, CA002-010)] was supplied to the pump, and then the pump was operated again. After confirming that the exosome circulation media from the inferior vena cava entered the original tube well, it was circulated for 1 hour. After 1 hour, the circulated media was centrifuged at 2000 xg for 10 minutes and 10,000 xg for 45 minutes, and exosomes were separated from the supernatant. The separated supernatant was concentrated to 500 μl using a 50 kDa membrane centrifugal concentrator (Sartorius, VS2032). Concentrated exosomes were loaded onto qEV original (Izon, qEVoriginal), a size exclusion chromatography (SEC). Additionally, 2.5 ml of PBS was added and passed, then discarded, and 1.5 ml of PBS was added again to obtain the passed exosome sample.

<Example 3> Hepatocyte separation

After connecting the mouse to the hepatic vascular occlusion system, After circulation for 1 hour, hepatocyte isolation buffer [Flushing Buffer (40ml), type I collagenase (7.5mg, Sigma Aldrich -C7661) - (Sigma Aldrich, C5138)] was connected to the pump and allowed to enter the liver. The flow rate was increased to 4 ml/min and the inferior vena cava was cut below the kidney to allow the buffer to come out. To allow collagenase to enter various parts of the liver, the upper part of the resected inferior vena cava was held with forceps to create counter pressure and released again. After 10 minutes, the pump was stopped, the liver was transferred to a petri dish, 5 ml of hepatocyte growth media was added, the liver membrane was opened with forceps, and the cells were separated by shaking.

The separated cells were passed through a 100μm mesh (SPL-93100) and placed in a 50ml tube (SPL-50050). This process was repeated with the remaining liver tissue until no more cells fell off. The tube containing the detached cells was centrifuged at 50 x g for 5 minutes, and the supernatant was discarded. Hepatocyte growth media again [Medium 199 - (Hyclone- SH30253.01), 10% FBS (Gendepot- F0600-050), 23 mM HEPES (Gibco-15630080), 10 nM Dexamethasone (Calbiochem-265005), 1x penicillin/streptomycin ( Add 20 ml of Gendepot-CA002-010), mix well, centrifuge at 50 x g for 5 minutes, and discard the supernatant.

Cells were placed back into 10 ml hepatocyte growth media. Add 7 ml of 42% percoll [6.3 ml Percoll - GE Healthcare, 17-0891-02, 700 μl 10x PBS, 8 ml Medium 199 - (Hyclone- SH30253.01)] to a new 50 ml tube and carefully incubate the cells. Raised 10ml. Percoll/cells were centrifuged at 300 x g for 5 minutes and the supernatant was discarded. The cells were put back into 2 ml hepatocyte growth media, the number of cells was counted, and the cells were placed in a collagen-coated dish (SPL-20100) or flask and cultured.

<Example 4> Isolation of exosomes from hepatic epithelial cell lines

인큐베이터 (Sanyo- MCO18AC)에 16시간 배양하였다. PBS로 2번 씻은 후, 엑소좀 순환 미디어 20 ml을 넣고 48시간 37℃인큐베이터에 48시간 배양하였다. 배양액을 2000 x g에서 10분간 원심 분리하고, 상층액을 0.2 μm syringe filter로 여과하였다. 여과한 배양액을 10,000 x g에서 45분간 원심 분리하고, 상층액을 새로운 튜브에 옮겼다. 옮긴 상층액을 50 kDa membrane 원심분리 농축기(Sartorius-VS2032)를 이용하여 500 μl까지 농축하였다. 농축된 엑소좀을 qEV original (Izon - qEVoriginal)에 로딩하였다. 추가로 2.5 ml PBS를 넣어 통과시켜 버리고 다시 1.5 ml PBS를 넣어 통과된 엑소좀 샘플을 획득하였다.Liver epithelial cells obtained from cell lines or animal livers were placed in a 150 mm cell culture dish (SPL-20150) filled with 70 to 80% culture media.

Cultured in an incubator (Sanyo-MCO18AC) for 16 hours. After washing twice with PBS, 20 ml of exosome circulation media was added and cultured in an incubator at 37°C for 48 hours. The culture was centrifuged at 2000 xg for 10 minutes, and the supernatant was filtered through a 0.2 μm syringe filter. The filtered culture was centrifuged at 10,000 xg for 45 minutes, and the supernatant was transferred to a new tube. The transferred supernatant was concentrated to 500 μl using a 50 kDa membrane centrifugal concentrator (Sartorius-VS2032). Concentrated exosomes were loaded into qEV original (Izon - qEVoriginal). Additionally, 2.5 ml of PBS was added and passed through, and 1.5 ml of PBS was added again to obtain the passed exosome sample.

<Experimental Example 1> Confirmation of whether TM4SF5 protein is loaded in exosomes derived from human liver epithelial cells

To confirm whether TM4SF5 protein is loaded in exosomes present in human liver epithelial cells, TM4SF5-APEX2 plasmid was transfected into liver epithelial cells SNU449 (Korea Cell Line Bank, Seoul, Korea). After 48 hours, DAB staining was performed, and the expression and location of TM4SF5 protein (TM4SF5-APEX2 protein) labeled with APEX2 (ascorbate peroxidase 2) was confirmed using a transmission electron microscope (Talos L120C, FEI, NICEM, Seoul National University). .

As a result, it was confirmed that TM4SF5-APEX2 protein, stained black, was surrounded by a membrane within the multivesicular body (MVB) of the liver epithelial cells (Figure 2a).

In addition, as a result of separating exosomes from the liver epithelial cells and confirming the presence of TM4SF5 protein through cryo-electron microscopy (cryo-EM), the TM4SF5-APEX2 protein stained black in the exosomes was found. confirmed (Figure 2b). This suggests that exosomes derived from liver epithelial cells are loaded with TM4SF5.

<Experimental Example 2> Size comparison between liver-derived exosomes and plasma-derived exosomes

<2-1> Confirmation of the presence of exosomes in samples obtained using the hepatic vascular occlusion circuit system

In order to confirm whether exosomes are present in the sample obtained through the hepatic vascular occlusion circuit system, the liver vascular occlusion circuit system of Example 1 was performed using the liver of a normal mouse (C57BL/6, Central Laboratory Animal, Seongnam-si). A sample was obtained by circulating the hepatic vascular occlusion circuit for 1 hour using the method of Example 2. The presence of exosomes in the obtained samples was confirmed through a transmission electron microscope (Talos L120C, FEI, NICEM, Seoul National University).

As a result, it was confirmed that exosomes existed in the sample, and exosomes were generally confirmed to have a diameter of 200 nm or less (Figure 3).

<2-2> Size comparison between liver-derived exosomes and plasma-derived exosomes

In order to confirm the difference in physical properties between typical exosomes obtained from plasma (serum) and liver-derived exosomes obtained by circulating in the liver vascular occlusion circuit for 1 hour, the sizes of liver-derived exosomes and plasma-derived exosomes were measured and compared. did.

Specifically, liver-derived exosomes were obtained from samples obtained by circulating the liver vascular occlusive circuit for 1 hour using the livers of three normal mice and five Tm4sf5 KO mice (Tm4sf5 ^-/- ). And exosomes were obtained from the plasma of 10 normal mice. The size of the obtained exosomes was measured through nanoparticle tracking analysis.

As a result, the average size of liver-derived exosomes in normal mice (WT) was 190.01 ± 28.64 nm (mean value ± standard deviation), the average size of liver-derived exosomes in KO mice was 210.23 ± 13.18 nm, and that of Alb-TG mice was 210.23 ± 13.18 nm. The average size of liver-derived exosomes was 162.02 ± 18.70 nm, that is, it was confirmed that the higher the expression of Tm4sf5 in liver epithelial cells, the smaller the size of liver epithelial cell-derived exosomes (Figures 4a and b).

On the other hand, the average size of exosomes obtained from the plasma of 10 normal mice was 131.59 nm, which was found to be relatively smaller than the liver-derived exosomes (Figure 4c).

<Experimental Example 3> Confirmation of TM4SF5 expression in animal liver epithelial cells, hepatocytes, and liver-derived exosomes

<3-1> Confirmation of TM4SF5 expression in hepatic epithelial cells and mouse liver-derived exosomes

To confirm the type and expression level of proteins present in exosomes in animal liver epithelial cells, hepatocytes, and liver-derived exosomes, protein electrophoresis and Western blot were performed.

Specifically, the TM4SF5-APEX2 plasmid, in which pcDNA3 APEX2 (Addgene) was linked to the TM4SF5 gene, was inoculated into SNU449 liver epithelial cells (Korean Cell Bank, Seoul, Korea) using lipofectamine 3000 (Thermo Fisher Scientific) according to the protocol provided by the company. Transfection was performed for a period of time. Exosomes obtained from transfected hepatic epithelial cells or isolated from the hepatic vascular occlusive circuit system of animals were washed twice with PBS and then incubated with lysis buffer [50 mM Tris-HCl, pH 7.4, 1% NP40, 0.25% sodium deoxycholate. , 150 mM NaCl, 1mM EDTA (based on 500 ml)], SDS, Na ₃ O ₄ V, and protease inhibitor cocktails (GenDepot) were added and treated at 4°C for 10 minutes. After quantifying the protein using BCA reagent (Thermo Scientifics), 4x sample buffer [100% glycerol 4 ml, Tris-HCl, pH 6.8, 2.4 ml, SDS 0.8 g, Bromophenol blue 4 mg, beta-mercaptoethaol 0.4 ml, H ₂ O 3.1 ml (based on 10 ml)] was added and boiled at 100°C for 5 minutes. After performing SDS-PAGE electrophoresis with the above sample, Nitrocellulose Membranes Protran ^?? After being transferred to a nitrocellulose membrane (Whatman), it was pretreated with 5% skim milk for 1 hour. After pretreatment, anti-TSG101 antibody (Santa Cruz Biotechnology Inc., sc-7964), anti-Alix antibody (Cell Signaling Technology, Inc., Cat # 2171), anti-CD63 antibody, anti-CD81 antibody, anti-CD9 antibody, and TM4SF5 primary antibody were used to react at 4°C for 15 hours. The next day, secondary antibodies were reacted and developed on X-ray film using ECL (Pierce, USA). Anti-mouse TM4SF5 (Accession number: NP_083636, epitope region of ¹¹⁷ clidnkwdyhfqetegaylrnd ¹³⁸ ) was custom-made by a professional antibody manufacturing company (Pro-Sci, Poway, CA, USA).

Isolated from Tm4sf5 KO mice (Tm4sf5 ^-/- ) and transgenic mice (Alb-Tm4sf5-FLAG) in which the Tm4sf5 gene is linked to the promoter of the albumin gene present in liver epithelial cells, causing the Tm4sf5 gene to be overexpressed only in the liver epithelial cells of the animal. As a result of Western blotting using a primary hepatocyte, it was confirmed that TM4SF5 protein was expressed in the primary hepatocyte (FIG. 5a).

In addition, Alb-Tm4sf5 mice were circulated through the hepatic vascular occlusion circuit for 1 hour using the method of Example 2, samples were separated, and Western blot was performed to confirm the proteins expressed in exosomes contained in the separated samples. As a result, the expression or presence of Alix, TSG101, and Flottlin proteins, which are well known as exosome markers, was confirmed (Figure 5b).

A control vector (EV, empty vector), a TM4SF5 expression vector (WT, N138A/N155Q mutation that does not allow N-glycosylation, or Cys2/6/9/74/74/74/6 that does not allow palmitoylation) were added to SNU449 human liver epithelial cells that do not express TM4SF5. In cases where expression was induced by injection into 75/79/80/84/189Ala mutant), exosomes were isolated and the presence of TM4SF5 (-Strep) and exosome markers CD54 and ALIX were confirmed through Western blot. The N138A/N155Q mutation, in which TM4SF5 glycosylation is impossible, was not present in exosomes. On the other hand, when TM4SF5 was present, it was confirmed that the presence of ALIX was significantly reduced (Figure 5c).

When injecting a control vector (EV, empty vector) or a TM4SF5 expression vector (TM4SF5) into SNU449 human liver epithelial cells that do not express TM4SF5, control siRNA (NS, non-specific) or siTM4SF5 (#4 sequence of TM4SF5) After controlling the expression of TM4SF5 by simultaneously injecting targeting siRNA), exosomes were isolated and the presence of GLUT1 and ALIX along with TM4SF5 was confirmed through Western blot.

The siRNA against TM4SF5 and shRNA against TM4SF5 used in the experiment and the primer sequence list are listed in Tables 1 to 3 below.

Number of siRNA targeting sequence against TM4SF5 Sequence (5'→3') siTM4SF5 #2 ACC AUG UGU ACG GGA AAA UGU GC siTM4SF5 #4 CCA UCU CAG CUU GCA AGU C siTM4SF5 #5 UGG ACC CAG AUG CUU AAU G siTM4SF5 #7 CCU CCU GCU GGU ACC UAA U siTM4SF5 #8 GCU UGC AAG UCU GGC UCA U siTM4SF5 #12 TGG ACC CAG ATG CTT AAT GAA

Number of shRNA targeting sequence against TM4SF5 Sequence (5'→3') shTM4SF5 #2 CCGGACCATGTGTACGGGAAAATGTGCCTCGA GGCACATTTTCCCGTACACATGGTTTTTTG shTM4SF5 #4 CCGGCCATCTCAGCTTGCAAGTCCTCGAGGACTTGCAAGCTGAGATGGTTTTTG shTM4SF5 #5 CCGGTGGACCCAGATGCTTAATGCTCGAGCA TTAAGCATCTGGGTCCATTTTTG shTM4SF5 #12 CCGGTGGACCCAGATGCTTAATGAACTCGAGT TCATTAAGCATCTGGGTCCATTTTTG

Primer Sense Anti-sense Mouse Glut1 #1 GCCUCUGCUG CUCAGUGUCA UCUUCAU AUGAAGAUGA CACUGAGCAG CAGAGGC Mouse Glut1 #2 GAUGAAAGAA GAGGGUCGGC AGAUGAU AUCAUCUGCC GACCCUCUUC UUUCAUC Mouse Glut2 #1 GGACAAACGGAAGGACAA UUGUCCUUCCGUUUGUCC Mouse Glut2 #2 GACGAAGGACAGGGACG CGUCCUGUCCUUCGUC Mouse Glut4 #1 GACUGGAACA CUGGUCCUAG CUGUAU AUACAGCUAG GACCAGUGUU CCAGUC Mouse Glut4 #2 GUUGCGGAUG CUAUGGGUCC UUACGU ACGUAAGGAC CCAUAGCAUC CGCAAC

As a result, when the presence of TM4SF5 in exosomes increased, the abundance of GLUT1 and ALIX decreased, and when the presence of TM4SF5 decreased (by siTM4SF5), the abundance of GLUT1 and ALIX did not decrease (Figure 5d).

This suggests that when TM4SF5 is present (loaded) in exosomes isolated from liver epithelial cells, it can replace the function of controlling the loading of various proteins into exosomes such as ALIX. In addition, when separated into exosomes, this means that the presence of GLUT1 may be reduced compared to the amount on the surface of hepatic epithelial cells.

In addition, as a result of confirming several proteins expressed in the primary hepatic epithelial cells and exosomes isolated through the hepatic vascular occlusive circuit system using Western blot, Tm4sf5(-FLAG), TSG101, and ALIX were expressed in the primary hepatic epithelial cells. Alternatively, it was confirmed that Tm4sf5, TSG101, ALIX, CD63, and CD81 were expressed or present in exosomes isolated through the hepatic vascular occlusion system (Figure 5e).

As described above, it was directly confirmed that exosomes obtained from the hepatic vascular occlusion circuit system using mice in which Tm4sf5 was expressed only in hepatic epithelial cells not only carried Tm4sf5 but also had several other exosomal markers. In addition, in the case of CD63 and CD81, it was confirmed that they were not found in small amounts in primary cells, but were clearly loaded in exosomes.

<3-2> Confirmation of increase in TM4SF5 loading of hepatocyte exosomes loaded with TM4SF5 due to glucose treatment outside the cells and confirmation of high energy state of hepatic epithelial cells

Huh7, a liver epithelial cell that naturally expresses TM4SF5, was maintained in a cell culture medium without glucose for 24 hours, then treated with glucose at a given concentration for 2 hours, and then cell extract (lysate) and exosomes were obtained and subjected to Western blotting. was carried out. As a result, the amount of TM4SF5 and CD63 loaded into exosomes gradually increased over time after glucose treatment, but ALIX showed a slight increase and then a tendency to decrease (Figure 5f).

Huh7 cells were maintained in a cell culture medium without glucose for 24 hours and then treated with glucose at a concentration of 25 mM for a given period of time. Cell extracts (lysates) and exosomes were obtained and Western blot was performed. As a result, the amount of TM4SF5 and CD63 loaded into exosomes gradually increased over time after glucose treatment, but ALIX showed a tendency to increase slightly (Figure 5g).

<3-3> Confirmation of binding of TM4SF5 and Glut1 in TM4SF5-loaded hepatocyte exosomes depending on the presence or absence of glucose treatment outside the cell

After transfecting HA-GLUT1 with CD151-strep or TM4SF5-strep expression vector into hepatic epithelial cells SNU449 cells, which do not naturally express TM4SF5, glucose was excluded from the cell culture medium and then treated with (-) 25 mM for 24 hours. After (+), cell extract (lysate) was obtained, CD151-Strep or TM4SF5-strep was pulled down with streptavidin beads, and the presence of HA-GLUT1 was confirmed by Western blot. As a result, it was confirmed that TM4SF5 binds well to GLUT1 when glucose is excluded from the outside of the cell, whereas when glucose is supplied (i.e., when TM4SF5-loaded exosomes are secreted), the binding decreases. In addition, it was confirmed that CD151 had minimal binding to GLUT1 under normal culture conditions. In addition, when serum (fetal bovine serum, FBS 10%) was excluded and then supplied, the binding between TM4SF5 and GLUT1 was reduced or inhibited, similar to the case of glucose. Therefore, it is judged that this suggests that the binding of TM4SF5 to GLUT1 becomes weaker or becomes less important as it is separated from liver epithelial cells into exosomes. However, it was confirmed that when treated without amino acid (AA) or arginine amino acid (Arg), the binding increased or did not decrease significantly. From this, it was assumed that TM4SF5 was loaded into exosomes, but less GLUT1 was loaded, depending on the level of extracellular glucose rather than amino acids (Figure 5h).

TM4SF5-Strep WT or several mutants were expressed in SNU449 cells, liver epithelial cells that do not natively express TM4SF5, and after excluding glucose from the cell culture medium, they were either left untreated (-) or treated (+) with 25 mM glucose for 24 hours. Then, the cell extract was obtained, pulled down with Strepavidin beads, and Western blot was performed. As a result, it was confirmed that WT, or Pro153Ala, and Val156Ala mutant TM4SF5 lost binding ability due to treatment of GLUT1 and extracellular glucose, but Thr157Ala mutant TM4SF5 maintained binding affinity, indicating that, unlike WT, loading of Thr157 amino acid into exosomes is important. It could be assumed that this could be done (Figure 5i).

<3-4> Confirmation of high energy status of liver epithelial cells depending on the presence or absence of glucose treatment outside the cells

After controlling the expression of TM4SF5 by injecting control shRNA (non-specific, NS) or shTM4SF5 (shRNA targeting sequence #4 or #8 of TM4SF5) into Hep3B liver epithelial cells that originally express TM4SF5, After excluding glucose for 4 hours, the cells were again treated with 25mM for a given time (min), then cell extracts were obtained and Western blot was performed. As a result, when TM4SF5 was expressed, compared to when its expression was suppressed, phosphorylation of S6K1 (ribosomal protein S6 kinase beta-1) increased and phosphorylation of AMPKα (catalytic subunit of AMP-activated protein kinase, AMPK) decreased. It was confirmed that a high energy state was secured (Figure 5j).

Huh7 hepatic epithelial cell line, which originally expresses TM4SF5, was treated with 25 mM glucose for a given period of time without excluding glucose from the culture medium (No starv.) for 24 hours. Then, cell extracts were obtained and subjected to Western blotting. carried out. As a result, the phosphorylation of AMPK and ACCα (acetyl CoA carboxylase α) decreased compared to the case when glucose was not treated and gradually over time, indicating a high energy state, and mTOR (mechanistic target of phosphorylation of rapamycin) and S6K1 increased, resulting in increased activation of protein metabolism (Figure 5k).

<3-5> Observation of changes in average particle size of TM4SF5-loaded hepatocyte exosomes according to extracellular glucose treatment

After regulating the expression of TM4SF5 by injecting control shRNA (non-specific, NS) or shTM4SF5 (shRNA targeting sequence #4 of TM4SF5) into Huh7 liver epithelial cells that originally express TM4SF5, the cells were incubated with RPMI. Exosomes were isolated after growing normally in culture medium (RPMI), excluding glucose in the cell culture medium for 24 hours, and then leaving the cells untreated (glu-) or treated (Glu+) with 25 mM for 24 hours. The average size of the isolated exosomes was analyzed using nanoparticle tracking analysis (NTA) and displayed in a graph. When TM4SF5 was expressed, the average size of exosomes decreased when treated again compared to when glucose was excluded, but such phenomenon was not observed when TM4SF5 expression was suppressed (Figure 5l).

In the case of Figure 5l, when the exosomes obtained were analyzed using turnable resistive pulse sensing (TRPS), it was confirmed that the size of the exosomes decreased with glucose treatment (Figure 5m).

In addition, the size and concentration of Huh7 exosomes that had undergone size-exclusion chromatography were confirmed using single angle dynamic light scattering (DLS). It was possible to analyze small particles of less than 100 nm. Therefore, TM4SF5-loaded exosomes secreted from liver epithelial cells are secreted when glucose is excluded from the outside of the cell and treated with high concentration roll, and the average size is much smaller. Secretion was confirmed (Figure 5n).

<3-6> Analysis of proteome present in hepatocyte exosomes loaded with TM4SF5

After controlling the expression of TM4SF5, control shRNA (non-specific, NS) or shTM4SF5 (shRNA targeting sequence #4 or #12 of TM4SF5) was injected into Huh7 liver epithelial cells that originally express TM4SF5, Exosomes were isolated. Proteins present in the isolated exosomes were analyzed using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrument. This takes advantage of the fact that exosomes are put into the inlet of the equipment, go through fragmentation and ionization steps, and then apply an electric field to move within the tube, and each moving ion fragment is classified by mass in a detector. PANDER (Protein Analysis Through Evolutionary Relationships, http://pantherdb.org) analysis to identify proteins present in TM4SF5-expressing or non-expressing exosomes and including gene ontology linked to biological function (PANDER-GO-slim) ) was performed. As a result, exosomes isolated from cells expressing TM4SF5 contained proteins related to biological adhesion [ITGB3 (Integrin Subunit Beta 3), FAM49B (amily with sequence similarity 49 member) compared to exosomes obtained from cells not expressing TM4SF5. B), ICAM2 (Intercellular Adhesion Molecule 2), PKP1 (Plakophilin 1), SIRPG (Signal Regulatory Protein Gamma), ITGBL1 (Integrin Subunit Beta Like 1)] were confirmed to be additionally present (Figure 5o).

