Opposite effects of genistein on the regulation of insulin-mediated glucose homeostasis in adipose tissue

BACKGROUND AND PURPOSE

Genistein is an isoflavone phytoestrogen found in a number of plants such as soybeans and there is accumulating evidence that it has beneficial effects on the regulation of glucose homeostasis. In this study we evaluated the effect of genistein on glucose homeostasis and its underlying mechanisms in normal and insulin-resistant conditions.

EXPERIMENTAL APPROACH

To induce insulin resistance, mice or differentiated 3T3-L1 adipocytes were treated with macrophage-derived conditioned medium. A glucose tolerance test was used to investigate the effect of genistein. Insulin signalling activation, glucose transporter-4 (GLUT4) translocation and AMP-activated PK (AMPK) activation were detected by Western blot analysis or elisa.

KEY RESULTS

Genistein impaired glucose tolerance and attenuated insulin sensitivity in normal mice by inhibiting the insulin-induced phosphorylation of insulin receptor substrate-1 (IRS1) at tyrosine residues, leading to inhibition of insulin-mediated GLUT4 translocation in adipocytes. Mac-CM, an inflammatory stimulus induced glucose intolerance accompanied by impaired insulin sensitivity; genistein reversed these changes by restoring the disturbed IRS1 function, leading to an improvement in GLUT4 translocation. In addition, genistein increased AMPK activity under both normal and inflammatory conditions; this was shown to contribute to the anti-inflammatory effect of genistein, which leads to an improvement in insulin signalling and the amelioration of insulin resistance.

CONCLUSION AND IMPLICATIONS

Genistein showed opposite effects on insulin sensitivity under normal and inflammatory conditions in adipose tissue and this action was derived from its negative or positive regulation of IRS1 function. Its up-regulation of AMPK activity contributes to the inhibition of inflammation implicated in insulin resistance.     

Long-term ethanol exposure inhibits glucose transporter 4 expression via an AMPK-dependent pathway in adipocytes

Aim:

The roles of AMP-activated protein kinase (AMPK) and myocyte enhancer factor 2 isoforms (MEF2A, D) as mediators of the effects of ethanol on glucose transporter 4 (GLUT4) expression are unclear. We studied the effects of ethanol in adipocytes in vivo and in vitro.

Methods:

Thirty-six male Wistar rats were divided into three groups and given ethanol in a single daily dose of 0, 0.5, or 5 g/kg for 22 weeks. The expression of AMPK, MEF2 isoforms A and D, and GLUT4 was measured and compared in the three groups. The existence of the AMPK/MEF2/GLUT4 pathway in adipocytes and the effects of ethanol on this pathway were studied in (a) epididymal adipose tissue from six male Wistar rats subcutaneously injected with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR, an AMPK activator) or with 0.9% NaCl (control); and (b) isolated rat and human adipocytes treated with or without ethanol, AICAR, and compound C (a selective AMPK inhibitor). Expression of AMPK, MEF2, and GLUT4 was measured by RT-PCR and Western blotting.

Results:

(1) Long-term ethanol exposure decreased activated AMPK, MEF2A, MEF2D, and GLUT4 expression in rat adipose tissue. (2) In rat and human adipocytes, AICAR-induced AMPK activation, with subsequent elevation of MEF2 and GLUT4 expression, was inhibited by compound C. (3) In vitro ethanol-treatment suppressed the AMPK/MEF2/GLUT4 pathway.

Conclusion:

The AMPK/MEF2/GLUT4 pathway exists in both rat and human adipocytes, and activated AMPK may positively regulate MEF2 and GLUT4 expression. Ethanol inhibition of this pathway leads to decreased GLUT4 expression, thus reducing insulin sensitivity and glucose tolerance.     

