Project description:Insulin resistance (IR) is likely to induce metabolic syndrome and type 2 diabetes mellitus (T2DM). Gluconeogenesis (GNG) is a complex metabolic process that may result in glucose generation from certain non-carbohydrate substrates. Chinese herbal medicine astragalus polysaccharides and berberine have been documented to ameliorate IR, and combined use of astragalus polysaccharide (AP) and berberine (BBR) are reported to synergistically produce an even better effect. However, what change may occur in the GNG signaling pathway of IR-HepG2 cells in this synergistic effect and whether AP-BBR attenuates IR by regulating the GNG signaling pathway remain unclear. For the first time, we discovered in this study that the optimal time of IR-HepG2 cell model formation was 48 h after insulin intervention. AP-BBR attenuated IR in HepG2 cells and the optimal concentration was 10 mg. AP-BBR reduced the intracellular H2O2 content with no significant effect on apoptosis of IR-HepG2 cells. In addition, a rapid change was observed in intracellular calcium current of the IR-HepG2 cell model, and AP-BBR intervention attenuated this change markedly. The gene sequencing results showed that the GNG signaling pathway was one of the signaling pathways of AP-BBR to attenuate IR in IR-Hepg2 cells. The expression of p-FoxO1Ser256 and PEPCK protein was increased, and the expression of GLUT2 protein was decreased significantly in the IR-HepG2 cell model, and both of these effects could be reversed by AP-BBR intervention. AP-BBR attenuated IR in IR-HepG2 cells, probably by regulating the GNG signaling Pathway.
Project description:<h4>Objectives</h4>Neuraminidase 1 (NEU1) cleaves terminal sialic acids of glycoconjugates during lysosomal catabolism. It also modulates the structure and activity of cellular surface receptors affecting diverse pathways. Previously we demonstrated that NEU1 activates the insulin receptor (IR) and that NEU1-deficient CathA<sup>S190A-Neo</sup> mice (hypomorph of the NEU1 activator protein, cathepsin A/CathA) on a high-fat diet (HFD) develop hyperglycaemia and insulin resistance faster than wild-type animals. The major objective of the current work was to reveal the molecular mechanism by which NEU1 desialylation activates the IR and to test if increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance.<h4>Methods</h4>To test if desialylation causes a conformational change in the IR dimer we measured interaction between the receptor subunits by Bioluminescence Resonance Energy Transfer in the HEK293T cells either overexpressing NEU1 or treated with the NEU1 inhibitor. The influence of NEU1 overexpression on insulin resistance was studied in vitro in palmitate-treated HepG2 cells transduced with NEU1-expressing lentivirus and in vivo in C57Bl6 mice treated with HFD and either pharmacological inducer of NEU1, Ambroxol or NEU1-expressing adenovirus. NEU1-deficient CathA<sup>S190A-Neo</sup> mice were used as a control.<h4>Results</h4>By desialylation of IR, NEU1 induced formation of its active dimer leading to insulin signaling. Overexpression of NEU1 in palmitate-treated HepG2 cells restored insulin signaling, suggesting that increased NEU1 levels may reverse insulin resistance. Five-day treatment of glycemic C57Bl6 mice receiving HFD with the activator of the lysosomal gene network, Ambroxol, increased NEU1 expression and activity in muscle tissue, normalized fasting glucose levels, and improved physiological and molecular responses to glucose and insulin. Ambroxol did not improve insulin sensitivity in obese insulin-resistant CathA<sup>S190A-Neo</sup> mice indicating that the Ambroxol effect is mediated through NEU1 induction. Sustained increase of liver NEU1 activity through adenovirus-based gene transfer failed to attenuate insulin resistance most probably due to negative feedback regulation of IR expression.<h4>Conclusion</h4>Together our results demonstrate that increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance suggesting that a pharmacological modulation of NEU1 activity may be potentially explored for restoring insulin sensitivity and resolving hyperglycemia associated with T2DM.
Project description:Activation of the hepatic cannabinoid type 1 receptor (CB1R) induces insulin resistance and gluconeogenesis via endoplasmic reticulum (ER) stress, thereby contributing to hyperglycemia. Gomisin N (GN) is a phytochemical derived from <i>Schisandra chinensis</i>. In the current study, we investigated the inhibitory effects of GN on hepatic CB1R-mediated insulin resistance and gluconeogenesis in 2-arachidonoylglycerol (AG; an agonist of CB1R)-treated HepG2 cells and in high-fat diet (HFD)-induced obese mice. Treatment with 2-AG induced the expression of ER stress markers, serine/threonine phosphatase <i>PHLPP1</i>, <i>Lipin1</i>, and ceramide synthesis genes, but reduced the expression of ceramide degradation genes in HepG2 cells. However, GN reversed 2-AG-mediated effects and improved the 2-AG-mediated impairment of insulin signaling. Furthermore, GN inhibited 2-AG-induced intracellular triglyceride accumulation and glucose production in HepG2 cells by downregulation of lipogenesis and gluconeogenesis genes, respectively. In vivo, GN administration to HFD obese mice reduced the HFD-induced increase in fasting blood glucose and insulin levels, which was accompanied with downregulation of HFD-induced expression of CB1R, ER stress markers, ceramide synthesis gene, and gluconeogenesis genes in the livers of HFD obese mice. These findings demonstrate that GN protects against hepatic CB1-mediated impairment of insulin signaling and gluconeogenesis, thereby contributing to the amelioration of hyperglycemia.
Project description:Insulin resistance (IR), caused by impaired insulin signal and decreased insulin sensitivity, is generally responsible for the pathophysiology of type 2 diabetes mellitus (T2DM). Sesquiterpene glycosides (SGs), the exclusive natural products from loquat leaf, have been regarded as potential lead compounds owing to their high efficacy in hypoglycemia and hypolipidemia. Here, we evaluated the beneficial effects of four single SGs isolated from loquat leaf, including SG1, SG2, SG3 and one novel compound SG4 against palmitic acid-induced insulin resistance in HepG2 cells. SG1, SG3 and SG4 could significantly enhance glucose uptake of insulin-resistant HepG2 cells at non-cytotoxic concentration. Meanwhile, Oil Red O staining showed the decrease of both total cholesterol and triglyceride content, suggesting the amelioration of lipid accumulation by SGs in insulin-resistant HepG2 cells. Further investigations found that the expression levels of phosphorylated AMPK, ACC, IRS-1, and Akt were significantly up-regulated after SGs treatment, on the contrary, the expression levels of SREBP-1 and FAS were significantly down-regulated. Notably, AMPK inhibitor Compound C (CC) blocked the regulative effects, while AMPK activator AICAR mimicked the effects of SGs in PA-treated insulin-resistant HepG2 cells. In conclusion, SGs (SG4>SG1?SG3>SG2) improved lipid accumulation in insulin-resistant HepG2 cells through the AMPK signaling pathway.
Project description:Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels