CN110151980B - Application of GLP-1 receptor agonist fusion protein in preparation of medicine for preventing or treating hyperlipidemia - Google Patents
Application of GLP-1 receptor agonist fusion protein in preparation of medicine for preventing or treating hyperlipidemia Download PDFInfo
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Abstract
The invention discloses an application of GLP-1 agonist fusion protein in preparing a medicament for preventing or treating hyperlipidemia including hypertriglyceridemia, high-low density lipoprotein cholesterol, high total cholesterol and related diseases such as non-alcoholic fatty liver, obesity, atherosclerosis and other cardiovascular and cerebrovascular diseases. The fusion protein is formed by connecting exenatide or a mutant thereof with a natural or optimized mutant Fc fragment of human immunoglobulin IgG1 through a connecting peptide or directly, can activate AMPK phosphorylation in liver tissues of golden yellow mice induced by high-fat diet, inhibit acetyl coenzyme A carboxylase activity, reduce expression of nSREBP-1C, down-regulate expression of fatty acid synthetase and stearoyl coenzyme A dehydrogenase, remarkably reduce plasma TG, TC and LDL-C levels, improve liver tissue morphology, reduce liver lipid accumulation, and reduce body weight and food intake.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to application of GLP-1 receptor agonist fusion protein in preparation of a medicament for preventing or treating hyperlipidemia and related diseases caused by hyperlipidemia.
Background
Atherosclerotic cardiovascular disease (CVD) is a serious health hazard to humans and its incidence is on an increasing trend year by year. The role of abnormal elevation of plasma low density lipoprotein cholesterol (LDL-C) in the pathogenesis of Atherosclerosis (AS) has been of great concern and has been identified AS a major risk factor for CVD induction (Nat Rev Dis polymers 2017, 3. In addition, increasing clinical trials have found that the risk of developing definitive risk factors such as LDL-C corrected coronary atherosclerotic Heart disease (CHD) remains, while elevated Triglyceride (TG) levels are significantly associated with increased patient mortality, myocardial infarction rate and recurrence of coronary events (Eur Heart J2011, 32 (11): 1345.). A large number of epidemiological studies have confirmed that TG levels are closely related to the occurrence and development of CVD, and high plasma TG concentrations have become independent risk factors for the induction of CVD in cardiovascular diseases such as atherosclerosis (Nat Rev Cardiol 2017 (7): 401). In addition, hypertriglyceridemia (TG) is closely related to chronic metabolic diseases such as nonalcoholic fatty liver (NAFLD), obesity, hepatic fibrosis, pancreatitis, and the like. Therefore, the development of the medicine for reducing triglyceride has important significance for preventing and treating chronic metabolic diseases such as atherosclerosis and other cardiovascular diseases (CVD), non-alcoholic fatty liver disease (NAFLD), obesity, hepatic fibrosis, pancreatitis and the like.
Exenatide (Exendin-4), an exogenous GLP-1 receptor polypeptide agonist found in the saliva of Exendin Blueda, north American, consists of 39 amino acids and has about 53% homology with the GLP-1 amino acid sequence. It has physiological functions similar to GLP-1 in mammals, and can stimulate glucose-dependent insulin secretion, i.e. it only acts when the body blood sugar concentration is high, but does not act when the blood sugar is normal or low. Exenatide was developed and marketed by Amylin and Eli Lilly in US at 4.2005 and marketed in China at 8.2009 for hypoglycemic treatment of type 2 diabetic patients. Compared with GLP-1, although exenatide is insensitive to dipeptidyl peptidase IV (DPP-4), the in vivo half-life of exenatide is prolonged to 3.3-4 hours (Barnet AH. Drugs Today (Barc) 41-78, kolterman OG. Am J Health Syst Pharm.2005, 173-81), but clinically, 2 injections per day are still needed, so that the prolongation of the in vivo half-life is beneficial to reducing the clinical dosage and frequency and improving the treatment effect.