Curcumin prevents leptin raising glucose levels in hepatic stellate cells by blocking translocation of glucose transporter-4 and increasing glucokinase

BACKGROUND AND PURPOSE

Hyperleptinemia is commonly found in obese patients, associated with non-alcoholic steatohepatitis and hepatic fibrosis. Hepatic stellate cells (HSCs) are the most relevant effectors during hepatic fibrogenesis. We recently reported that leptin stimulated HSC activation, which was eliminated by curcumin, a phytochemical from turmeric. This study was designed to explore the underlying mechanisms, focusing on their effects on intracellular glucose in HSCs. We hypothesized that leptin stimulated HSC activation by elevating the level of intracellular glucose, which was eliminated by curcumin by inhibiting the membrane translocation of glucose transporter-4 (GLUT4) and inducing the conversion of glucose to glucose-6-phosphate (G-6-P).

EXPERIMENTAL APPROACH

Levels of intracellular glucose were measured in rat HSCs and immortalized human hepatocytes. Contents of GLUT4 in cell fractions were analysed by Western blotting analyses. Activation of signalling pathways was assessed by comparing phosphorylation levels of protein kinases.

KEY RESULTS

Leptin elevated the level of intracellular glucose in cultured HSCs, which was diminished by curcumin. Curcumin suppressed the leptin-induced membrane translocation of GLUT4 by interrupting the insulin receptor substrates/phosphatidyl inositol 3-kinase/AKT signalling pathway. Furthermore, curcumin stimulated glucokinase activity, increasing conversion of glucose to G-6-P.

CONCLUSIONS AND IMPLICATIONS

Curcumin prevented leptin from elevating levels of intracellular glucose in activated HSCs in vitro by inhibiting the membrane translocation of GLUT4 and stimulating glucose conversion, leading to the inhibition of HSC activation. Our results provide novel insights into mechanisms of curcumin in inhibiting leptin-induced HSC activation.     

Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation

Aim:

To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS), extracted from Astragalus membranaceus Bunge, in L6 myotubes in vitro.

Methods:

APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[³H]-D-glucose method. The adenine nucleotide contents in the cells were measured by HPLC. The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis. The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach.

Results:

Treatment of L6 myotubes with APS (100−1600 μg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/mL) for 36 h. The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C, a selective AMPK inhibitor or in the cells overexpressing AS160-4P. Treatment of L6 myotubes with APS strongly promoted the activation of AMPK. We further demonstrated that either Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes, and the increased cellular AMP: ATP ratio was also involved. Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160, which was significantly attenuated by pretreatment with Compound C.

Conclusion:

Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.     

β(2)-Adrenoceptors increase translocation of GLUT4 via GPCR kinase sites in the receptor C-terminal tail

BACKGROUND AND PURPOSE

β-Adrenoceptor stimulation induces glucose uptake in several insulin-sensitive tissues by poorly understood mechanisms.

EXPERIMENTAL APPROACH

We used a model system in CHO-K1 cells expressing the human β₂-adrenoceptor and glucose transporter 4 (GLUT4) to investigate the signalling mechanisms involved.

KEY RESULTS

In CHO-K1 cells, there was no response to β-adrenoceptor agonists. The introduction of β₂-adrenoceptors and GLUT4 into these cells caused increased glucose uptake in response to β-adrenoceptor agonists. GLUT4 translocation occurred in response to insulin and β₂-adrenoceptor stimulation, although the key insulin signalling intermediate PKB was not phosphorylated in response to β₂-adrenoceptor stimulation. Truncation of the C-terminus of the β₂-adrenoceptor at position 349 to remove known phosphorylation sites for GPCR kinases (GRKs) or at position 344 to remove an additional PKA site together with the GRK phosphorylation sites did not significantly affect cAMP accumulation but decreased β₂-adrenoceptor-stimulated glucose uptake. Furthermore, inhibition of GRK by transfection of the βARKct construct inhibited β₂-adrenoceptor-mediated glucose uptake and GLUT4 translocation, and overexpression of a kinase-dead GRK2 mutant (GRK2 K220R) also inhibited GLUT4 translocation. Introducing β₂-adrenoceptors lacking phosphorylation sites for GRK or PKA demonstrated that the GRK sites, but not the PKA sites, were necessary for GLUT4 translocation.

