Insulin-induced Akt Activation Research Articles

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Mitochondrial involvement in the metabolic defects evoked by hepatocyte-specific BBSome deficiency

The BBSome, a protein complex of 8 Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as an important regulator of metabolic function. BBSome deficient human and mice display several metabolic features including obesity and type 2 diabetes. Here, we show that mice lacking the BBSome specifically in liver which were generated by crossing the Bbs1fl/fl mice with liver-specific (albumin) AlbCre mice exhibit significantly higher lipid droplets in liver, by Oil Red-O staining, and in primary cultured hepatocytes (5.7%±0.4% vs 2.4%±0.6%, control), and by Transmission electron microscope (TEM). AlbCre/Bbs1flox also have elevated fasting glucose (169± 14 vs 136±8 mg/dL, p<0.05) and impaired insulin sensitivity. In addition, insulin-induced activation of AKT was significantly decreased in the liver (2.6±0.07 vs 6.6± 0.34 AU, p<0.05), but not in white adipose tissue and skeletal muscle, of AlbCre/Bbs1flox mice. Using immunohistochemistry, we found that insulin receptor level in plasma membrane was decreased by 24.2±6.8% in primary cultured hepatocytes derived from AlbCre/Bbs1flox mice which explains the insulin resistance. Our previous work implicating the BBSome in the regulation of mitochondria led us to hypothesize that mitochondria dysfunction contributes to the development of metabolic defects in AlbCre/Bbs1flox mice. TEM studies revealed that mitochondria were significantly larger and longer in hepatocytes of AlbCre/Bbs1flox mice than controls. This was associated with decreased levels of Dynamin-related protein 1 (DRP1), which critically regulates mitochondrial function through fission. Next, we asked whether the mitochondrial defects may explain the insulin receptor mistraffcking in BBSome deficient hepatocytes. To test this, hepatocytes were infected with feline immunodeficiency virus that express an active mutant form of DRP1 (FIV-DRP1S637A). Interestingly, insulin receptor localization in the plasma membrane was significantly higher in hepatocytes of Bbs1fl/fl/AlbCre­ mice infected with FIV-DRP1S637A pointing to mitochondrial defects as the underlying cause of insulin receptor mislocalization induced by hepatic BBSome deficiency. Taken together, these findings demonstrate that BBSome regulation of metabolic function involve mitochondria. NIH and VA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo

ObjectiveDefining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. MethodsA whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. ResultsAlthough the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. ConclusionUbe4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

A purinergic mechanism underlying metformin regulation of hyperglycemia

Metformin, created in 1922, has been the first-line therapy for treating type 2 diabetes mellitus for almost 70 years; however, its mechanism of action remains controversial, partly because most prior studies used supratherapeutic concentrations exceeding 1mM despite therapeutical blood concentrations of metformin being less than 40μM. Here we report metformin, at 10-30μM, blocks high glucose-stimulated ATP secretion from hepatocytes mediating its antihyperglycemic action. Following glucose administration, mice demonstrate increased circulating ATP, which is prevented by metformin. Extracellular ATP through P2Y2 receptors (P2Y2R) suppresses PIP3 production, compromising insulin-induced AKT activation while promoting hepatic glucose production. Furthermore, metformin-dependent improvements in glucose tolerance are abolished in P2Y2R-null mice. Thus, removing the target of extracellular ATP, P2Y2R, mimics the effects of metformin, revealing a new purinergic antidiabetic mechanism for metformin. Besides unraveling long-standing questions in purinergic control of glucose homeostasis, our findings provide new insights into the pleiotropic actions of metformin.

Consequences of in vitro benzyl butyl phthalate exposure for blubber gene expression and insulin-induced Akt activation in juvenile grey seals

Plastic and plasticiser pollution of marine environments is a growing concern. Although phthalates, one group of plasticisers, are rapidly metabolised by mammals, they are found ubiquitously in humans and have been linked with metabolic disorders and altered adipose function. Phthalates may also present a threat to marine mammals, which need to rapidly accumulate and mobilise their large fat depots. High molecular weight (HMW) phthalates may be most problematic because they can accumulate in adipose. We used blubber explants from juvenile grey seals to examine the effects of overnight exposure to the HMW, adipogenic phthalate, benzyl butyl phthalate (BBzP) on expression of key adipose-specific genes and on phosphorylation of Akt in response to insulin. We found substantial differences in transcript abundance of Pparγ, Insig2, Fasn, Scd, Adipoq and Lep between moult stages, when animals were also experiencing differing mass changes, and between tissue depths, which likely reflect differences in blubber function. Akt abundance was higher in inner compared to outer blubber, consistent with greater metabolic activity in adipose closer to muscle than skin, and its phosphorylation was stimulated by insulin. Transcript abundance of Pparγ and Fasn (and Adipoq in some animals) were increased by short term (30 min) insulin exposure. In addition, overnight in vitro BBzP exposure altered insulin-induced changes in Pparγ (and Adipoq in some animals) transcript abundance, in a tissue depth and moult stage-specific manner. Basal or insulin-induced Akt phosphorylation was not changed. BBzP thus acted rapidly on the transcript abundance of key adipose genes in an Akt-independent manner. Our data suggest phthalate exposure could alter seal blubber development or function, although the whole animal consequences of these changes are not yet understood. Knowledge of typical phthalate exposures and toxicokinetics would help to contextualise these findings in terms of phthalate-induced metabolic disruption risk and consequences for marine mammal health.

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.

Investigation into the Role of the Skeletal Muscle BBSome in Insulin Sensitivity

The BBSome is a protein complex composed of eight Bardet‐Biedl syndrome (BBS) proteins including BBS1. Humans and mice lacking a functional BBSome display impaired insulin sensitivity, hyperglycemia, and type 2 diabetes, highlighting the importance of this protein complex in the control of insulin action and glucose homeostasis. However, the contribution of the BBSome in insulin sensitive tissues such as the skeletal muscle to the control of insulin sensitivity is not clear. We hypothesized that BBSome deficiency in the skeletal muscle will lead to insulin resistance and diabetes. To test this, we generated a novel conditional knockout mouse model lacking the Bbs1 gene specifically in the skeletal muscle. This was achieved by crossing mice expressing floxed alleles of the Bbs1 gene (Bbs1fl/fl) with mice expressing Cre‐recombinase in the skeletal muscle (MCKCre). These mice were further bred with tdTomato reporter mice to validate Cre‐recombinase using red tdTomato protein fluorescence. Body weight, glucose handling (measured using glucose tolerance test, GTT) and insulin sensitivity (measured using insulin tolerance test, ITT) of Bbs1fl/fl/MCKCre mice were compared to littermate controls. Furthermore, we used Western blot to test the ability of insulin to activate Akt, a key signaling pathway associated with the insulin receptor. We found that both male and female Bbs1fl/fl/MCKCre mice have no significant alteration in body weight relative to wild type controls (31.4±1.5 vs 31.1±1.0 g in males and 24.1±2.2 vs 23.0±1.7 g in females at 16 weeks of age). There was also no significant change in glucose handling (GTT AUC: 30608±2106 vs 30741±3480 in males and 28836±4466 vs 26530±3334 AUC in females) and insulin sensitivity (ITT AUC: 11017±1285 vs 9935±1080 in males and 8223±792 vs 5803±978 AUC in females) between Bbs1fl/fl/MCKCre mice and controls. This was further confirmed by the lack of difference in insulin‐induced Akt activation in skeletal muscle, liver, and adipose tissue. These findings suggest that BBS1 protein in skeletal muscle is not required for insulin sensitivity. Next, we assessed whether challenging the mice with high fat/high sucrose diet (HFHSD), starting at weaning, could reveal a role for the skeletal muscle BBSome in insulin sensitivity. The weight gain evoked by HFHSD was comparable between Bbs1fl/fl/MCKCre mice and controls (32.3±3.1 vs 33.3±1.0 g in males at 10 weeks of age). Once they reach 16 weeks of age, the HFHSD mice will be subject to glucose tolerance and insulin sensitivity tests to determine if obesity exaggerates hyperglycemia and insulin resistance in Bbs1fl/fl/MCKCre mice.