CN201610678121.0 discloses a long-acting hypoglycemic fusion protein with high activity, which is formed by connecting a mutant exenatide with Fc fragment of optimized mutated human immunoglobulin IgG1 through a connecting peptide or directly. The fusion protein has long-acting hypoglycemic effect and can perform soluble expression in Escherichia coli E.
Disclosure of Invention
The present inventors have disclosed in earlier studies a high-activity long-acting hypoglycemic fusion protein (chinese patent document CN 201610678121.0) which is formed by connecting a high-activity exenatide mutant with a linker peptide or directly with an Fc fragment of an optimized mutated human immunoglobulin IgG1, has a long-acting hypoglycemic effect, and can be expressed in e.coli in a soluble manner.
Based on the preliminary research, the invention further researches the pharmacological activity of the fusion protein, and unexpectedly discovers that the fusion protein can more remarkably reduce plasma hypertriglyceridemia (TG), high-low-density lipoprotein cholesterol (LDL-C) and high Total Cholesterol (TC) compared with exenatide, has the advantage of long half life in vivo, and can be applied to the preparation of medicines for treating hyperlipidemia and related diseases caused by hyperlipidemia.
The specific technical scheme of the invention is as follows:
application of GLP-1 receptor agonist fusion protein in preparation of medicines for preventing or treating hyperlipidemia and related diseases caused by hyperlipidemia.
Preferably, the GLP-1 receptor agonist fusion protein is exenatide or a mutant thereof, which is formed by connecting the exenatide or the mutant thereof with a natural or optimized mutant human immunoglobulin IgG Fc fragment through a connecting peptide or directly. More preferably, the amino acid sequence of the GLP-1 receptor agonist is shown in SEQ ID NO:1 (or SEQ ID NO: 2).
The hyperlipemia of the invention comprises one or more of high triglyceride, high low density lipoprotein cholesterol and high total cholesterol. The related diseases caused by the hyperlipidemia comprise cardiovascular and cerebrovascular diseases such as non-alcoholic fatty liver, obesity, atherosclerosis and the like.
The invention discloses a new application of a long-acting fusion protein constructed by exenatide or a mutant thereof, wherein the fusion protein is constructed by the exenatide or the mutant thereof through a connecting peptide or directly with an Fc fragment of optimized mutated human immunoglobulin IgG, can effectively inhibit the expression of triglyceride synthetase in golden hamster livers induced by high-fat diet, obviously reduce the levels of high Triglyceride (TG), high-low density lipoprotein cholesterol (LDL-C) and high Total Cholesterol (TC) in golden hamster serum induced by the high-fat diet, and has the advantage of long half life in vivo, so that the fusion protein can be used for preparing medicaments for treating the high Triglyceride (TG), the high-low density lipoprotein cholesterol (LDL-C) and related diseases thereof such as non-alcoholic fatty liver, obesity, atherosclerosis and other cardiovascular and cerebrovascular diseases.
The long-acting GLP-1 receptor stimulant fusion protein can be prepared into pharmaceutically acceptable dosage forms by being mixed with pharmaceutically acceptable auxiliary materials.
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FIG. 1 is a survey of the improvement of palmitic acid-induced lipid accumulation in HepG2 cells by the fusion protein of the present invention. FIGS. 1A-1C show the cell viability after Exendin-4, EX4-Fc and EX-L21K-Fc administration in that order; panel D shows the results of staining of cell lipid accumulation with oil red O at microscopic magnifications of 200 and 400; FIG. E is the absorbance value A of the staining solution after the dye is dissolved 510nm (E)。 #### p<0.0001 compared to an untreated control; *** p<0.001, **** p<0.0001 compared to the PA group (n =3,means ± SEM).
FIG. 2 results of studies on the activation of AMP-dependent protein kinase (AMPK) phosphorylation by the fusion protein of the present invention. (FIGS. 2A-2E show the expression levels of p-AMPK/T-AMPK, nSREBP-1c, FAS, p-ACC/ACC, SCD-1 protein in HepG2 cells after administration, in order. # p<0.05, ## p<0.01 in comparison to untreated controls; * p<0.05, ** p<0.01, *** p<0.001 compared to PA group (n =8, means ± SEM).