CONCLUSIONS AND IMPLICATIONS

Glucose uptake in response to activation of β₂-adrenoceptors involves translocation of GLUT4 in this model system. The mechanism is dependent on the C-terminus of the β₂-adrenoceptor, requires GRK phosphorylation sites, and involves a signalling pathway distinct from that stimulated by insulin.     

Astragalus polysaccharides alleviates glucose toxicity and restores glucose homeostasis in diabetic states via activation of AM PK

Aim:

To establish the mechanism underlying the improvement of glucose toxicity by Astragalus polysaccharide (APS), which occurred via an AMP activated protein kinase (AMPK)-dependent pathway.

Methods:

In vivo and in vitro effects of APS on glucose homeostasis were examined in a type 2 diabetes mellitus (T2DM) rat model. The T2DM rat model was duplicated by a high-fat diet (58% fat, 25.6% carbohydrate, and 16.4% protein) and a small dose of streptozotocin (STZ, 25 mg/kg, ip). After APS therapy (700 mg·kg⁻¹·d⁻¹, ig) for 8 weeks, blood glucose, glycosylated hemoglobin, and serum insulin were measured. Insulin sensitivity was evaluated by the comprehensive analysis of oral glucose tolerance tests (OGTT) and HOMA IR index. Hepatic glycogen was observed by the PAS staining method. The expression and activity of skeletal muscle AMPKα and acetyl-CoA carboxylase (ACC), and the phosphorylation of hepatic glycogen synthase (GS), the glycogen synthase (GS),were measured by Western blotting. Glucose uptake was measured with the 2-deoxy-[³H]-D-glucose method in C2C12 cells.

Results:

The hyperglycemia status, insulin sensitivity, glucose uptake, and activation level of AMPK in diabetic rats were improved in response to APS administration. APS could also alleviate glucose toxicity in cultured mouse cells by the activation of AMPK.

Conclusion:

APS can alleviate glucose toxicity by increasing liver glycogen synthesis and skeletal muscle glucose translocation in the T2DM rat model, via activation of AMPK.     

Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy

Background and Purpose

Insulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin.

Experimental Approach

Total internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein.

Key Results

Using our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR.

Conclusions and Implications

Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.Table of Links

Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

BACKGROUND AND PURPOSE

mTOR inhibitors are currently used as immunosuppressants in transplanted patients and as promising anti-cancer agents. However, new-onset diabetes is a frequent complication occurring in patients treated with mTOR inhibitors such as rapamycin (Sirolimus). Here, we investigated the mechanisms associated with the diabetogenic effects of chronic Sirolimus administration in rats and in in vitro cell cultures.

EXPERIMENTAL APPROACH

Sirolimus was administered to rats fed either a standard or high-fat diet for 21 days. Metabolic parameters were measured in vivo and in ex vivo tissues. Insulin sensitivity was assessed by glucose tolerance tests and euglycaemic hyperinsulinaemic clamps. Rapamycin effects on glucose metabolism and insulin signalling were further evaluated in cultured myotubes.

KEY RESULTS

Sirolimus induced a decrease in food intake and concomitant weight loss. It also induced specific fat mass loss that was independent of changes in food intake. Despite these beneficial effects, Sirolimus-treated rats were glucose intolerant, hyperinsulinaemic and hyperglycaemic, but not hyperlipidaemic. The euglycaemic hyperinsulinaemic clamp measurements showed skeletal muscle is a major site of Sirolimus-induced insulin resistance. At the molecular level, long-term Sirolimus administration attenuated glucose uptake and metabolism in skeletal muscle by preventing full insulin-induced Akt activation and altering the expression and translocation of glucose transporters to the plasma membrane. In rats fed a high-fat diet, these metabolic defects were exacerbated, although Sirolimus-treated animals were protected from diet-induced obesity.

CONCLUSIONS AND IMPLICATIONS

Taken together, our data demonstrate that the diabetogenic effect of chronic rapamycin administration is due to an impaired insulin action on glucose metabolism in skeletal muscles.     

