Insulin-stimulated Glucose Consumption Research Articles

Irisin improves insulin resistance by inhibiting autophagy through the PI3K/Akt pathway in H9c2 cells.

As an important complication of diabetes mellitus, diabetic cardiomyopathy (DCM) is thought to arise as a result of insulin resistance (IR) in cardiomyocytes. Improving IR in cardiomyocytes may therefore be a way to treat DCM. A recently discovered myokine, irisin, has been shown to be significantly associated with increased insulin sensitivity both in clinical and pre-clinical studies of diabetes mellitus. Based on previously research, we hypothesized that irisin may be a potential candidate for increasing the insulin sensitivity of cardiomyocytes. The aim of the present study was to examine the ability of irisin to affect IR induced by treatment of rat cardiomyocyte H9c2 cells with palmitic acid (PA) and to explore its underlying mechanism. Differentiated H9c2 cells were treated with 500μM PA, 200ng/mL irisin, and 500μM PA+200ng/mL irisin with or without 100nM rapamycin (RAP) for 24h. We found that coincubation with 200ng/mL irisin for 24h significantly increased insulin-stimulated glucose consumption compared to the 500μM PA group alone. Additionally, coincubation with irisin significantly alleviated the degree of autophagy compared to the 500μM PA group alone as evidenced by monodansylcadaverine (MDC) fluorescence, the LC3II/LC3I protein levels ratio, and the protein levels of Atg5 and Atg7. Coincubation with irisin increased the levels of PI3Kp110α, pAkt and Akt compared to the 500μM PA group alone. All these effects of irisin were reversed by RAP. Our results indicate that irisin improves IR in H9c2 cells, possibly in part by inhibiting autophagy through activating the PI3K/Akt pathway.

A colorimetric bioassay for quantitation of both basal and insulin-induced glucose consumption in 3T3-L1 adipose cells

IntroductionThe quantitation of glucose consumption in animal cell cultures is mainly based on the use of radiolabeled or fluorescent analogues, resulting in expensive and tedious procedures, requiring special equipment and, sometimes, with potential health and environmental risks. ObjectivesThe objective of this work was to evaluate the application of a blood plasma colorimetric assay to quantify glucose consumption in in vitro cultures of adipose cells. MethodsWe worked with 3T3-L1 adipose cells differentiated by 7–8 days, which were exposed to different initial glucose concentrations (5.5, 2.8 and 1.4 mM) for variable times, either in the absence or the presence of 100 nM insulin. Using a commercial colorimetric glucose assay, extracellular glucose was determined, and glucose uptake was calculated as the difference between the initial and final glucose concentration. ResultsThe colorimetric assay allowed us to quantify glucose uptake in our cell model, observing a linear response over time (r2≥0.9303) to the different glucose concentrations, both in the basal and insulin-induced condition. The insulin-stimulated glucose consumption was higher than basal consumption at all glucose concentrations evaluated, but significant differences were observed at 120-, 360- and 480-min in glucose 5.5 mM (p ≤ 0.01, n = 5), and 240 min in glucose 1.4 mM (p ≤ 0.01, n = 5). A Vmax of 4.1 and 5.9 nmol/ml/min (basal and insulin-induced, respectively) and a Km of 1.1 mM (same in basal vs insulin-stimulated) were calculated. The bioassay was also useful in a pharmacological context: in glucose 1.4 mM, glucose consumption showed an effect that depended on insulin concentration, with a calculated EC50 of 18.4 ± 1.1 nM. ConclusionsA simple and low-cost bioassay is proposed to quantify glucose consumption in 3T3-L1 adipose cells.

3-Deoxyglucosone interferes with insulin signaling and attenuates insulin action on glucose-induced GLP-1 secretion in the enteroendocrine L cell line STC-1.

