How Does Metformin Increase Insulin Sensitivity
Background. Type two diabetes has become ane of the most common diseases worldwide, causing a serious social burden. Equally a starting time-line handling for diabetes, metformin tin effectively improve insulin resistance. It has been reported that 12α-hydroxylated BA (mainly CA) is associated with insulin resistance. The purpose of this study was to clarify the changes in CA and possible signaling mechanisms in diabetic rats subsequently metformin intervention. Methods. HepG2 cells were cultured after adding different concentrations of metformin. The prison cell viability was measured using CCK8 kit, and the expression of FXR, MAFG, and CYP8B1 in cells was detected by WB. The rat models of type 2 diabetes were induced by low-dose streptozotocin by feeding a high-fat diet, and the control rats (CON) were fed on normal food; the diabetic rats (DM) were given a high-fat diet without supplementation with metformin, while the metformin-treated diabetic rats (DM + MET) were given a high-fat diet and supplemented with metformin. Biochemical parameters were detected at the end of the test. Expression levels of FXR, CYP8B1, and MAFG were assessed by WB. Serum CA were measured using an enzyme-linked immunosorbent analysis (ELISA). Results. In HepG2 cells, metformin dose-dependently enhanced the transcriptional activity of FXR and MAFG and inhibited the expression of CYP8B1. Metformin-treated DM rats showed improved glucose and bile acrid metabolism. In add-on, significantly increased FXR and MAFG and decreased CYP8B1 were observed in DM + MET rats. At the same time, the CA content of metformin-treated rats was lower than that of diabetic rats. Conclusion. Changes in CA synthesis later on metformin handling may be associated with inhibition of CYP8B1. These results may play an of import function in improving insulin sensitivity after metformin treatment.
1. Introduction
T2DM is a common circuitous metabolic disorder. Insulin resistance and relatively insufficient insulin secretion are major features of type 2 diabetes. As a first-line antidiabetic drug, metformin mainly reduces hyperglycemia and improves glucose uptake and insulin sensitivity by inhibiting gluconeogenesis. Studies have shown that insulin resistance may be associated with 12α-hydroxylated bile acids [ane]. The compositional ratios and related principles of 12α-hydroxylated BA afterwards metformin treatment may play a major role in alleviating insulin resistance.
Sterol 12α-hydroxylase (CYP8B1) is an essential enzyme that promotes the synthesis of 12α-hydroxylated bile acids [two]. Different CYP7A1, which is the rate-limiting enzyme of the classical pathway of bile acrid metabolism, CYP8B1 is mainly responsible for the synthesis of 12α-hydroxylated bile acids and controls the ratio of cholic acid in bile to that of chenodeoxycholic acid, among which cholic acrid is the most arable 12α-hydroxylated bile acid in liver tissue [3]. Farnesol X receptor (FXR) is an activated transcriptional regulator of bile acrid and glucose metabolism [iv]. In addition, FXR-mediated SHP inhibits CYP7A1 expression [v]. In contrast to the detailed study of the mechanism by which metformin inhibits CYP7A1 [6, 7], the mechanism by which metformin inhibits CYP8B1 and cholic acid synthesis remains unclear. Recent studies have confirmed that MAFG is a target of FXR and a key transcriptional repressor of bile acid synthesis and metabolism [viii–11]. MAFG has been shown to reduce the transcription level of CYP8B1 in the liver [12].
Bile acid contour is a key regulator of metabolic pathways. Metformin inhibits the expression of the enzyme (CYP8B1) required for the synthesis of 12α-hydroxylated bile acid and inhibits the synthesis of cholic acid, which may be effective targets for the treatment of type ii diabetes [13].
ii. Materials and Methods
2.1. Reagents and Antibodies
DMEM medium (HyClone Corp.), fetal bovine serum (Sijiqing), metformin hydrochloride (Sigma, United states), CCK8 kit (Sigma, USA), FXR antibody (Biorbyt), CYP8B1 antibody (Abcam), CYP7A1 antibody (Abcam), and MAFG antibody (Abcam) were used in the experiments.