Sitagliptin, an dipeptidyl peptidase-4 inhibitor, does not alter the pharmacokinetics of the sulphonylurea, glyburide, in healthy subjects

AIMS

Sitagliptin, a dipeptidyl peptidase-4 inhibitor, is an incretin enhancer that is approved for the treatment of Type 2 diabetes. Sitagliptin is mainly renally eliminated and not an inhibitor of CYP450 enzymes in vitro. Glyburide, a sulphonylurea, is an insulin sensitizer and mainly metabolized by CYP2C9. Since both agents may potentially be co-administered, the purpose of this study was to examine the effects of sitagliptin on glyburide pharmacokinetics.

METHODS

In this open-label, randomized, two-period crossover study, eight healthy normoglycaemic subjects, 22–44 years old, received single 1.25-mg doses of glyburide alone in one period and co-administered with sitagliptin on day 5 following a multiple-dose regimen for sitagliptin (200-mg q.d. ×6 days) in the other period.

RESULTS

The geometric mean ratios and 90% confidence intervals [(glyburide + sitagliptin)/glyburide] for AUC_0–∞ and C_max were 1.09 (0.96, 1.24) and 1.01 (0.84, 1.23), respectively.

CONCLUSION

Sitagliptin does not alter the pharmacokinetics of glyburide in healthy subjects.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• No data are available on the potential drug interaction of sitagliptin and glyburide.
• Sitagliptin belongs to a new class of drugs called DPP-4 inhibitors recently approved for the treatment of Type 2 diabetes.

WHAT THIS STUDY ADDS

• Glyburide is a commonly used sulphonylurea medication to treat Type 2 diabetes.
• Combination therapy is often required to achieve adequate glucose control in Type 2 diabetes.
• Sitagliptin does not appear to interfere with glyburide pharmacokinetics and therefore may be potentially co-administered with glyburide for the treatment of Type 2 diabetes.

     

A sesquiterpene quinone,dysidine, from the sponge Dysidea villosa,activates the insulin pathway through inhibition of PTPases

Aim:

The sesquiterpene hydroquinones/quinones belong to one class of marine sponge metabolites, and they have received considerable attention due to their varied biological activities, including anti-tumor, anti-HIV, and anti-inflammatory action. In order to probe the potential anti-diabetic effect of the sesquiterpene hydroquinones/quinones, the effect of dysidine on the insulin pathway was studied.

Methods:

The promotion of glucose uptake by dysidine was studied in differentiated 3T3-L1 cells. The increase in membrane-located GLUT4 by dysidine was studied in CHO-K1/GLUT4 and 3T3-L1 cells by immuno-staining. The activation of the insulin signaling pathway by dysidine was probed by Western blotting. The inhibition of PTPases by dysidine was detected in vitro.

Results:

Dysidine, found in the Hainan sponge Dysidea villosa in the Chinese South Sea, effectively activated the insulin signaling pathway, greatly promoted glucose uptake in 3T3-L1 cells, and showed strong insulin-sensitizing activities. The potential targets of action for dysidine were probed, and the results indicated that dysidine exhibited its cellular effects through activation of the insulin pathway, possibly through the inhibition of protein tyrosine phosphatases, with more specific inhibition against protein tyrosine phosphatase 1B (PTP1B).

Conclusion:

Our findings are expected to expand understanding of the biological activities of sesquiterpene hydroquinones/quinones, and they show that dysidine could be a potential lead compound in the development of an alternative adjuvant in insulin therapy.     

Uncoupling protein-2 mediates the protective action of berberine against oxidative stress in rat insulinoma INS-1E cells and in diabetic mouse islets

BACKGROUND AND PURPOSE

Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets.

EXPERIMENTAL APPROACH

Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by elisa. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy.

KEY RESULTS

Incubation of INS-1E cells and rat islets with HG (30 mmol·L⁻¹; 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m⁺ mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine.

CONCLUSIONS AND IMPLICATIONS

Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine.     

Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition,a possible mechanism of action?

Aim:

The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study.

Materials and Methods:

The effects of extract on glucose uptake, insulin receptor-β (IR-β), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days.

Results:

The effect of extract promoted glucose uptake, IR-β and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC₅₀ 17.4 μg/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P<0.05 after treatment compared to before treatment).