Maintenance of glucose homeostasis is reciprocally regulated by insulin and glucagon-like peptide-1 (GLP-1). We previously reported that GLP-1 secretion in response to anoral glucose load was impaired following an administration of 3-deoxyglucosone (3DG), an independent factor associated with the development of pre-diabetes. Here we investigated the effects of 3DG on insulin signaling and insulin-induced GLP-1 secretion under high-glucose conditions in the enteroendocrine L cell line STC-1. STC-1 cells were exposed to 3DG (80, 300, and 1000ng/ml) in the presence of 10-7M insulin and 25mM glucose. GLP-1 secretion was determined by ELISA, glucose uptake was monitored with 2-NBDG (2-(N(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose), glucose consumption was detected by glucoseoxidase, and protein expression of insulin signaling molecules was examined by western blot. Results showed a decrease in insulin-induced GLP-1 secretion and insulin receptor phosphorylation after 3DG treatment. Concomitantly, 3DG treatment inhibited insulin-induced phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway activation. In the presence, but not absence, of insulin, 3DG treatment decreased insulin-stimulated glucose consumption. Inhibition of PI3K with Wortmannin attenuated insulin-induced increment in glucose transporter 2 (GLUT2) expression and 2-NBDG uptake. Accordingly, insulin-induced increase in GLUT2 expression and 2-NBGD uptake was significantly inhibited by 3DG treatment. 3DG-mediated reduction in GLUT2 expression contributes to the attenuation of insulin-induced GLP-1 secretion under high-glucose conditions in part through the insulin-PI3K/Akt/GLUT2 pathway in STC-1 cells. We conclude that 3DG interferes with insulin signaling and attenuates insulin action on glucose-induced GLP-1 secretion in STC-1 cells.

EFFECTS OF EXENDIN-4 ON ENDOPLASMIC RETICULUM STRESS-MEDIATED INSULIN RESISTANCE IN 3T3-L1 ADIPOCYTES.

Endoplasmic reticulum stress (ERS) is suspected as an important factor in the initiation of insulin resistance. To explore the effects of exendin-4 (Ex-4) on the endoplasmic reticulum stress (ERS)-mediated insulin resistance in 3T3-L1 adipocytes. In our study, 3T3-L1 adipocytes were pre-treated with ERS inhibitors tauroursodeoxycholic acid (TUDCA), Ex-4 and an ERS inducer tunicamycin (TM) then induced insulin resistance. Glucose consumption of the adipocytes was measured. Western blots determined the protein levels of ERS markers and insulin signaling pathway. TM treatment reduced insulin-stimulated glucose consumption by 19.7% in 3T3-L1 adipocytes. This repression was blunted by 24h pre-treatment with TUDCA or Ex-4. Ex-4 augmented insulin-stimulated glucose consumption in adipocytes by 14.9%. Western blotting showed that TM treatment significantly increased the ER stress markers including p-IRE, p-JNK, p-PERK, p-eIF2a and ATF6 expression, whereas 24h pre-treatment of adipocytes with TUDCA or Ex-4 alleviated the ER stress. Ex-4 alleviates ERS-induced insulin resistance by upregulating the expression of phosphorylated Akt. ERs mediates insulin resistance in 3T3-L1 adipocytes, and exendin-4 significantly improves this insulin resistance.

Macrophage-Mediated Effects of Airborne Fine Particulate Matter (PM2.5) on Hepatocyte Insulin Resistance in Vitro.

Fine particulate matter (PM2.5) pollution poses significant health risks worldwide, including metabolic syndrome-related diseases with the characteristic feature of insulin resistance. However, the mechanism and influencing factors of this effect are poorly understood. In this serial in vitro study, we aimed at testing the hypothesis that macrophage-mediated effects of PM2.5 on hepatic insulin resistance depend on its chemical composition. Mouse macrophages were exposed to PM2.5 that had been collected during summer or winter in Beijing, which represented different compositions of PM2.5. Thereafter, hepatocytes were treated with macrophage-conditioned medium (CM). PM2.5 induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 expression and secretion in macrophages, particularly after winter PM2.5 exposure. Correspondingly, winter CM weakened hepatocellular insulin-stimulated glucose consumption. Further investigation revealed that the normal insulin pathway was suppressed in winter CM-treated hepatocytes, with increased phosphorylation of insulin receptor substrate 1 at serine residue 307 (Ser307) and decreased phosphorylation of protein kinase B (PKB/AKT) and forkhead box transcription factor O1 (FoxO1). Moreover, c-Jun N-terminal kinase, a key moderator of the sensitivity response to insulin stimulation, was activated in hepatocytes treated with winter CM. Although further studies are warranted, this preliminary study suggested an association between PM composition and insulin resistance, thus contributing to our understanding of the systemic toxicity of PM2.5.

The Compound of Mangiferin-Berberine Salt Has Potent Activities in Modulating Lipid and Glucose Metabolisms in HepG2 Cells.