These results lead us to assume that exosomes loaded with TM4SF5 will be able to approach target cells, attach and fuse more efficiently, and deliver the contents of the exosomes to target cells.

Organs or target cells that can consume glucose in the blood may include adipose tissue and muscle tissue. At this time, it is divided into white adipose tissue (WAT), whose main function is fat storage, and brown adipose tissue, whose main function is heat generation through fat burning, and these express GLUT4 (Glucose Transporter Type 4). By doing so, it activates glycolysis through absorption of glucose in the blood, thereby promoting lowering of glucose in the blood or diffusion into fat/muscle tissue. Therefore, we sought to confirm the binding between TM4SF5 and GLUT4.

For this purpose, control Strep-vector (empty vector, EV) or Strep-linked TM4SF1, TM4SF4, TM4SF5, TM4SF18, TM4SF20, CD9, and CD151 were injected together with HA-GLUT4 to express each other in HEK293FT cell line, and then cell extract was obtained. did. Each Strep-linked protein from the cell extract was pulled down with streptavidin-beads, and then Western blot was performed on HA-GLUT4 and Strep.

As a result, it was confirmed that TM4SF5 binds well to GLUT4, unlike TM4SF18 and CD151, with TM4SF1, TM4SF4, TM4SF20, and CD9 (Figure 5p). This suggests that TM4SF5-loaded exosomes derived from liver epithelial cells can approach target cells in adipose tissue or muscle tissue and attach more efficiently and bind to GLUT4 present in the target cells.

<Experimental Example 4> Confirmation of physical changes in liver-derived exosomes according to blood sugar status

<4-1> Measurement of physical changes in liver-derived exosomes according to glucose blood concentration

If we check whether the liver-derived exosomes change depending on the blood concentration of glucose, this can be an important indicator of the patient's physical condition or the liver's ability to remove glucose from the blood. To confirm this, the liver blood vessels of Example 1 were tested. Physical changes in exosomes according to glucose blood concentration were measured using a closed-circuit system.

Specifically, liver-derived exosomes were isolated from three normal mice under normal dietary conditions and two normal mice starved by restricting food for 24 hours through the method of Example 2, and using the same method as disclosed in Experimental Example 2. The size of exosomes was confirmed.

As a result, the average size of liver-derived exosomes isolated from three normal mice under normal diet conditions was 179.433 ± 11.272 nm, while the average size of liver-derived exosomes isolated from two normal mice under feed-restricted conditions was 276.9 nm. With an average size of ±3.818 nm, it was confirmed that the size distribution of exosomes was very wide compared to exosomes from mice under normal diet conditions (Figure 6a).

In addition, when perfusing exosome circulation media in the same normal mouse, the size of the exosomes obtained by flowing media containing glucose for 1 hour and the size of exosomes obtained by flowing media without glucose afterward were calculated using glucose. As a result of checking depending on the presence of , it was confirmed that the size distribution of exosomes widened when switching from the state in which glucose was present to the state in which it was not present (Figure 6b). Therefore, abnormalities in the size distribution of exosomes or inhibition of TM4SF5 expression can be important indicators for diagnosing problems with glucose control ability.

<4-2> Confirmation of glucose absorption and activation of glycolysis by binding between TM4SF5 and GLUT1 expressed in liver epithelial cells

After injecting control Strep-vector (empty vector, EV) or Strep-TM4SF5 together with HA-GLUT1, HA-GLUT2, HA-GLUT3, HA-GLUT4, and HA-GLUT9 into the HEK293FT cell line and expressing it, glucose in the cell culture medium was expressed. After exclusion (-) or treatment with 25 mM for 24 hours (+), cell extracts were obtained. After pulling down each Strep-TM4SF5 from the cell extract with streptavidin-beads, Western blotting was performed on HA and Strep. As a result, it was confirmed that TM4SF5 binds to GLUT1, GLUT4, and GLUT9. At this time, GLUT1 binds well when not treated with glucose, but when treated with glucose, the binding decreases, but GLUT9 showed the opposite pattern. , Meanwhile, GLUT4 was confirmed to bind well regardless of glucose treatment (Figure 6c).

After TM4SF5-FLAG and HA-GLUT1 were simultaneously injected and expressed in the SNU449 cell line, the distribution locations of TM4SF5-FLAG (red) and HA-GLUT1 (green) were confirmed through fluorescence immunostaining. As a result, red and green were confirmed. By confirming the overlapped yellow appearance, it was confirmed that TM4SF5 and GLUT1 can bind to each other in the liver epithelial cell membrane (Figure 6d).

Expression of TM4SF5 was controlled by injecting control shRNA (non-specific, NS) or shTM4SF5 (shRNA targeting sequence #4 or #8 of TM4SF5) into Huh7 liver epithelial cells, or injecting 100 μM fasentin (GLUT1/ After treatment with 4 inhibitor, SIGMA, Cas number 392721-37-8), glucose uptake into the cells was measured. As a result, glucose absorption was decreased when TM4SF5 expression was suppressed or when GLUT1/4 inhibitor was treated (Figure 6e).

After injecting control vector (EV) or TM4SF5-HA into AML12 cells, which are normal mouse liver epithelial cells, and expressing them, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells were measured in a 24-well plate format. Using a Seahorse A glycolytic stress test was performed by measuring ECAR while processing sequentially. As a result, it was confirmed that the level of ECAR was much higher in cells expressing TM4SF5, thereby enabling glycolysis to be significantly activated (Figure 6f).

AML12 cells, which are normal liver epithelial cells in mice, were injected and expressed with control vector (EV) or TM4SF5-HA, treated with 1.0 μM of oligomycin and 0.5 mM of glucose, and after about 30 minutes, the concentration of glucose was increased to 1.0, Glucose sensitivity was confirmed by measuring ECAR while sequentially increasing it to 5.0 and 10.0. As a result, it was confirmed that in cells expressing TM4SF5, the degree of ECAR was much higher even when treated with low glucose, indicating that the glycolysis process could be activated much more sensitively (Figure 6g).

As described above, it was confirmed that when TM4SF5 is expressed in liver epithelial cells, compared to when it is not expressed, it binds to GLUT1 and promotes the absorption of glucose into liver epithelial cells, thereby activating glycolysis.

<4-3> Confirmation of absorption of glucose and activation of glycolysis by targeting brown adipose tissue and BAT cells of exosomes loaded with liver epithelial cell-derived TM4SF5 and binding to GLUT4

After controlling the expression of each Glut by injecting control siRNA (NS, non-speific) or siRNA for Glut1, Glut2, or Glut4 mouse genes into AML12 cells stably expressing control vector (EV) or TM4SF5, exosomes was separated. The isolated exosomes (4 x 10 ⁸ parts) were reacted with brown adipose tissue cells from normal mice, differentiated for 7 days, and then mixed. Afterwards, ECAR was measured while glucose, oligomycin, and 2-DG were sequentially added. As a result, exosomes (sEV ^Tm4sf5 ) obtained from cells expressing TM4SF5 and injected with control siRNA (NS) or siGlut2 were treated with exosomes obtained from cells not expressing TM4SF5 or AML12-TM4SF5 cells in which siGlut1 or siGlut4 was suppressed. The degree of ECAR was higher than in the other case, confirming that glycolysis was activated. In other words, it was confirmed that exosomes secreted when TM4SF5 and Glut1/Glut4 are expressed in liver epithelial cells can cause activation of glycolysis in target cells (brown adipose tissue cells) (Figure 6h).

Under the same conditions as shown in Figure 6h, exosomes were treated with differentiated brown adipose tissue cells, and then treated with 0.5 mM glucose along with 1.0 μM oligomycin, increasing the concentration of glucose to 1.0, 2.0, and 5.0 about every 30 minutes. While adding, ECAR was measured to confirm glucose sensitivity. As a result, the glycolysis of brown adipose tissue cells was activated by exosomes derived from liver epithelial cells expressing TM4SF5, but when the expression of Glut1 in the liver epithelial cells was suppressed, the activation of glycolysis was lost, and the expression of Glut4 was suppressed and was irrelevant. In other words, it was confirmed that Glut4 in liver epithelial cells is not required for the function of activating target cell glycolysis of exosomes derived from liver epithelial cells expressing TM4SF5 (Figure 6i).

Exosomes were isolated from AML12 cells expressing control vector (EV) or Tm4sf5-Strep. Then, the exosomes were treated with primary BAT (brown adipose tissue cells) differentiated from normal mice at a concentration of 4 x 10 ⁸ partcle/condition for 24 hours. When treating exosomes (sEV ^Tm4sf5-Strep ) obtained from cells expressing TM4SF5, the TM4SF5-specific inhibitor TSAHC was left untreated at 2 μM or treated for 24 hours. Afterwards, the cell extract was obtained and pulled-done with streptavidin beads, and then Western blot was performed using anti-Strep antibody or Glut4.

As a result, when exosomes (sEV ^Control ) obtained from AML12 cells that do not express Tm4sf5 were treated, Glut4 could not bind to Tm4sf5-Strep, and when sEV ^Tm4sf5-Strep was treated, Glut4 from BAT was unable to bind to exosomes. It bound to Tm4sf5-Strep that existed in the moth, and it was confirmed that this was reduced by TSAHC treatment. As a result, it was confirmed that liver epithelial cell-derived Tm4sf5-loaded exosomes attached to BAT target cells were capable of binding Tm4sf5-Strep and Glut4. In addition, it was confirmed that this effect was related to the activity of Tm4sf5 (Figure 6j).

Exosomes isolated under the same conditions as in Figure 6j were treated with differentiated primary BAT cells. At this time, exosomes of various concentrations were treated. Subsequently, as a result of measuring glucose uptake into BAT cells, it was confirmed that when treated with hepatic epithelial cell-derived exosomes expressing Tm4sf5, glucose uptake increased, especially in a concentration-dependent manner (Figure 6k).

<Experimental Example 5> Confirmation of the effect of liver-derived exosomes loaded with TM4SF5 on improving glucose metabolism and blood sugar level

<5-1> Confirmation of blood sugar control effect in animals according to liver expression of Tm4sf5

Normal mice (WT), liver epithelial cell-specific Tm4sf5 overexpressing mice (Alb-TG ^Tm4sf5 or alb-TG), and Tm4sf5 ^-/- KO mice (n = 10) were used as a single cohort to repeatedly measure blood glucose levels. . Starting from 8 weeks of age, glucose was injected at concentrations of 2.0, 4.0, 1.0, and 0.5 g/kg every week until 11 weeks of age, and then 45 minutes later, an intraperitoneal glucose tolerance test was performed each time. Blood sugar level was measured through IPGTT). As a result, it was confirmed that WT and alb-TG mice had lower blood glucose levels than Tm4sf5 ^-/- KO mice.

24 hours after the last blood glucose level was measured in 11-week-old mice, the same WT and KO mice were injected with AAV8-Tbg-Tm4sf5-HA (mouse Tm4sf5-HA gene into the liver (epithelial cell)-specific Tbg (Thyroxine binding globulin) Adeno-associated virus subtype 8), which expresses a gene fragment linked to a promoter, was injected intravenously once at a concentration of 2 × 10 ¹⁰ vg/mouse. After 5 weeks, glucose was again injected IP at 2.0 or 4.0 g/kg every week, and then GTT was performed. As a result, it was confirmed that there was no significant difference in blood sugar levels between KO mice and WT mice in which Tm4sf5 expression was restored with AAV8 (FIG. 7a).

<5-2> Confirmation of the effect of liver-derived exosomes loaded with TM4SF5 on improving glucose metabolism and blood sugar level

To confirm the effect of liver-derived exosomes loaded with TM4SF5 on glucose uptake and blood sugar level control in various cells or tissues, the Tm4sf5 gene was overexpressed in the liver using the albumin promoter (Alb-Tm4sf5). Exosomes obtained through the liver vascular occlusion circuit system were separated through size exclusion chromatography, and liver-derived exosomes loaded with TM4SF5 were administered to normal mice (WT) or Tm4sf5 KO mice (Tm4sf5 ^-/- ). After doing so, the results were confirmed.

Specifically, exo obtained through the liver vascular occlusion circuit system of Example 1 in a mouse (Alb-Tm4sf5) in which the Tm4sf5 gene, which is specifically expressed in liver epithelial cells and is present in the exosome membrane, was overexpressed in the liver using an albumin promoter. The exosome sample was passed through size exclusion chromatography, and 1.5 ml of the passed exosome sample was concentrated to 200 μl using a 50 kDa membrane centrifugal concentrator. Afterwards, Tm4sf5 KO mice fasted for 16 hours were anesthetized using 30% isoflurane [Piramal - Terrell??, solvent is polyethylene glycol-200 (Sigma Aldrich -8.07483)], and the concentrated exosomes were injected into the tail. 200 μl was injected via intravascular injection. 5 minutes later, when the mouse woke up from anesthesia, the first blood sugar level was measured through tail blood sampling. Next, 20% glucose (Sigma Aldrich - G8270) was injected intraperitoneally at 2g (glucose)/kg (mouse body weight), and blood sugar was measured using a blood glucose meter (Johnson and johnson - One touch ultra) for 30, 60, and 120 minutes after injection. Changes were measured. On the other hand, in the control group, PBS was administered to normal mice.

In general, Tm4sf5 ^-/- mice exhibit a phenotype in which the ability to process glucose in the blood is lower than that of normal mice for a certain period of time after glucose injection (Figure 7b), whereas exosomes derived from the liver of Alb-Tm4sf5 mice were injected into normal mice. In contrast, when injected into Tm4sf5 ^-/- mice, the level of glucose in the blood was lowered, confirming that the glucose processing ability was significantly improved compared to the control group (Figure 7c). Therefore, TM4SF5-loaded exosomes secreted from liver epithelial cells can control blood sugar levels in the body, and ultimately promote glucose absorption into the blood by muscle tissue or fat tissue or cells of such tissues, which is positive for homeostatic regulation. It was confirmed that it can play a role.

<Experimental Example 6> Confirmation of tissue and organ absorption and distribution of liver-derived exosomes

To confirm the extent to which liver-derived exosomes obtained through the hepatic vascular occlusion system migrate to tissues and organs, liver-derived exosomes were injected into normal mice and observed using in vivo animal imaging equipment.