FIG. 3 the effect of the administration of the fusion proteins of the invention on body weight and feed intake of HFD fed LVG syrian golden hamster. FIG. 3A shows the weight change of golden hamsters measured weekly on HFD and during the administration; FIG. 3B shows the average food intake of golden hamster before and during administration. (n =8, means ± SEM).
FIG. 4 Effect of subcutaneous administration of the fusion proteins of the invention on TG, TC, LDL-C and HDL-C in serum of HFD-fed LVG syrian golden yellow hamster. FIG. 4A serum Triglyceride (TG) levels; FIG. 4B serum Total Cholesterol (TC) levels; FIG. 4C serum low density lipoprotein cholesterol (LDL-C) levels; FIG. 4D serum high density lipoprotein cholesterol (HDL-C) levels. ### p<0.001 compared to Chow + PBS group; * p<0.05, ** p<0.01, **** p<0.0001 compared to the HFD + PBS group (n =8, means ± SEM).
FIG. 5 LVG Syria golden yellow fed to HFD after administration of the fusion protein of the inventionMorphology of the liver and effects of liver lipid accumulation in mice. FIG. 5A is a graph showing a reduction in hepatic body index of HFD-fed LVG Syria golden hamsters; FIG. 5B, amelioration of pathological hypertrophy of liver; FIG. 5C shows improvement of pathological liver changes (pathological liver sections after eosin-hematoxylin staining, microscopic magnification 200 times); FIG. 5D shows the reduction of TG content in liver tissue; FIG. 5E reduction of TC content in liver tissue. ## p<0.01 compared to the Chow + PBS group; * p<0.05, ** p<0.01 compared to the HFD + PBS group (n =3-8, means. + -. SEM).
FIG. 6 Effect of the fusion proteins of the invention on AMPK phosphorylation in liver tissue of HFD fed LVG Syria golden yellow mice after administration. FIGS. 6A-6E show the expression levels of p-AMPK/T-AMPK, nSREBP-1c, FAS, p-ACC/ACC, and SCD-1 protein in sequence. # p<0.05, ## p<0.01, #### p<0.0001 compared to Chow + PBS group; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 compared to the HFD + PBS group (n =8, means ± SEM).
Detailed Description
The following examples are provided to illustrate specific steps of the present invention, but are not intended to limit the scope of the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
The present invention is further illustrated by the following specific examples.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Long-acting fusion proteins improve palmitic acid-induced lipid accumulation in HepG2 cells
The long-acting fusion proteins EX4-Fc (SEQ ID NO: 1) and EX-L21K-Fc (SEQ ID NO: 2) used in the invention are the abbreviations of EX-hIgG1Fc and EX-L21K-mhIgG1Fc in the Chinese patent CN201610678121, and are prepared according to the method described in the patent, and the molar concentration calculation method is also obtained according to the single-chain molecular weight calculation.
Human liver cancer HepG2 (purchased from Shanghai cell Bank of Chinese academy of sciences) was resuspended in 10-vol% FBS (Cat: 10099141, gibco) MEM medium (41500034, gibco), and then the suspension was incubated at 5X 10 5 Inoculating each cell/well into a 96-well cell culture plate for culture, treating with a serum-free opti-MEM culture medium for 4h when the confluence rate reaches 70%, removing the culture medium, adding 200. Mu.L of 0-40 nM Exendin-4 and fusion protein thereof for culture for 24h, adding 20. Mu.L of MTT into each well, continuing to culture for 4h, removing the original culture medium, adding 150. Mu.L of dimethyl sulfoxide, shaking on a shaking table at 37 ℃ for 10min at low speed to fully dissolve crystals, and determining the concentration of A in each well 570nm And (4) an absorbance value. MTT experimental results (FIGS. 1A-C) show that the HepG2 cells treated by 0-40 nM of drug have no significant difference in cell viability among groups (p)>0.05 Shows that the drug treatment does not produce cytotoxicity in the concentration range, and can be used for subsequent drug treatment experiments.