The mangiferin-berberine (MB) salt was synthesized by ionic bonding of mangiferin (M) and berberine (B) at an equal molecular ratio. This study aimed to investigate the activities of MB salt in modulating lipid and glucose metabolisms in HepG2 cells. After 24 h treatment of the studying compounds, cellular AMP-activated protein kinase α (AMPKα)/acetyl-CoA carboxylase (ACC) protein levels and carnitine palmitoyltransferase (CPT) 1 activities, intracellular lipid contents, mRNA expression levels of target genes, glucose consumption, and glucose production amounts were determined. Compound C (CC) was used in the blocking experiments. Our results showed that MB salt increased p-AMPKα (Thr172)/p-ACC (Ser79) levels and CPT1 activity and suppressed oleic acid- (OA-) induced lipid accumulation and upregulation of lipogenic genes potently in HepG2 cells. The above activities of MB salt were AMPK dependent and were superior to those of M or B when administered at an equal molar concentration. MB salt enhanced basal and insulin-stimulated glucose consumption and suppressed gluconeogenesis more potently than M or B alone. The inhibiting activity of MB salt on cellular gluconeogenesis was AMPK dependent. Our results may support MB salt as a new kind of agent for the development of novel lipid or glucose-lowering drugs in the future.

Purification and Characterization of Aporphine Alkaloids from Leaves of Nelumbo nucifera Gaertn and Their Effects on Glucose Consumption in 3T3-L1 Adipocytes

Aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn are substances of great interest because of their important pharmacological activities, particularly anti-diabetic, anti-obesity, anti-hyperlipidemic, anti-oxidant, and anti-HIV’s activities. In order to produce large amounts of pure alkaloid for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Without any initial cleanup steps, four main aporphine alkaloids, including 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were successfully purified from the crude extract by HSCCC in one step. The separation was performed with a simple two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (5:3:3:2.5:5, v/v/v/v/v). In each operation, 100 mg crude extracts was separated and yielded 6.3 mg of 2-hydroxy-1-methoxyaporphine (95.1% purity), 1.1 mg of pronuciferine (96.8% purity), 8.5 mg of nuciferine (98.9% purity), and 2.7 mg of roemerine (97.4%) respectively. The chemical structure of four aporphine alkaloids are identified by means of electrospray ionization MS (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Moreover, the effects of four separated aporphine alkaloids on insulin-stimulated glucose consumption were examined in 3T3-L1 adipocytes. The results showed that 2-hydroxy-1-methoxyaporphine and pronuciferine increased the glucose consumption significantly as rosiglitazone did.

Genipin ameliorates age-related insulin resistance through inhibiting hepatic oxidative stress and mitochondrial dysfunction

Insulin resistance (IR) increases with age and plays a key role in the pathogenesis of type 2 diabetes mellitus. Oxidative stress and mitochondrial dysfunction are supposed to be major factors leading to age-related IR. Genipin, an extract from Gardenia jasminoides Ellis fruit, has been reported to stimulate insulin secretion in pancreatic islet cells by regulating mitochondrial function. In this study, we first investigated the effects of genipin on insulin sensitivity and the potential mitochondrial mechanisms in the liver of aging rats. The rats were randomly assigned to receive intraperitoneal injections of either 25mg/kg genipin or vehicle once daily for 12days. The aging rats showed hyperinsulinemia and hyperlipidemia, and insulin resistance as examined by the decreased glucose decay constant rate during insulin tolerance test (kITT). The hepatic tissues showed steatosis and reduced glycogen content. Hepatic malondialdehyde level and mitochondrial reactive oxygen species (ROS) were higher, and levels of mitochondrial membrane potential (MMP) and ATP were lower as compared with the normal control rats. Administration of genipin ameliorated systemic and hepatic insulin resistance, alleviated hyperinsulinemia, hyperglyceridemia and hepatic steatosis, relieved hepatic oxidative stress and mitochondrial dysfunction in aging rats. Furthermore, genipin not only improved insulin sensitivity by promoting insulin-stimulated glucose consumption and glycogen synthesis, inhibited cellular ROS overproduction and alleviated the reduction of levels of MMP and ATP, but also reversed oxidative stress-associated JNK hyperactivation and reduced Akt phosphorylation in palmitate-treated L02 hepatocytes. In conclusion, genipin ameliorates age-related insulin resistance through inhibiting hepatic oxidative stress and mitochondrial dysfunction.