에서 보관하였다. 정상 마우스는 3일전 defined diet(AIN93G) 섭취하도록 먹이를 교체하였다. 상기 마우스를 이소플루란으로 마취한 뒤 200 μl의 엑소좀을 꼬리 정맥주사하였다. 24시간 후에 마우스를 해부하여 각 장기를 분리 후 IVIS[엑스선형광분석기(IVIS®SpectrumCT)]를 이용하여 Max excitation wavelength 784nm, Max emission wavelength 806nm로 촬영하였다.Specifically, exosomes obtained from Alb-Tm4sf5 TG mice and Tm4sf5 ^-/- KO mice were purified by size exclusion chromatography using the hepatic vascular occlusion circuit system of Example 1-1, and then purified by size exclusion chromatography. It was concentrated to 500 μl using a membrane centrifugal concentrator. At this time, the amount of exosomes was ~ ^2x109 particles/ml. 1 μl Near infrared labeling dye (System Biosciences, EXOGV900A-1) was added to the concentrated exosomes and reacted at room temperature for 1 hour. After reaction, 9 ml of PBS was added, 2 ml of 20% sucrose was added to the bottom of an ultracentrifuge tube (Beckman Coulter, 344059), and the diluted exosomes were added to the top. Samples were centrifuged at 110,000 xg for 70 minutes at 4°C. Remove all supernatants and add exosomes to the bottom in 200 μl Dulbecco's phosphate buffer saline (DPBS) (Sigma, MO, USA).

It was stored in . Normal mice were fed a defined diet (AIN93G) 3 days ago. The mouse was anesthetized with isoflurane and then 200 μl of exosomes were injected into the tail vein. After 24 hours, the mouse was dissected, each organ was separated, and images were taken using IVIS [X-ray fluorescence spectrometer (IVIS®SpectrumCT)] at a Max excitation wavelength of 784 nm and a Max emission wavelength of 806 nm.

As a result, there was no significant difference between exosomes derived from the liver of Alb-Tm4sf5 TG mice or exosomes derived from the liver of Tm4sf5 ^-/- KO mice, but exosomes derived from the liver of Tm4sf5 ^-/- KO mice were expressed in the lungs and liver during 24-hour treatment. It was confirmed that exosomes derived from the liver of Alb-Tm4sf5 TG mice loaded with TM4SF5 reached further into the leg bones and were absorbed into the brown adipose tissue (BAT) tissue on the back of Tm4sf5 ^-/-. (Figure 8). This means that exosomes derived from the liver of mice containing TM4SF5 can be absorbed into brown adipose tissue (BAT) and can be absorbed better than exosomes not loaded with TM4SF5, and are absorbed into brown adipocytes. This suggests that, if possible, it could have a positive effect on efficient energy metabolism and blood sugar utilization.

<Experimental Example 7> Confirmation of the effect of liver-derived exosomes on sugar metabolism of brown adipose tissue adipocytes

<7-1> Separation and differentiation of brown adipose tissue adipocytes

After euthanizing an 8-week-old male normal mouse, it was laid down with its back facing upward, an incision was made at the back of the neck of the mouse, the fat layer was confirmed, and the dense brown adipose tissue (BAT) in the middle was separated from the white adipose tissue. The isolated brown adipose tissue was placed in a Petri dish placed on ice along with PBS. After moving to the biosafety bench and removing as much PBS as possible, the tissue was chopped into pieces of 0.5 mm ³ to 1 mm ³ using a razor or surgical knife. Transfer the tissue to a 15 ml conical tube and add 1 ml collagenase buffer (1 mg/ml collagenase type II (Sigma, C6885), 123.0mM NaCl, 5.0mM KCl, 1.3mM CaCl ₂ , 5.0mM Glucose, 100.0mM HEPES, 4% BSA and 1x penicillin/streptomycin (P/S), pH 7.4) were added, and 1 ml PBS was added. Vortex for 10 seconds and shake at 37°C for 30 minutes. After the enzyme reaction, 400 μl FBS was added and mixed well. The sample was passed through a 100 μm nylon mesh and centrifuged at 600 xg for 5 minutes. The supernatant was removed, resuspended in 1 ml growth media (DMEM/F12 (Hyclon, SH30023.01), 15% FBS, 1x penicillin-streptomycin, 10,000 unit/ml), and cultured in a 6 well plate. After 24 hours, the media and blood cells were removed and new media (2 ml) was added. Media was changed daily until cells grew 90%. When the cells grew to 90%, the media was removed, washed once with PBS, cells were picked out with accutase (Sigma, A6964), and the cells were newly cultured on a plate suitable for the purpose of the experiment. Thereafter, the media was changed daily until the cells grew 100%.

After cells were grown to 100%, induction media (0.5 mM 3-Isobutyl-l-methylxanthine (IBMX, Sigma, I5879), 1 μM Dexamethasone (Sigma, D4902), 1 μg/ml Insulin (Sigma, I0516), 1 μM Rosiglitazone ( Sigma, R2408), 1 nM 3,3',5-Triiodo-L-thyronine (T3, Sigma, T0281), 15% FBS, DMEM/F12, 1x penicillin-streptomycin, 0.2 μm filtered) for 2 days. Two days later, it was replaced with maintenance media (1 μg/ml Insulin, 1 nM T3, 15% FBS, DMEM/F12, 1x penicillin-streptomycin, 0.2 μm filtered). For 6 to 7 days, the media was changed every two days and differentiated.

<7-2> Glycolysis stress test of brown adipose tissue (BAT) adipocytes by exosome treatment

Exosomes isolated from the murine normal liver epithelial cell AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector were treated with adipocytes isolated from BAT and differentiated, and various drugs were administered using Seahorse equipment. By measuring the acidification of the extracellular solution of BAT adipocytes in the added state, a glycolysis stress test of adipocytes by exosome treatment was performed.

Specifically, mouse liver epithelial AML12 cells were cultured in 150 mm dishes at 1.5 x 10 ⁶ , three 150 mm dishes for each group. 16 hours later, 15 μg empty vector (EV) or TM4SF5-HA gene was infected with 45 μl of 1 mg/ml polyethyleneimine (PEI, Santa Cruz Biothechnology, SC-507159). After 48 hours, the culture medium was obtained, exosomes were isolated, and 500 μl of differentiated brown adipocytes were replaced with Seahorse media (Agilent, 103334-100) adjusted to pH 7.4 at 37°C. At this time, ^4.0 F5557) was treated together in the necessary wells. Afterwards, the cells were incubated for 45 minutes in a 37°C incubator without CO ₂ . Into the seahorse sensor cartridge that had been hydrated a day before, A port-Glucose (10mM, Sigma, G7021), B-port Oligomycin A (1 uM, Sigma, 75351), C-port 2-DG (100mM, Alfa Aesar, L07338). was added and calibrated, and then Glycolysis stress assay was performed.

As a result of the glycoslysis stress test, it was confirmed that when treated with exosomes loaded with Tm4sf5, the glycolysis capacity of adipocytes was significantly increased compared to the control group treated with exosomes without Tm4sf5 (Figure 9a). When exosomes loaded with Tm4sf5 were treated together with an inhibitor of Glut1/4 (fasentin), the extracellular acidification rate (ECAR) was confirmed to be low, confirming the effect of activating glycolysis by treatment of exosomes loaded with Tm4sf5. It was confirmed that it depends on the presence and activation of Glut4 in adipocytes. Through this, it can be assumed that exosomes derived from liver epithelial cells loaded with Tm4sf5 target adipocytes depending on the activity of Glut4, ultimately helping to activate glycolysis so that adipocytes can utilize glucose well.

<7-3> Glucose sensitivity test of BAT adipocyte glycolysis by treatment with exosomes derived from liver epithelial cells

Exosomes isolated from the murine normal hepatic epithelial cell AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector were treated with adipocytes isolated from BAT and differentiated, and then mixed at various concentrations using Seahorse equipment. The degree of activation of the glycolysis function was measured by measuring the acidification of the extracellular solution due to glycolysis while additionally adding glucose.