HepG2 cells are evenly paved in a six-hole cell culture plate for culture until the confluency reaches 70%, and after the HepG2 cells are treated by a serum-free opti-MEM culture medium for 4 hours, 400 mu M Palmitic Acid (PA) is added to induce fat accumulation of the HepG2 cells, and meanwhile, 20nM drugs are respectively added to treat the cells for 24 hours. Washing with 4 deg.C precooled PBS for 3 times, fixing with 4% paraformaldehyde for 30min, dyeing with oil red O at room temperature for 1h, washing with distilled water, and observing the dyeing result with microscope (200 × 400). Drying, adding 1mL of isopropanol into each well, dissolving the dye by shaking at 37 ℃ for 10min, and determining A 510nm Absorbance. The results (FIGS. 1D-E) show that the PA model group stained more oil red O than the untreated control group, indicating that lipid packing had formed and molding was successful in PA-induced HepG2 cells. Compared with the PA model group, the administration group HepG2 cells have reduced oil red O staining, improved lipid accumulation condition and most obvious improvement effect of the PA + EX-L21K-Fc group **** p<0.0001vs.PAgroup)。
Example 2 Long-acting fusion proteins activate AMP-dependent protein kinase (AMPK) phosphorylation in HepG2 cells, which inactivates the phosphorylation of its substrate, acetyl-CoA carboxylase, while AMPK phosphorylation activation reduces the levels of nuclear cholesterol regulatory element-binding protein 1c (nSREBP-1 c), and down-regulates the expression of TG synthesis-associated Fatty Acid Synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA dehydrogenase (SCD-1).
AMP-dependent protein kinase (AMPK) is a key molecule for bioenergy regulation and can regulate various metabolic pathways including carbohydrate and lipid metabolism. p-AMPK (i.e., phosphorylated AMP-dependent protein kinase) can phosphorylate ACC to p-ACC, rendering it inactive, as the p-ACC/ACC ratio increases, reducing lipid synthesis.
After HepG2 cell treatment according to example 1, the p-AMPK/AMPK ratio in PA group cells was significantly reduced compared to normal control group ((A)) ## p<0.01). Compared with PA group, the fusion proteins EX-Fc and EX-L21K-Fc can obviously promote AMPK phosphorylation and obviously improve the ratio of p-AMPK/T-AMPK ( ** p<0.01, *** p<0.001vs.PA group)<PA group (FIG. 2A), and fusion protein EX-L21K-Fc activated AMPK phosphorylation more efficiently than EX-Fc fusion protein.
Sterol-regulatory element binding protein 1c (SREBP-1 c), which is the major SREBP-1 subtype in the liver of adults, is a key transcription factor for regulating lipid synthesis genes, and SREBP-1c (nSREBP-1 c) in the nucleus can be combined with Sterol Regulatory Element (SRE) to enhance the expression of various lipid synthesis related genes such as Fatty Acid Synthase (FAS), acetyl CoA Carboxylase (ACC) and stearoyl CoA dehydrogenase (SCD-1).
After treatment of the cells as described in example 1, the p-ACC/ACC ratio was decreased in HepG2 cells of the PA group and the expression levels of nSREBP-1c, FAS and SCD-1 were increased (respectively) ## p<0.01, # p<0.05, ## p<0.01 FIGS. 2B-2E). Compared with the PA group, the p-ACC/ACC ratio is increased to different degrees in HepG2 cells in the drug group, and the increased level of the PA + EX-L21K-Fc group is most remarkable (the PA + EX-L21K-Fc group is the most remarkable) * p<Pa group) 0.05vs. Compared to the PA group, the fusion proteins EX-Fc and EX-L21K-Fc groups nSREBP-1c, FAS and SCD-1 were significantly reduced in level, and the PA + EX-L21K-Fc group was the most significantly elevated (FIGS. 2B-E). The results demonstrate that lipid accumulation is induced in PAIn the HepG2 cell, the fusion proteins EX-Fc and EX-L21K-Fc can phosphorylate ACC to p-ACC through AMPK signal pathway to inactivate the ACC, and at the same time, the ratio of p-ACC/ACC is increased, and the level of a key transcription factor nSREBP-1c for TG synthesis caused by PA induction is obviously reduced, thereby obviously reducing the expression of enzymes FAS and SCD-1 related to TG synthesis.