Metformin from Bristol-Myers Squibb and STZ from Sigma (USA), likewise as metformin and compound C from Sigma-Aldrich Co. (St Louis, MO, United states of america) were employed in the experiments.
two.ii. Prison cell Culture
Human hepatoma HepG2 cells were acquired from the Cell Banking concern of Shandong Academy of Medical Sciences (Shandong, Cathay). HepG2 cells were cultured in high glucose-DMEM containing 10% fetal bovine serum and one% penicillin/streptomycin. The cells were incubated in a moist temper of 5% COtwo at 37°C and passaged every 3 days by trypsinization.
2.3. Cell Proliferation Assay
The cytotoxic effect of metformin was evaluated using a CCK8 assay. HepG2 cells were sowed into 96-well plates at a density of 1 × 10four per well and were incubated at 37°C for 24 h in a humidified temper of 5% CO2. Then unlike concentrations (0, 0.v, 1, i.5, and 2 mM) of metformin were added. Subsequently 24 h of culture, a cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8) (Sigma, USA) co-ordinate to the manufacturer's illustrations. The absorbance was read at 450 nm with a microplate reader (Bio-Rad).
2.four. Cultured Cells
In order to prove the influences of metformin intervention on FXR, MAFG, and CYP8B1 expression, metformin was added to the medium in half dozen-well plates containing HepG2 (5 × ten5 cells/ml) with the following ultimate concentrations: 0, 0.5, 1, ane.five, and 2 mM for 24 h. In addition, cells were cultured with 1 mM metformin for 0, 12, 24, or 48 h. The cells were harvested for western blotting.
2.v. Animal Experiments
Male person Wistar rats, weighing 200–230 g, were obtained from Vital River Laboratory Animal Engineering Co.Ltd. (Beijing, China), at 8 weeks of age and housed at 23 ± two°C with a 12-h cycle of light/dark. H2o and food were given advert libitum. All animal methods in the experiment were approved by the Animal Research Commission of Shandong Academy. Subsequently 1 week adaptation period, sixty Wistar rats were randomly distributed into three groups: control rats (CON) grouping (n = 20), diabetic rats (DM) group (n = 20), and metformin-treated diabetic rats (DM + MET) group (n = 20). For the CON group, rats were fed with a standard diet (6% fat, 64% carbohydrate, and 23% poly peptide). For the DM and DM + MET groups, rats were fed with a HFD (25% fat, 48% carbohydrate, and 20% poly peptide). After HFD feeding for ten weeks, DM and DM + MET rats were injected intraperitoneally 35 mg/kg body weight streptozotocin (STZ) dissolved in citrate buffer (pH 4.2) to induce a type 2 diabetes model. CON rats were given an equal volume of saline intraperitoneally. Seventy-ii hours after STZ injection, the fasting blood glucose (FBG) level was measured by a glucometer. The FBG ≧eleven.i mmol/Fifty was regarded equally successful consecration of diabetes and selected for further studies.
DM + MET rats were administered metformin (500 mg·kg−1·d−1) intragastrically for 12 weeks subsequently diabetes model consecration; CON rats and DM rats were given an equal book of saline intraperitoneally. FBG levels of the three groups of rats were recorded every forenoon, and the mental country, food intake, water intake, and body mass were measured. At 32 weeks, all rats were fasted one dark before serum and tissue collection. The serum samples and tissues obtained were stored at −80°C.
2.6. Nutrient and H2o Intake Measured
5 rats were housed in each cage, and 200 g of feed was weighed and placed in a muzzle. At the same time on the second day, the remaining feed was taken out on the tray and the remaining value was read. The amount of feed added minus the remaining amount is the total nutrient intake of the rat. The total food intake of the rats was divided past the number of rats, ie., the average food intake per rat in the cage.
Two drinking bottles were placed in each cage , each containing 150 ml of water. At the aforementioned fourth dimension on the next day, the water was collected into a graduated cylinder and the remaining water was read. The corporeality of water added minus the remaining amount is the total amount of water in the caged rats. The total corporeality of water in the cage was divided by the number of rats, which is the average water intake per rat in the cage.
two.7. Oral Glucose Tolerance Test (OGTT), Glucose Concentration, and Insulin Concentration
At 24 weeks, an OGTT was performed subsequently an overnight fast (12 h). A solution of fifty% glucose (two.5 g/kg) was administered orally, and four claret samples were obtained from the retrobulbar venous plexus at 0, 30, lx, and 120 minutes for the measurement of glucose and insulin, respectively. Glucose concentration was evaluated using a glucometer (Roche, Basel, Switzerland). Insulin concentration was measured with an ELISA kit purchased from Mercodia (Uppsala, Sweden).