Specifically, mouse liver epithelial AML12 cells were cultured in 150 mm dishes at 1.5 x 10 ⁶ , three 150 mm dishes for each group. 16 hours later, 15 μg empty vector or TM4SF5-HA gene was infected with 45 μl of 1 mg/ml polyethyleneimine (PEI, Santa Cruz Biothechnology, SC-507159). After 48 hours, the culture medium was obtained and exosomes were isolated as described above. Adipocytes isolated and differentiated from BAT isolated from normal mice were distributed at 1.5x10 ⁴ in a 24 well seahorse plate. Cultured. After 16 hours, the culture medium was changed to 500 μl with Seahorse media (Agilent, 103334-100) adjusted to pH 7.4 at 37°C. At this time, 4.0 x 10 ⁸ exosomes obtained from AML12 cells were added. Afterwards, the cells were incubated for 45 minutes in a 37°C incubator without CO ₂ . A seahorse sensor cartridge that had been hydrated a day before was calibrated by adding 0.1 mM Glucose, 1 μM Oligomycin A for A port, 0.5 mM Glucose for B port, 1 mM Glucose for C port, and 5mM Glucose for D-port, and then performing a glucose sensitivity assay. .

As a result, when exosomes isolated from AML12 cells expressing TM4SF5 were processed into BAT adipocytes, the function of adipocyte glycolysis was improved under various glucose addition conditions compared to exosomes isolated from cells not expressing TM4SF5. was confirmed to improve (Figure 9b). As a result, it was confirmed that exosomes loaded with TM4SF5 can increase sensitivity to glucose in activating glycolysis in target fat cells. In other words, hepatic epithelial cell-derived exosomes loaded with Tm4sf5 can ultimately help targeted fat cells utilize glucose well.

<7-4> Effect of blood sugar control in animals according to Tm4sf5 expression

Exosomes were isolated from AML12 cells expressing HA-Tm4sf5. Then, the exosomes (AML12-sEV ^HA-Tm4sf5 ) were treated with primary BAT (brown adipose tissue cells) isolated and differentiated from normal mice at a concentration of 4 x 10 ⁸ particles/condition for 24 hours. Then, the DNA of BAT cells was stained with DAPI (blue), Glut1 (left) or Glut4 (right) was stained red, and HA-Tm4sf5 was stained green, and observed using a confocal microscope. As a result, it was confirmed that AML12-sEV ^HA-Tm4sf5 coexisted with Glut1 or Glut4 around the nucleus or cell surface of BAT cells and was stained yellow (Figure 9c).

Three weeks after injection of the control virus AAV8-EV or AAV8-Tbg-Tm4sf5-HA expressing the Tm4sf5-HA gene, H&E and immunohistochemistry were performed using BAT isolated from normal WT mice or KO mice to determine the degree of HA-Tm4sf5 staining. was observed. As a result, the BAT of animals injected with the Tm4sf5-HA expressing AAV8 virus had smaller and fewer fat droplets than the BAT of animals injected with AAV8-EV, resulting in improved thermogenesis. It was confirmed that this was done (Figure 9d).

In order to confirm the signaling factors related to the improved heat generation in FIG. 9D, a tissue extract was obtained from a portion of the BAT tissue in FIG. 9D and Western blot was performed. As a result, it was confirmed that the expression level of uncoupling protein 1 (UCP1), a protein that disrupts the connection between the mitochondrial proton gradient and the ATP synthesis process, does not change in the BAT tissue of each animal unrelated to different thermogenesis. It was confirmed that UCP1 was unrelated to the improvement of thermogenesis, and that mTOR phosphorylation increased (p=0.1220) in animals injected with Tm4sf5-HA-expressing AAV8 virus compared to AAV8-EV virus injection into KO mice (p=0.1220). Figure 9e).

Since it is known that glucose uptake in BAT increases by phosphorylation of mTOR (Molecular Metabolism. 2017. 6(6):611-619), the above results indicate that the expression of Tm4sf5 in hepatic epithelial cells is positive for glucose uptake in BAT. It can be assumed that it can have an impact and play a role in appropriately lowering excessive blood sugar levels.

<Experimental Example 8> Confirmation of the effect of liver-derived exosomes on glucose metabolism in muscle cells

<8-1> Analysis of glucose uptake in C2C12 muscle cells

To determine the effect of liver-derived exosomes on glucose metabolism in muscle cells, exosomes isolated from the murine normal hepatic epithelial AML12 cell line expressing Tm4sf5-HA or the AML12 cell line expressing only the control vector, C2C12 Muscle cells were treated and the degree of glucose absorption into muscle cells was analyzed.

Specifically, AML12 cells were cultured at 1.5 x 10 ⁶ in 150 mm dishes, 3 for each group. 16 hours later, 15 μg empty vector (EV) or TM4SF5-HA gene was infected with 45 μl of 1 mg/ml polyethyleneimine (PEI, Santa Cruz Biothechnology, SC-507159). 48 hours later, after washing twice with PBS, the culture medium was replaced with 20 ml DMEM/F12, 10% Ultrafiltered FBS (exosome free-FBS). After 48 hours, the culture medium was obtained and exosomes were isolated. C2C12 cells, which are muscle cells, were cultured at 1.5 x 10 ⁴ in a 96-well plate. After 16 hours _, the cells were washed twice with PBS, added to 50 μl glucose free RPMI1640 along with ^4.0 1mM 2-DG was added and left at room temperature for 10 minutes. After that, 25 μl of stop buffer was added to the Glucose uptake-Glo assay kit (Promega, J1343) and shaken, then 25 μl of neutralize buffer was added and shaken, and then 100 μl of luciferase buffer was added and shaken. After reacting at room temperature for 1 hour, luciferase luminescence was measured.

As a result, it was confirmed that when exosomes isolated from the AML12 cell line or the AML12 cell line expressing only the control vector were treated, the amount of glucose uptake increased compared to when the exosomes were not treated. In particular, the exosome loaded with Tm4sf5 When treated, it was confirmed that the degree of glucose absorption was higher compared to when treated with exosomes not loaded with Tm4sf5 (FIG. 10a). This suggests that glucose absorption into muscle cells can be enabled by Tm4sf5-loaded exosomes so that a large amount of glucose in the blood can be utilized.

<8-2> A204 Glucose sensitivity test of muscle cell glycolysis

In addition, TM4SF5-exosomes (Huh7-Exo-from cells in RPMI) were isolated and obtained from human liver epithelial cells Huh7, which originally express TM4SF5, while cultured in normal RPMI-1640 culture medium, and then cultured in glucose-excluded culture medium. The isolated TM4SF5-exosomes (Huh7-Exo-from cells in glu-media) and the isolated TM4SF5-exosomes (Huh7-Exo-from cells in glu+ media) were cultured in a culture medium containing 25 mM glucose, respectively, at A204. After treating muscle cells for 45 minutes, the sensitivity of glycolysis to various glucose concentrations was confirmed.