Conclusion:

The results suggest that Annona squamosa hexane extract exerts its action by modulating insulin signaling through inhibition of PTP1B.KEY WORDS: Annona squamosa, type 2 diabetes mellitus, insulin mimetics, PTP1B inhibitor, OGTT, ob/ob mice     

Yhhu981, a novel compound,stimulates fatty acid oxidation via the activation of AMPK and ameliorates lipid metabolism disorder in ob/ob mice

Aim:

Defects in fatty acid metabolism contribute to the pathogenesis of insulin resistance and obesity. In this study, we investigated the effects of a novel compound yhhu981 on fatty acid metabolism in vitro and in vivo.

Methods:

The capacity to stimulate fatty acid oxidation was assessed in C2C12 myotubes. The fatty acid synthesis was studied in HepG2 cells using isotope tracing. The phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) was examined with Western blot analysis. For in vivo experiments, ob/ob mice were orally treated with yhhu981 acutely (300 mg/kg) or chronically (150 or 300 mg·kg⁻¹·d⁻¹ for 22 d). On the last day of treatment, serum and tissue samples were collected for analysis.

Results:

Yhhu981 (12.5–25 μmol/L) significantly increased fatty acid oxidation and the expression of related genes (Sirt1, Pgc1α and Mcad) in C2C12 myotubes, and inhibited fatty acid synthesis in HepG2 cells. Furthermore, yhhu981 dose-dependently increased the phosphorylation of AMPK and ACC in both C2C12 myotubes and HepG2 cells. Compound C, an AMPK inhibitor, blocked fatty acid oxidation in yhhu981-treated C2C12 myotubes and fatty acid synthesis decrease in yhhu981-treated HepG2 cells. Acute administration of yhhu981 decreased the respiratory exchange ratio in ob/ob mice, whereas chronic treatment with yhhu981 ameliorated the lipid abnormalities and ectopic lipid deposition in skeletal muscle and liver of ob/ob mice.

Conclusion:

Yhhu981 is a potent compound that stimulates fatty acid oxidation, and exerts pleiotropic effects on lipid metabolism by activating AMPK.     

Karanjin from Pongamia pinnata induces GLUT4 translocation in skeletal muscle cells in a phosphatidylinositol-3-kinase-independent manner

Insulin-stimulated glucose uptake in skeletal muscle is decreased in type 2 diabetes due to impaired translocation of insulin-sensitive glucose transporter 4 (GLUT4) from intracellular pool to plasma membrane. Augmenting glucose uptake into this tissue may help in management of type 2 diabetes. Here, the effects of an identified antihyperglycemic molecule, karanjin, isolated from the fruits of Pongamia pinnata were investigated on glucose uptake and GLUT4 translocation in skeletal muscle cells. Treatment of L6-GLUT4myc myotubes with karanjin caused a substantial increase in the glucose uptake and GLUT4 translocation to the cell surface, in a concentration-dependent fashion, without changing the total amount of GLUT4 protein and GLUT4 mRNA. This effect was associated with increased activity of AMP-activated protein kinase (AMPK). Cycloheximide treatment inhibited the effect of karanjin on GLUT4 translocation suggesting the requirement of de novo synthesis of protein. Karanjin-induced GLUT4 translocation was further enhanced with insulin and the effect is completely protected in the presence of wortmannin. Moreover, karanjin did not affect the phosphorylation of AKT (Ser-473) and did not alter the expression of the key molecules of insulin signaling cascade. We conclude that karanjin-induced increase in glucose uptake in L6 myotubes is the result of an increased translocation of GLUT4 to plasma membrane associated with activation of AMPK pathway, in a PI-3-K/AKT-independent manner.     

Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

Aim:

To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.

Methods:

HepG2 and C2C12 cells were used. Cell viability was determined using MTT assay. Real-time PCR was performed to measure the gene expression. Western blotting assay was applied to investigate the protein phosphorylation level. Enzymatic assay kits were used to detect the total cholesterol (TC), triglyceride (TG) and glucose contents.

Results:

Danthron (0.1, 1, and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in both HepG2 and C2C12 cells. Meanwhile, danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions, and the TC and TG levels. In addition, danthron treatment efficiently increased glucose consumption. The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.

Conclusion:

Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.     

Alagebrium attenuates acute methylglyoxal-induced glucose intolerance in Sprague-Dawley rats

Background and purpose:

Alagebrium is a breaker of cross-links in advanced glycation endproducts. However, the acute effects of alagebrium on methylglyoxal (MG), a major precursor of advanced glycation endproducts have not been reported. MG is a highly reactive endogenous metabolite, and its levels are elevated in diabetic patients. We investigated whether alagebrium attenuated the acute effects of exogenous MG on plasma MG levels, glucose tolerance and distribution of administered MG in different organs in Sprague-Dawley rats.

Experimental approach:

We measured MG levels (by HPLC), glucose tolerance, adipose tissue glucose uptake, GLUT4, insulin receptor and insulin receptor substrate 1 (IRS-1) protein expression, and phosporylated IRS-1 in rats treated with MG at doses of either 17.25 mg·kg⁻¹ i.p. (MG-17 i.p.) or 50 mg·kg⁻¹ i.v. (MG-50 i.v.) with or without alagebrium, 100 mg·kg⁻¹ i.p.

Key results:

Alagebrium attenuated the increased MG levels in the plasma, aorta, heart, kidney, liver, lung and urine after MG administration. In MG-treated rats, glucose tolerance was impaired, plasma insulin levels were higher and insulin-stimulated glucose uptake by adipose tissue was reduced, relative to the corresponding control groups. In rats treated with MG-50 i.v., GLUT4 protein expression and IRS-1 tyrosine phosphorylation were decreased. Alagebrium pretreatment attenuated these effects of MG. In an in vitro assay, alagebrium reduced the amount of detectable MG.

Conclusions and implications:

Alagebrium acutely attenuated MG-induced glucose intolerance, suggesting a possible preventive role for alagebrium against the harmful effects of MG.     

Long-term baicalin administration ameliorates metabolic disorders and hepatic steatosis in rats given a high-fat diet

Aim:

Baicalin, one of the major flavonoids in Scutellaria baicalensis, possesses antioxidant and anti-inflammatory properties. However, the effects of baicalin on metabolic disorders and hepatic steatosis have not been investigated.

Methods:

Body weight was examined in high-fat diet (HFD)-fed rats with or without baicalin treatment. At the end of the experiment, serum biochemical parameters, liver histology and lipid profile were analyzed to assess whether the animals were suffering from metabolic disorders or hepatic steatosis. In the liver, the phosphorylation of AMP activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and the gene expression of some enzymes involved in lipogenesis were examined. The effects of baicalin on the phosphorylation of AMPK and lipid accumulation induced by high glucose in human hepatoma HepG2 cells were also examined.

Results:

Baicalin (80 mg/kg) administered ip for 16 weeks suppressed body weight gain in HFD-fed rats. Weight reduction was accompanied by the reduction of visceral fat mass. Baicalin significantly decreased the elevated serum cholesterol, free fatty acid and insulin concentrations caused by the HFD. Baicalin also suppressed systemic inflammation by reducing the serum level of tumor necrosis factor α. Baicalin reduced hepatic lipid accumulation, enhanced the phosphorylation of AMPK and ACC and down-regulated genes involved in lipogenesis, including fatty acid synthase and its upstream regulator SREBP-1c. In HepG2 cells, baicalin (5 and 10 μmol/L) increased the phosphorylation of AMPK and decreased lipid accumulation following the addition of high glucose.

Conclusion:

Our study suggests that baicalin might have beneficial effects on the development of hepatic steatosis and obesity-related disorders by targeting the hepatic AMPK.     

Metformin and male reproduction: effects on Sertoli cell metabolism

Background and Purpose

Metformin is commonly used to treat type 2 diabetes (T2D). While new clinical applications have been ascribed to metformin, including treatment of anovulatory infertility, its effects on male reproduction have not been investigated. The Sertoli cell (SC) is crucial for germ cell development, exerting metabolic control of spermatogenesis, therefore, we investigated the effects of metformin on SC metabolism.