2.viii. Biochemical Analysis
At the end of the exam, the rats were fasted for xvi hours. Blood samples from the eye angular vein were analyzed to evaluate the levels of parameters of total bile acids (TBAs), depression-density lipoprotein cholesterol (LDL-C), full cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG). They were tested by cobas 8000 automatic biochemistry analyzer (Roche, Basel, Switzerland). Glucose level was tested past using a glucometer. Insulin was detected with ELISA kits. The steady-state model assessment of insulin resistance (HOMA-IR) index was calculated as [FBG (mmol/L) × FIN (μU/mL)]/22.5.
2.ix. Western Blotting
Homogenates of rat liver or HepG2 cell lysates were prepared for western absorb assay. Samples were centrifuged at 10,000 rpm for ten min at 4°C, and the supernatant was collected. Total protein concentration was measured by BCA. The proteins were loaded equally on 12% SDS-Folio and transferred onto PVDF membranes (0.2 mm, Millipore, Billerica, MA, USA). Membranes were blocked in PBST/v% nonfat dry out milk pulverisation and were incubated overnight at 4°C with the primary antibodies against FXR, MAFG, CYP8B1, CYP7A1, and β-actin. The secondary antibodies binded to HRP were then incubated with the membranes at room temperature for 45 min. After removal of the secondary antibodies, the blots were washed by an ECL detection kit (Millipore, Billerica, MA, The states) and imaged on an automated gel imaging analysis system. Image J (National Institutes of Wellness, Bethesda, Doc, USA) was used for quantification of immunoblots.
2.10. Detection of Cholic Acid in Liver Tissue by ELISA
i thousand of liver tissue sample was weighed, 9 g of PBS with a pH of seven.2–7.iv was added, and the sample was homogenized by hand or a homogenizer. Centrifuge for approximately 20 min (2000–3000 r/min), and collect the supernatant carefully. A portion was packed for testing and the rest was frozen for later use. If a precipitate forms during storage, it should exist centrifuged once again. Cholic acrid was detected using an ELISA kit.
ii.11. Statistical Analysis
Data were calculated from at least three independent experiments and were expressed as mean ± SEM. Comparisons among three groups were obtained via i-fashion assay of variance (ANOVA) followed past Dunnett's test. was considered statistically pregnant. GraphPad Prism .6.0 (GraphPad Software Inc., San Diego, CA) was used to perform the data analysis.
3. Results
3.ane. Result of Various Doses of Metformin (Met) on Cell Viability
In order to assess the effect of metformin on HepG2 cells, we treated HepG2 cells with various concentrations of metformin for up to 96 hours. We so determined cell growth using the CCK8 analysis. Equally the dose of metformin increased, we observed a pregnant decrease in the activeness of HepG2 cells (Figure 1(a)).
iii.2. Effects of Various Concentrations of Metformin on FXR, MAFG, and CYP8B1 at HepG2 Jail cell Level
In social club to study the effects of metformin on FXR, MAFG, and CYP8B1 at the jail cell level, the cells were incubated with metformin at dissimilar concentrations (0, 0.v, 1, ane.5, and ii mM) for 24 hours or with 1.five mM metformin for various durations (0, 12, 24, or 48 h). As shown in Figure 1, the protein expression levels of FXR, MAFG, and CYP8B1 were measured later on incubation with various metformin concentrations for different durations. Western blot analysis showed that metformin promoted the expression of FXR (Figures 1(b) and 1(e)) and MAFG (Figures 1(c) and i(f)) in a fourth dimension- and dose-dependent way, while inhibiting the protein expression level of CYP8B1 (Figures ane(d) and ane(g)). These results suggest that metformin intervention can enhance the sensitivity of FXR and the transcriptional activity of MAFG in a dose-dependent manner and inhibit the expression of CYP8B1 in HepG2.