Example 3 reduction of weight and food intake in golden hamster after administration of Long-acting fusion protein
LVG Syrian Golden yellow rats (LVG Golden Syrian Hamsters) 40 were purchased only from Beijing Wintonli model animals, inc., 6-7 weeks old, male, body mass (134 + -4) g, laboratory animal production license number SCXK (Jing) 2016-0011, strain code 501. Golden hamster is bred in an SPF animal house, the room temperature is controlled at 24-26 ℃, the humidity is 40-60%, the brightness is 12h each, the food and water source are sufficient, and the experiment is carried out according to the random grouping of the body weight after the golden hamster is bred adaptively for 1 week. Group 8, normal diet; another group, 32, was given a High Fat Diet (HFD). After feeding for 4 weeks, hypertriglyceridemia and liver fat accumulation are formed in the high fat diet group. Subsequently, the golden hamster of the high-fat diet group was randomly divided into 4 groups by weight, and subcutaneously injected with 20 nmol/kg -1 5 mL/kg dose volume -1 . The Chow + PBS group, HFD + PBS group and HFD + EX4 group were administered 1 time per day, the HFD + EX4-Fc group and HFD + EX-L21K-Fc group were administered 2 times per week for 4 weeks, and 2 weight and food intake were recorded per week. After 4 weeks of administration, golden hamster is fasted for 8 hours, blood is taken after euthanasia, and liver middle lobe is taken for fixation and embedding. Meanwhile, a part of liver tissues are taken and added with a homogenate medium, after mechanical homogenate, supernatant is taken to determine the content of TG in the liver tissues, and the rest part is immediately placed in a liquid nitrogen tank for freeze preservation and used for the subsequent detection of the expression level of TG synthesis related enzyme.
As shown in fig. 3A and table 1, the weight average of golden mice in the first 4 weeks normal diet group and the high fat diet group steadily increased, and the weight of the high fat diet group increased more rapidly than that of the normal diet group. After the model is formed by the induction of high fat diet for 4 weeks, the body weights (g) of the HFD + PBS group, the HFD + EX4-Fc group and the HFD + EX-L21K-Fc group are 163.72 +/-4.43, 163.07 +/-5.11, 162.47 +/-2.57 and 161.46 +/-4.71 respectively, and have very significant difference compared with the normal diet group of 146.33 +/-1.95 g (the weight ratio is very significant) (( #### p<0.0001 Indicate that the group of high fat diets has formedHigh lipid phenotype, without significant difference between HFD groups. After 4 weeks of administration, the weight average of the HFD + EX4 group, the HFD + EX4-Fc group and the HFD + EX-L21K-Fc group was significantly reduced, with the weight reduction of golden hamster being most significant in the HFD + EX-L21K-Fc group.
The change of food intake before and after administration is shown in fig. 3B, the food intake of golden hamster in normal diet group is stable in the whole experiment period, the food intake of golden hamster in HFD + EX4 group, HFD + EX4-Fc group and HFD + EX-L21K-Fc group is sharply reduced after administration and then slowly increased to a stable state, and the food intake of golden hamster in HFD + EX-L21K-Fc group is most obviously reduced after administration.
The results show that the Exendin-4, the fusion proteins EX4-Fc and EX-L21K-Fc can effectively inhibit the weight increase of golden hamster in high-fat diet group, and reduce the food intake, and the effect of EX-L21K-Fc group is more obvious.