Kiwifruit (Actinidia chinensis) extract annuls chronic insulininduced insulin resistance in l6 skeletal muscle cells

Kiwi fruit has long been believed to be beneficial to human health due to its numerous biological actions; however the pharmacological mechanisms associated with these benefits are incompletely understood. In the present study, the effects of ‘Halla Gold’ (Actinidia chinensis var.), a new kiwifruit cultivar bred by the Jeju Rural Development Administration (RDA), on chronic insulin-induced insulin resistance in L6 skeletal muscle cells was investigated. Chronic insulin reduced phosphorylation of Akt and GSK-3α/β, and increased phosphorylation of glycogen synthase (GS). Furthermore, glucose consumption and glucose transporter 4 (Glut4) translocation to the plasma membrane were inhibited. However, ‘Halla Gold’ extract (HGE) increased phosphorylation of Akt and GSK-3α/β concomitantly with decreased phosphorylation of GS. Moreover, HGE improved insulin-stimulated glucose consumption by facilitating Glut4 translocation and this stimulation was suppressed by inhibition of PI3 kinase. Together, our data suggest that ‘Halla Gold’ extract abolishes chronic insulin-induced insulin resistance through the PI3K/Akt pathway.

An epigenetic modifier enhances the production of anti-diabetic and anti-inflammatory sesquiterpenoids from Aspergillus sydowii

The addition of a DNA methyltransferase inhibitor, 5-azacytidine, to Aspergillus sydowii fungus culture broth changed its secondary metabolites profile. Analysis of the culture broth extract led to the isolation of three new bisabolane-type sesquiterpenoids: (7S)-(+)-7-O-methylsydonol (1), (7S,11S)-(+)-12-hydroxysydonic acid (2) and 7-deoxy-7,14-didehydrosydonol (3), along with eight known compounds. The isolated compounds were evaluated for their anti-diabetic and anti-inflammatory activities. Among the isolates, (S)-(+)-sydonol (4) did not only potentiate insulin-stimulated glucose consumption but also prevented lipid accumulation in 3T3-L1 adipocytes. Additionally, (S)-(+)-sydonol (4) exhibited significant anti-inflammatory activity through inhibiting superoxide anion generation and elastase release by fMLP/CB-induced human neutrophils. This is the first report on isolating a secondary metabolite with anti-diabetic and anti-inflammatory activities from microorganisms.

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor-α (TNF- α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H2 S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF-α-induced insulin resistance is involved in endogenous H2 S generation. The aim of the present study is to investigate the role of endogenous H2 S in TNF-α-induced insulin resistance by studying 3T3-L1 adipocytes. We found that treatment of 3T3-L1 adipocytes with TNF-α leads to deficiency in insulin-stimulated glucose consumption and uptake and increase in endogenous H2 S generation. We show that cystathionine γ-lyase (CSE) is catalysed in 3T3-L1 adipocytes to generate H2 S and that CSE expression and activity are upregulated by TNF-α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF-α-induced insulin resistance in 3T3-L1 adipocytes, whereas H2 S treatment of 3T3-L1 adipocytes impairs insulin-stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H2 S system contributes to TNF-α-caused insulin resistance in 3T3-L1 adipocytes. Our findings suggest that modulation of CSE/H2 S system is a potential therapeutic avenue for insulin resistance.

Effect of Calcium-Regulating Hormones and Calcium Channel Modulators on Glucose Consumption by Muscle and Adipose Tissues In Vivo and In Vitro

We studied the effects of calcitonin, parathyrin, and Ca(2+) channel antagonist isoptin and agonist Bay-K-8644 on glucose consumption by muscle (diaphragm) and adipose (epididymal) tissues and insulin-stimulated glucose consumption in vivo and in vitro. Calcitonin and parathyrin did not alter glucose consumption; parathyrin did not affect, while calcitonin completely abolished the stimulating effect of insulin in vivo and in vitro. Isoptin significantly increased glucose consumption in vivo and in vitro, while Bay-K-8644 in vitro had no effect glucose consumption. Isoptin did not affect, while Bay-K-8644 significantly reduced the stimulating effect of insulin on glucose consumption by the muscle and adipose tissues. Isoptin did not affect the stimulating effect of insulin against the background of parathyrin administration and completely blocked the inhibitory effect of calcitonin on insulin-stimulated glucose consumption by the muscle and adipose tissues in vivo and in vitro, while Bay-K-8644 potentiated this effect of calcitonin in vitro.