3.3. Effect of Metformin on Nutrient and Fluid Intake and Body Weight of Diabetic Rats
Nosotros divided the experiment into ii parts: before and afterwards the formation of the diabetes model; the food and fluid intake and body weight of each group of rats were recorded every day (Table 1). During the experiment, HFD/STZ induced blazon ii diabetes (DM), the rats were junior, the body was thin, and the glaze was not shiny. Information technology is expressed as a diet and the corporeality of drinking water is significantly increased, which is consistent with the characteristics of blazon 2 diabetes. After treatment with metformin, the diabetic rats improved their spirits, and the amount of water and nutrient intake decreased compared with the DM group, but still more the normal control group.
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| Afterward STZ injection, metformin decreased food intake and body weight gain in rats. Data are hateful ± SEM for twenty animals in each grouping. a versus CON rats. b vs. DM rats. CON rats: control rats group; DM rats: HFD/STZ induced diabetic rats grouping; DM + METF rats: HFD/STZ-induced diabetic rats supplemented with metformin treatment. | ||||||||||||||||||||||||||||
The fluid and food intake as well as body weight of high-fat nutrition DM and DM + MET rats were significantly higher than those of the control rats before modeling. There was no significant difference in fluid and nutrient intake and trunk weight betwixt the DM rats and the DM + MET rats. Later on successful modeling, DM rats showed weight loss compared to command rats, and DM + MET rats lost weight compared to DM rats later on treatment with metformin. Our results signal that metformin can reduce the body weight of diabetic rats. Inhibition of ambition and reduction of nutrient intake in diabetic rats are its main mechanism.
3.4. Furnishings of Metformin on FBG, Insulin, and Biochemical Parameters of Diabetic Rats
Based on previous studies, nosotros established a T2DM model by feeding a HFD and intraperitoneal injection of a small dose of STZ. The levels of FBG and insulin were measured from sera nerveless past puncturing the retro-orbital plexus at week 24. The results demonstrated that DM rats had loftier levels of FBG and high fasting insulin levels compared to the CON rats (Figures ii(a) and two(b); ). Oral glucose tolerance test (OGTT) showed that glucose concentration in DM rats was higher than that in CON rats at whatsoever fourth dimension point (Figures 2(a)) and insulin secretion was delayed. DM rats showed severe insulin resistance, which was consequent with the pathophysiological characteristics of type two diabetes. Administering metformin to DM + MET rats for 12 weeks effectively reduced FBG levels, and insulin secretion tiptop appeared before. This suggests that metformin tin can meliorate insulin resistance nether the current experimental conditions.
(a)
(b)
As shown in Table two, TC, TG, TBAs, and LDL-C were markedly increased in the DM rats, suggesting that DM rats accept disordered claret lipids and bile acids. Afterwards 12 weeks of treatment with metformin, the levels of TC, TG, TBAs, and LDL-C were significantly lower than those of DM rats ( ). The administration of metformin results in a decrease in bile acid reabsorption. Since bile acids are converted from cholesterol in the liver, cholesterol tin can be excreted into the intestine through bile acrid secretion and finally excreted in the carrion, thereby promoting the excretion of bile acids in the torso.
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| The trunk weight (BW), fasting plasma glucose (FPG), fasting insulin (FIN), full cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), depression-density lipoprotein-cholesterol (LDL-C), total bile acids (TBAs), and triglycerides (TG) levels were analyzed using cobas 8000 automatic biochemistry analyzer. HOMA-IR = [FBG(mmol/50) × FINS(μU/mL)]/22.v. Data are expressed as hateful ± SEM. a , aa vs. CON rats. b vs. DM rats. CON group: control rats group; DM group: HFD/STZ-induced diabetic rats grouping; DM + METF rats: HFD/STZ-induced diabetic rats supplemented with metformin handling. | ||||||||||||||||||||||||||||||||||||||||||||||||||||
3.v. Effect of Metformin on the Expression of Genes Involved in Cholic Acrid Synthesis in Diabetic Rats
It has been establish that metformin affects FXR, MAFG, and CYP8B1 at the cellular level. Nosotros further investigated whether metformin affects FXR, MAFG, CYP8B1, and CYP7A1 in liver tissue levels of diabetic rats. At 32 weeks of age, FXR (Effigy 3(a)) and MAFG (Figure 3(b)) levels were reduced in DM rats, while CYP8B1 (Figure three(c)) and CYP7A1 (Figure three(e)) were increased. In dissimilarity, after metformin treatment, nosotros detected an increase in FXR (Effigy 3(a)) and MAFG (Figure iii(b)) and a subtract in CYP8B1 (Effigy 3(c)) and CYP7A1 (Figure 3(eastward)) in DM + MET rats compared with DM rats.
3.half-dozen. Outcome of Metformin on Cholic Acid Synthesis in Diabetic Rat
We quantified cholic acid in liver tissue equally nosotros observed decrease in the expression of CYP8B1 reflecting cholic acid synthesis and an increment in the expression of MAFG and FXR in CYP8B1 regulation after metformin handling. It occurred that the concentration of cholic acid (Figure 3(d)) in DM rats was obviously more than that in CON rats, and the cholic acid (Figure 3(d)) content was lower than that of DM rats after metformin intervention.
4. Give-and-take
In the enquiry, we established a rat model with T2DM by HFD and small-scale-dose STZ injection. Information technology is reported that HFD causes insulin resistance, STZ is toxic to islet β cells, and injection of large doses of STZ can damage most islet β cells to induce T1DM, while our laboratory demonstrated that low-dose STZ tin can be used to set up T2DM models [14, fifteen].
In this study, we proved that metformin inhibits the expression of CYP8B1, thereby inhibiting the synthesis of CA, as a novo target for the treatment of type ii diabetes. The activity of Cyp8b1 was decreased in DM rats after metformin administration, which in turn led to a subtract in the level of CA. At the same time, we observed a decrease in weight gain and an improvement in insulin resistance in diabetic rats subsequently metformin treatment. This may be due to a correlation between 12α-hydroxylated BA levels (mainly CA) and insulin sensitivity [1].
A growing number of research is interested in the furnishings of metformin on endogenous BA, and whether BA signaling can explain its effect on improving insulin sensitivity, so we determined that metformin regulates the pathway of sterol 12α-hydroxylase CYP8B1. Metformin is effective in lowering blood glucose [16], reducing body weight [17], and improving insulin resistance [18] and has been widely studied for its importance in the treatment of diabetes [19]. The master pathway for cholesterol catabolism is the formation of bile acids [20]. CYP7A1 is the beginning rate-limiting enzyme in the classical pathway for bile acid synthesis, producing ii major bile acids, cholic acrid, and chenodeoxycholic acid. CYP8B1 catalyzes the synthesis of CA and plays a central role in intestinal cholesterol absorption and cholesterol gallstones, dyslipidemia, and the pathogenesis of diabetes [21]. FXR is idea to be the master regulator of this steady-state process, inhibiting bile acid synthesis, while increasing bile acid secretion in hepatocytes and regulating bile acid content in the liver [3]. By modulating the FXR signal, bile acids themselves can act every bit signaling molecules that touch blood sugar and lipid metabolism [20]. In our report, FXR was activated afterwards metformin intervention and showed a drib in blood glucose. The machinery may be that activation of FXR increases glycogen synthesis and reduces glycolysis. In add-on, FXR protects islet beta cell activity and affects glucose regulation [15]. In the liver, FXR activation induces SHP and leads to a decrease in CYP7A1 activeness. Our experiments also confirmed this, activation of FXR after metformin administration resulted in decreased expression of CYP7A1 (Figure iii(due east), ). In the intestine, bile acrid stimulates FXR to upregulate the expression of FGF19 (FGF15 in rodents) in intestinal epithelial cells [22, 23]. We observed a decrease in CYP8B1 expression after metformin administration, but the principle is nonetheless indeterminate. de Aguiar Vallim et al. [8, 9] discovered a new FXR-regulated transcriptional inhibitor, MAFG, which inhibits BA metabolism by inhibiting CYP8B1 expression. The MAFG gene is reported to exist a direct target of FXR, which in turn inhibits many genes involved in bile acid synthesis and metabolism. In particular, de Aguiar Vallim et al. [8] indicated that overexpression of MAFG inhibited the activity of hepatic CYP8B1, thereby reducing the contents of CA and increasing the contents of MCA. In addition, knocking out the MAFG factor increases the contents of CYP8B1 and CA [8, 9].