TABLE 1 Effect of Long-acting fusion proteins on HFD-fed LVG Syria golden hamsters body weight
### p＜0.001， #### p＜0.0001vs.Chow+PBSgroup； **** p＜0.0001vs.HFD+PBSgroup； ΔΔΔ p＜0.001，p＜0.001， ΔΔΔΔ p＜0.0001vs.HFD+EX4group(n＝8，values are means±SEM).
Example 4 significant reduction of plasma TG, TC and LDL-C levels in LVG syrian golden hamster following administration of Long-acting fusion proteins
After the golden hamster obtained after the model preparation and the drug administration in example 3 is killed, a fresh blood sample is taken and kept still in a refrigerator at 4 ℃ for 4h, and then centrifuged at 3500rpm at 4 ℃ for 10min to obtain upper serum, and the contents of TG, TC, LDL-C and HDL-C are measured by using a biochemical detection kit (Nanjing institute of bioengineering research). The results showed that the serum content of TG, TC and LDL-C was significantly higher than that of the normal control group, but the HDL-C content was not affected (FIG. 4D). After 4 weeks of administration, the serum TG, TC and LDL-C contents of golden hamster are reduced to different degrees compared with model groupAnd the HFD + EX-L21K-Fc group showed a very significant difference in TG reduction: ( **** p < 0.0001vs. HFD + PBS group) (FIG. 4A), also showed significant difference in lowering TC and LDL-C: ( * p＜0.05， ** p < 0.001vs. HFD + PBS group) (FIGS. 4B, C).
Example 5 Long-acting fusion proteins improve the morphology of the liver and reduce lipid accumulation in liver tissues in golden hamster
After the mice with golden yellow color obtained after the drug administration was molded as in example 3 were sacrificed, fresh livers were taken, weighed and the liver weights were recorded, and the liver body index was calculated. As shown in FIG. 5A, the liver body indices of the HFD + PBS group were significantly increased compared to the Chow + PBS group (II) ((III)) #### p is less than 0.0001), which indicates that the livers of the golden hamster are more pathologically enlarged than the normal golden hamster after the HFD feeding. After administration, liver hypertrophy is relieved to different degrees, wherein the hepatic body index of HFD + EX-L21K-Fc group is reduced most remarkably (the hepatic body index of the HFD + EX-L21K-Fc group is reduced) *** p is less than 0.001), which indicates that the Exendin-4 and the fusion protein can effectively improve the pathological hypertrophy state of the liver, and the GLP-1 receptor agonist fusion protein EX-L21K-Fc has the most obvious effect.
As shown in FIG. 5B, the liver of Chow + PBS group golden hamster is darker and the liver surface is fine and smooth, while the livers of HFD + PBS group, HFD + EX4-Fc group and HFD + EX-L21K-Fc group are hypertrophic and blunted to different degrees, and the liver surface is rougher and whitish. However, compared with the HFD + PBS group, the liver morphologies of the HFD + EX4 group, the HFD + EX4-Fc group and the HFD + EX-L21K-Fc group were improved to different degrees, and the liver morphologies of the HFD + EX-L21K-Fc group were improved more significantly.
After the paraffin section H & E of the liver tissue is stained, the cell nucleus is purple blue, the cytoplasm is red, and fat vacuoles are formed at the intracellular lipid accumulation positions, so that the degree of fat accumulation in the liver cells can be judged according to the size of the intracellular fat vacuoles. The result is shown in a figure of 5C, in which the liver cells are arranged regularly in a strip shape by Chow + PBS, and the nucleus structure is complete; the arrangement of the gold hepatocytes in the HFD + PBS group is obviously disordered, the phenomenon of nucleus fragmentation can be seen, and the fat vacuoles in the cells are large, so that the liver lipid accumulation of the group is obviously higher than that of the Chow + PBS group; the HFD + EX4 group and the HFD + EX4-Fc group have reduced fat vacuole number and size compared with the HFD + PBS group, which indicates that liver lipid accumulation is improved; compared with the HFD + PBS group, the HFD + EX-L21K-Fc group has the advantages of obviously regular arrangement of liver cells, obviously improved nucleus fragmentation phenomenon and obviously reduced lipid vacuole number and size.