Experimental Approach

Rat SCs were cultured in the absence and presence of metformin (5, 50 and 500 μM). mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK 1), lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by quantitative PCR and Western blot respectively. LDH activity was assessed and metabolite production/consumption determined by ¹H-NMR.

Key Results

Metformin (50 μM) decreased mRNA and protein levels of GLUT1, GLUT3, MCT4 and PFK 1 but did not affect LDH mRNA or protein levels. However, although glucose consumption was maintained in metformin-treated cells, LDH activity, lactate and alanine production were increased, indicating an enhanced glycolytic flux. No metabolic cytotoxicity was detected in SCs exposed to supra-pharmacological concentration of metformin.

Conclusions and Implications

Our results indicate that metformin: (i) decreases mRNA and protein levels of glycolysis-related transporters in SCs but increases their activity; and (ii) stimulates alanine production, which induces antioxidant activity and maintains the NADH/NAD⁺ equilibrium. The increased lactate in metformin-treated SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells. Thus, metformin can be considered as a suitable antidiabetic drug for male patients of reproductive age with T2D.     

Salidroside ameliorates insulin resistance through activation of a mitochondria-associated AMPK/PI3K/Akt/GSK3β pathway

Background and Purpose

Recent reports have suggested that salidroside could protect cardiomyocytes from oxidative injury and stimulate glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase (AMPK). The aim of this study was to evaluate the therapeutic effects of salidroside on diabetic mice and to explore the underlying mechanisms.

Experimental Approach

The therapeutic effects of salidroside on type 2 diabetes were investigated. Increasing doses of salidroside (25, 50 and 100 mg·kg⁻¹·day⁻¹) were administered p.o. to db/db mice for 8 weeks. Biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of salidroside. Primary cultured mouse hepatocytes were used to further explore the underlying mechanisms in vitro.

Key Results

Salidroside dramatically reduced blood glucose and serum insulin levels and alleviated insulin resistance. Hypolipidaemic effects and amelioration of liver steatosis were observed after salidroside administration. In vitro, salidroside dose-dependently induced an increase in the phosphorylations of AMPK and PI3K/Akt, as well as glycogen synthase kinase 3β (GSK3β) in hepatocytes. Furthermore, salidroside-stimulated AMPK activation was found to suppress the expression of PEPCK and glucose-6-phosphatase. Salidroside-induced AMPK activation also resulted in phosphorylation of acetyl CoA carboxylase, which can reduce lipid accumulation in peripheral tissues. In isolated mitochondria, salidroside inhibited respiratory chain complex I and disturbed oxidation/phosphorylation coupling and moderately depolarized the mitochondrial membrane potential, resulting in a transient increase in the AMP/ATP ratio.

Conclusions and Implications

Salidroside exerts an antidiabetic effect by improving the cellular metabolic flux through the activation of a mitochondria-related AMPK/PI3K/Akt/GSK3β pathway     

Depletion of insulin receptor substrate 2 reverses oncogenic transformation induced by v-src

Aim:

To investigate the role of insulin receptor substrate 2 (IRS-2) in oncogenic transformation induced by v-src.

Methods:

IRS-2 gene was silenced using small interfering RNAs (siRNAs). Nuclear translocation and interaction of IRS-2 with v-src was determined using subcellular fractionation, confocal microscopy, and immunoprecipitation. The activity of the cyclin D1 promoter and r-DNA promoter was measured with a luciferase assay.

Results:

Depletion of IRS-2 inhibited R-/v-src cell growth and reverse the oncogenic transformation. IRS-2 bound to src via its two PI3-K binding sites, which are critical for activities involved in the transformation. Nuclear IRS-2 occupied the cyclin D1 and rDNA promoters. The combination of IRS-2 and v-src increased the activity of the two promoters, especially the rDNA promoter.

Conclusion:

Depletion of insulin receptor substrate 2 could reverse oncogenic transformation induced by v-src.