We found that metformin administration may touch the pathways involving FXR, MAFG, and CYP8B1. In vitro, nosotros detected the expression of FXR, MAFG, and CYP8B1 in HepG2 cells after metformin administration and found that metformin increased the expression of FXR and MAFG in HepG2 cells in a time- and dose-dependent manner, while decreasing the expression of CYP8B1.
When completed in vivo experiments, HFD/STZ-induced blazon 2 diabetic models showed elevated blood sugar and delayed peak serum insulin at all time points. Therefore, the diabetic rat model induced past HFD/STZ was in an insulin resistant state and was even so able to secrete large amounts of insulin. Metformin significantly improved FBG and insulin resistance in diabetic rats. In addition, lipid mass spectrometry such as LDL-C and TG was ameliorated after metformin intervention. In vivo, we demonstrate that FXR and MAFG are reduced and CYP8B1 is elevated in HFD/STZ-treated diabetic rats (Effigy 3, all ), which resemble to previous enquiry studies [7]. It has been reported that the level of Cyp8b1 mRNA and enzyme activity is increased in streptozotocin-induced diabetic rats [1]. Loftier cholesterol diet in diabetic rats further increased cholesterol to bile acid conversion and CA/CDCA ratio [24, 25].
Our current study showed that FXR and MAFG expression was increased while CYP8B1 expression was decreased in DM + MET rats after metformin administration. Our results contradict a research which found that metformin-activated AMPK downregulates FXR transcriptional activity, thereby disrupting bile acid homeostasis and impairing the liver [26]. Nosotros hypothesize that this is because our animal model is unlike from the cholestasis model.
To assess the effect of metformin on bile acid metabolism, we measured serum bile acids that reflect systemic BA levels. In this piece of work, we surveyed elevated serum BA levels in diabetic rats, suggesting a disorder of bile acid metabolism, which is consequent with our previous studies [xv]. The decrease in total serum BA levels in the DM + MET grouping after metformin treatment was due to the decreased expression of CYP7A1, a cardinal enzyme involved in bile acid synthesis, which inhibited the synthesis of BA. The major products of the BA synthesis pathway in humans are CDCA and CA. In rodents, most CDCA is converted to MCA [27]. CA produces secondary bile acid DCA with the effect of the intestinal microbiota. CA, DCA, TDCA, and TCA are 12α-hydroxylated BAs catalyzed past CYP8B1 [27]. Biddinger and Kahn [28] observed an increase in BA levels catalyzed by serum 12α-hydroxylated BA compared to the control grouping. Haeusler'south research [29] showed that insulin resistance is associated with increased levels of serum 12α-hydroxylated BA. Kaur et al. demonstrated that loss of CA resulted in an improvement in insulin resistance in Cyp8b1−/− mice [thirty]. In decision, at that place is a striking relativity between 12α-hydroxylated BA and insulin resistance. Based on CA being the most abundant 12α-hydroxylated BA in liver tissue, nosotros used an ELISA kit to measure the CA content of liver tissue in a diabetic rat model. Nosotros found that the CA content of diabetic rats increased significantly, and the CA content decreased after metformin administration. These results are consequent with the expression of CYP8B1 at the genetic level. After assistants of metformin, the expression of CYP8B1 is decreased and the CA content is reduced. The higher up findings may exist important for metformin to meliorate insulin resistance and farther explore the novel mechanism of action of metformin. Our research is based primarily on animal studies, and the clinical impact of metformin on bile acid metabolism homeostasis remains to exist adamant in humans.
Information Availability
The information used to back up the findings of this study are included inside the commodity.
Conflicts of Interest
The authors declare that they take no conflicts of involvement.
Acknowledgments
This study was supported by Shandong Province Key R&D Plan (Grant no. 2022GSF201019) and Jinan Science and Technology Innovation Plan of Clinical Medicine (Grant no. 202205072).
Copyright
Copyright © 2022 Mengsiyu Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original piece of work is properly cited.
How Does Metformin Increase Insulin Sensitivity,
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