Accurately weighing the weight of the liver tissue of the golden hamster, adding absolute ethyl alcohol as a homogenizing medium according to the proportion that the weight (g) of the liver is that the volume (ml) =1, mechanically homogenizing under an ice bath condition, centrifuging at 2500rpm for 10min at 4 ℃, taking the supernatant, and detecting the content of TG and TC in the liver tissue by using a biochemical detection kit. The results show that EX-L21K-Fc can significantly reduce the TG (figure 5D) and TC (figure 5E) contents in liver tissues, which shows that EX-L21K-Fc has obvious effects on reducing the TG content in liver tissues and improving liver lipid accumulation, and the effect is better than Exendin-4.
Example 6 activation of AMPK phosphorylation in liver tissue of golden hamster after administration of Long-acting fusion protein, inhibition of nSREBP-1c expression, and further inhibition of TG synthetase Gene expression
To further verify the mechanism of action of the novel fusion protein in improving HTG, the expression levels of the genes involved in the regulation of TG synthesis and oxidative degradation in the liver of high-fat diet-fed golden hamster were examined again (fig. 6). As can be seen from the figure, the expression level of the related gene in the liver cells is consistent with the content trend of the related gene in HepG2 cells, and the ratio of p-AMPK/AMPK in the liver is significantly reduced in the HFD + PBS group golden hamsters compared with the Chow + PBS group (the ratio of p-AMPK/AMPK in the liver is significantly reduced in the HFD + PBS group to golden hamsters in golden hamsters compared with the Chow + PBS group) (( #### p<0.0001 Exendin-4 and its fusion protein can promote AMPK phosphorylation and increase p-AMPK/AMPK ratio (FIG. 6A). After the HFD + PBS group golden yellow hamster is fed with high fat diet, the expression levels of nSREBP-1c, FAS and SCD-1 are increased compared with the normal group (respectively ## p<0.01, ## p<0.01, # p<0.05 p-ACC/ACC ratio decreased, after the administration of the HFD + EX4 group, the HFD + EX4-Fc group, and the HFD + EX-L21K-Fc group, decreased expression levels of nSREBP-1c, FAS and SCD-1, increased p-ACC/ACC ratios to different extents, and most significantly increased levels in the HFD + EX-L21K-Fc group ( ** p<0.01 (FIGS. 6B-E).
Sequence listing
<110> university of Chinese pharmacy
Application of GLP-1 receptor agonist fusion protein in preparation of medicine for preventing or treating hyperlipidemia
<160> 2
<170> PatentIn version 3.5
<130> EX4-Fc
<210> 1
<211> 277
<212> PRT
<213> Artificial sequence
<400> 1
Met His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
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Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
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Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Val Glu Pro Lys
35 40 45
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
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Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
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Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
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Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
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Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
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Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
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Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
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Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
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Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
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Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
195 200 205
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
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Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
225 230 235 240
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
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Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
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<130> EX-L21K-Fc
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<211> 277
<212> PRT
<213> Artificial sequence
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Met His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
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Glu Glu Ala Val Arg Lys Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
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Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly
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Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
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Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
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Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
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Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
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Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
115 120 125
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
130 135 140
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
145 150 155 160
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
165 170 175
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
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Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
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Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
210 215 220
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
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Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
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Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
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275
Claims (2)
- An application of GLP-1 receptor stimulant fusion protein in preparing a medicine for preventing or treating hyperlipidemia and related diseases caused by hyperlipidemia, wherein the amino acid sequence of the GLP-1 receptor stimulant fusion protein is shown as SEQ ID NO:2, and the related diseases caused by hyperlipidemia are nonalcoholic fatty liver or obesity.
- 2. The use according to claim 1, wherein the hyperlipidemia comprises one or more of hypertriglyceridemia, high low density lipoprotein cholesterol, and high total cholesterol.
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| CN1347327A (en) * | 1999-01-14 | 2002-05-01 | 安米林药品公司 | Methods for glucagon suppression |
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