Shilianhua extract inhibits GSK-3β and promotes glucose metabolism

The extract of plant Shilianhua (SLH; Sinocrassula indica Berge) is a component in a commercial product for control of blood glucose. However, it remains to be investigated whether the SLH extract enhances insulin sensitivity in a model of type 2 diabetes. To address this question, the SLH crude extract was fractionated into four parts on the basis of polarity, and bioactivities of each part were tested in cells. One of the fractions, F100, exhibited a strong activity in the stimulation of glucose consumption in vitro. Glucose consumption was induced significantly by F100 in 3T3-L1 adipocytes, L6 myotubes, and H4IIE hepatocytes in the absence of insulin. F100 also increased insulin-stimulated glucose consumption in L6 myotubes and H4IIE hepatocytes. It increased insulin-independent glucose uptake in 3T3-L1 adipocytes and insulin-dependent glucose uptake in L6 cells. The glucose transporter-1 (GLUT1) protein was induced in 3T3-L1 cells, and the GLUT4 protein was induced in L6 cells by F100. Mechanism study indicated that F100 induced GSK-3beta phosphorylation, which was comparable with that induced by insulin. Additionally, the transcriptional activity of NF-kappaB was inhibited by F100. In RAW 264.7 macrophages, mRNA expression of NF-kappaB target genes (TNFalpha and MCP-1) was suppressed by F100. In KK.Cg-A(y)/+ mice, F100 decreased fasting insulin and blood glucose and improved insulin tolerance significantly. We conclude that the F100 may be a bioactive component in the SLH plant. It promotes glucose metabolism in vitro and in vivo. Inhibition of GSK-3beta and NF-kappaB may be the potential mechanism.

Inhibitory effects of insulin-like growth factor (IGF)-binding proteins-1 and -3 on IGF-activated glucose consumption in mouse BALB/c 3T3 fibroblasts

We have examined the biological effects of insulin-like growth factor-binding proteins (IGFBPs) on insulin-like growth factor (IGF)-activated glucose consumption in a BALB/c 3T3 subline. The method employed was a colorimetric measurement of glucose consumption, allowing the detection of changes from the initial glucose concentration in conditioned medium, following the addition of IGFs and IGFBPs. Human IGFBP-1, purified from amniotic fluid, inhibited IGF-activated glucose consumption, although it had no effect on insulin-activated glucose consumption. The median effective dose (ED50) of IGFBP-1 to cause inhibitory effects on IGF-activated glucose consumption was 100-200 micrograms/l and was similar for both IGF-I and IGF-II at a concentration of 1.0 microgram IGF/l. Therefore, at IGF concentrations of comparable activity, the inhibitory effects of IGFBP-1 were greater for IGF-I than for IGF-II, because of the higher activity of IGF-I in this assay. Recombinant human IGFBP-3 also inhibited IGF-activated glucose consumption, without affecting insulin-stimulated glucose consumption. The inhibitory effects of IGFBP-3 were greater for IGF-II than for IGF-I when IGFBP-3 was coincubated with either of the IGFs, at both IGF concentrations of comparable activity and equivalent molar concentrations. Thus, it became clear that the inhibitory effects of these IGFBPs on IGF biological action depended primarily upon their affinity for the specific IGF ligand and molar ratio of IGFBP/IGF peptide. Interestingly, when cells were pretreated with IGFBP-3, prior to the simultaneous addition of IGFs and IGFBP-3, the inhibitory effect was higher for IGF-I than for IGF-II. Either no effect or a minor inhibitory effect on IGF-activated glucose consumption was detected with IGFBP pretreatment alone. When the ED50 for inhibition of IGF action by IGFBPs in this in-vitro assay was compared with the physiological concentrations of IGFs and IGFBPs in normal human serum and in amniotic fluid, it was estimated that the IGFBP-1 concentration present in serum was not sufficient to modulate IGF action effectively while the concentration in amniotic fluid was enough for effective suppression. IGFBP-3 exhibited an ED50 low enough to suppress IGF-II and possibly IGF-I action when cells were pretreated with IGFBP-3. Thus, our data suggested that IGFBP-1 in amniotic fluid and IGFBP-3 in serum could be a potent inhibitor for IGF action. IGFBP-1 in serum, however, may not be able to function as a direct inhibitor under physiological conditions but, rather, may modulate IGF action together with other IGFBPs.