Specifically, hepatic epithelial Huh7 cells were cultured at 1.5 x 10 ⁶ in 150 mm dishes, 3 cells per group. 48 hours later, after washing twice with PBS, the culture medium was replaced with 20 ml DMEM, 10% Ultrafiltered FBS (exosome free-FBS). The culture medium was normally RPMI containing 10% FBS, FBS was cultured to remove glucose using a 100 kDa membrane centirfugal concentrator (Satorius -vs 2041) (glu-media), and glucose was added to the Glu ^- media at 25mM. Culture was performed using the added culture medium (glu ⁺ media). After 48 hours, the cultures were obtained and exosomes were isolated as described above. A204 cells, which are myoepithelial cells, were grown at 1.5 x 10 ⁴ cells/well in a 24 well seahorse plate. Cultured. After 16 hours, the culture medium was changed to 500 μl with Seahorse media (Agilent, 103334-100) adjusted to pH 7.4 at 37 degrees. At this time, 4.0 x 10 ⁸ exosomes obtained from Huh7 cells were added. Afterwards, the cells were incubated for 45 minutes in a 37°C incubator without CO ₂ . A seahorse sensor cartridge that had been hydrated a day before was calibrated by adding 0.1mM Glucose for A port, 1 μM Oligomycin A, 0.5mM Glucose for B-port, 1mM Glucose for C-port, and 5mM Glucose for D-port, and then running a glucose sensitivity assay.

As a result, TM4SF5-exosomes isolated from culture medium containing 25 mM glucose were more effective in A204 muscle cells than TM4SF5-exosomes isolated from Huh7 cells cultured in RPMI media without glucose control or culture medium from which glucose was removed. When treated with , it was confirmed that the sensitivity of glycolysis to glucose was high (FIG. 10b). This indicates that when TM4SF5-exosomes isolated from a culture medium containing 25mM of glucose are treated with muscle cells, they can increase the sensitivity of muscle cells to glucose and further activate glycolysis efficiently.

Claims (22)

In the liver vascular occlusion circuit system for extracting liver-derived exosomes,
A peristaltic pump for circulating the hepatic vascular occlusive circuit;
Exosome circulating media and flushing buffer circulated along the hepatic vascular occlusive circuit by the peristaltic pump;
A three-way connector including a switch to control circulation of the exosome circulation media and flushing buffer;
a first tubing connecting the inlet of the peristaltic pump with the three-way connector and connecting the outlet of the peristaltic pump with a third tubing;
A second tubing connecting the exosome circulation media and the flushing buffer with the three-way connector, respectively;
A first catheter inserted into the subject's portal vein;
a third tubing connecting the first catheter and the first tubing;
A first connector connecting the first catheter and the third tubing;
a second catheter inserted into the subject's inferior vena cava;
A fourth tubing connecting the second catheter and the exosome circulation media;
A second connector connecting the second catheter and the fourth tubing; and
A fifth tubing connecting the exosome circulation media and flushing buffer with an oxygen tank;
A hepatic vascular occlusive circuit system comprising a.
According to paragraph 1,
A hepatic vascular occlusion circuit system, characterized in that the flushing buffer is contained in a constant temperature water bath.
According to paragraph 1,
The hepatic vascular occlusive circuit system, characterized in that the exosome circulating media and the flushing buffer are connected to an oxygen tank.
According to paragraph 2,
A hepatic vascular occlusion circuit system, characterized in that the constant temperature water tank maintains a temperature of 40°C to 45°C.
It relates to a method of extracting liver-derived exosomes using the liver vascular occlusion circuit system of any one of claims 1 to 4,
inserting the first catheter into the subject's portal vein;
Operating the peristaltic pump;
Connecting the first catheter and a first connector connected to the pump;
inserting the second catheter into the inferior vena cava of the subject;
Connecting the second catheter to a fourth tubing and connecting the other end of the fourth tubing to the exosome circulating media tube;
stopping the peristaltic pump and turning the switch on the three-way connector to supply the exosome circulating media to the pump;
Connecting the exosome circulation media and flushing buffer with an oxygen tank;
Reactivating the peristaltic pump to circulate the exosome circulation media;
centrifuging the circulated exosome circulation media to separate the supernatant;
Concentrating the separated supernatant; and
A liver-derived exosome extraction method comprising: separating exosomes from the concentrated supernatant.
According to clause 5,
A method of extracting liver-derived exosomes, characterized in that the step of circulating the exosome circulation media is circulated for 30 to 90 minutes.
According to clause 5,
The step of centrifuging the circulated exosome circulation media to separate the supernatant includes operating the centrifuge at a centrifugal force of 2,000 xg for 5 to 20 minutes and a centrifugal force of 10,000 xg for 30 to 60 minutes. , liver-derived exosome extraction method.

According to clause 5,
The step of concentrating the separated supernatant is characterized in that it is concentrated to 5x10 ⁹ particles/ml by centrifuging 100 to 1,000 μl using a membrane centrifugal concentrator.
According to clause 5,
A method of extracting liver-derived exosomes, wherein the step of separating the liver-derived exosomes is performed using size exclusion chromatography.
According to clause 5,
The liver-derived exosomes are characterized in that the distribution area of the average size is narrowed to 200 nm or less or includes a size of 200 nm or less.
A pharmaceutical composition for preventing or treating diabetes-related diseases, containing exosomes containing TM4SF5 (Transmembrane 4 L6 family member 5) protein.
According to clause 11,
The exosome is used for preventing or treating diabetes-related diseases, characterized in that it is derived from one or more selected from the group consisting of liver tissue, liver tissue/extracellular fluid, liver epithelial cells (hepatocytes), and hepatocytes. Pharmaceutical composition.
According to clause 11,
The exosome is a pharmaceutical composition for preventing or treating diabetes-related diseases, characterized in that the distribution area of the average size is narrowed to 200 nm or less or is an exosome containing a size of 200 nm or less.
According to clause 11,
The exosomes are characterized in that they are secreted according to the change in level or presence of one or more selected from the group consisting of glucose, amino acid, or serum in liver tissue or liver tissue/extracellular fluid. A pharmaceutical composition for preventing or treating diabetes-related diseases.
According to clause 11,
The exosome is a pharmaceutical composition for preventing or treating diabetes-related diseases, characterized in that it binds to TM4SF5 and GLUT1, GLUT4, or GLUT9.
According to clause 11,
The exosomes include ITGB3 (Integrin Subunit Beta 3), FAM49B (Family with sequence similarity 49 member B), ICAM2 (Intercellular Adhesion Molecule 2), PKP1 (Plakophilin 1), SIRPG (Signal Regulatory Protein Gamma), and ITGBL1 (Integrin Subunit Beta). A pharmaceutical composition for preventing or treating diabetes-related diseases, comprising one or more proteins selected from the group consisting of Like 1).

According to clause 11,
The exosome is a pharmaceutical composition for preventing or treating diabetes-related diseases, characterized in that phosphorylation of AMPK or ACC is reduced or phosphorylation of mTOR, S6K1 or AKT is increased.

According to clause 11,
The exosome is characterized in that it binds to GLUT4 in muscle, adipose tissue or cells, activates glycolysis, increases sensitivity to glucose levels outside the tissue/cell, or increases phosphorylation of UCP1 and mTOR. , Pharmaceutical composition for preventing or treating diabetes-related diseases.
According to clause 11,
The diabetes-related disease is characterized by one or more diseases selected from the group consisting of fatty liver, steatohepatitis, diabetes, hyperlipidemia, hypertension, obesity, microvascular damage, nerve damage, ketoacidosis, arteriosclerosis, cardiovascular disease, and cerebrovascular disease. A pharmaceutical composition for preventing or treating diabetes-related diseases.
According to clause 11,
The exosome is a pharmaceutical composition for preventing or treating diabetes-related diseases, characterized in that it regulates blood sugar levels.
According to clause 11,
The exosome is a pharmaceutical composition for preventing or treating diabetes-related diseases, characterized in that it regulates homeostasis by promoting glucose absorption into the blood of muscle cells, fat cells, muscle tissue, or fat tissue.
According to clause 11,
The exosomes require the activity of Glut4, lower blood glucose levels, promote absorption into cells, promote thermogenesis, or regulate blood glucose homeostasis. Characterized by a pharmaceutical composition for preventing or treating diabetes-related diseases.