Lithium enhances exercise-induced glycogen breakdown and insulin-induced AKT activation to facilitate glucose uptake in rodent skeletal muscle

The purpose of this study was to investigate the effect of lithium on glucose disposal in a high-fat diet-induced type 2 diabetes mellitus (T2DM) and streptozotocin-induced type 1 diabetes mellitus (T1DM) animal model along with low-volume exercise and low-dose insulin. Lithium decreased body weight, fasting plasma glucose, and insulin levels when to treat with low-volume exercise training; however, there were no adaptive responses like an increase in GLUT4 content and translocation factor levels. We discovered that lithium enhanced glucose uptake by acute low-volume exercise-induced glycogen breakdown, which was facilitated by the dephosphorylation of serine 473-AKT (Ser473-AKT) and serine 9-GSK3β. In streptozotocin-induced T1DM mice, Li/low-dose insulin facilitates glucose uptake through increase the level of exocyst complex component 7 (Exoc7) and Ser473-AKT. Thus, lithium enhances acute exercise-induced glycogen breakdown and insulin-induced AKT activation and could serve as a candidate therapeutic target to regulate glucose level of DM patients.

A New Treatment Strategy for Diabetic Dyslipidemia?

The increased cardiovascular disease (CVD) risk associated with type 2 diabetes (T2D) is likely due in part to diabetic dyslipidemia—a condition characterized by elevated plasma levels of triglyceride‐rich lipoproteins (TRLs), smaller denser LDL particles, and decreased HDL cholesterol (1,2). Lifestyle modifications and statins are first-line interventions for CVD risk reduction in individuals with T2D, but such individuals remain at considerable risk for cardiovascular events (3). This residual CVD risk appears to be linked to abnormal metabolism of TRLs (3)—chylomicrons, VLDL, and their respective remnant lipoprotein particles (RLPs), many of which are present as intermediate-density lipoproteins (4). Thus, there is a need for developing effective and safe alternative approaches/medications to prevent and reduce diabetic dyslipidemia. In this issue of Diabetes , Roger et al. (5) report a potential new therapeutic strategy to simultaneously reduce LDL and VLDL levels by using a hybrid compound that demonstrates dual inhibition of cannabinoid-1 receptor (CB1R) and inducible nitric oxide synthase (iNOS) in a mouse model. The elevated TRLs in T2D are generally attributed to hepatic overproduction of apolipoprotein B (APOB)-containing VLDL, increased intestinal secretion of APOB48 (a truncated form of APOB100 in chylomicrons and their remnants), and dysregulated clearance of TRLs (1,2,6,7), as shown by Fig. 1. Insulin resistance and the resulting elevated fatty acids are believed to be responsible for the abnormal TRL metabolism in T2D (1,2,7). For example, inhibition of the transcription factor FoxO1 by insulin-induced Akt activation represses expression of microsomal triglyceride transfer …

Abscisic Acid: A Conserved Hormone in Plants and Humans and a Promising Aid to Combat Prediabetes and the Metabolic Syndrome.

Abscisic acid (ABA) is a hormone with a very long evolutionary history, dating back to the earliest living organisms, of which modern (ABA-producing) cyanobacteria are likely the descendants, well before separation of the plant and animal kingdoms, with a conserved role as a signal regulating cell responses to environmental challenges. In mammals, nanomolar ABA controls the metabolic response to glucose availability by stimulating glucose uptake in skeletal muscle and adipose tissue with an insulin-independent mechanism and increasing energy expenditure in the brown and white adipose tissues. Activation by ABA of AMP-dependent kinase (AMPK), in contrast to the insulin-induced activation of AMPK-inhibiting Akt, is responsible for stimulation of GLUT4-mediated muscle glucose uptake, and for the browning effect on white adipocytes. Intake of micrograms per Kg body weight of ABA improves glucose tolerance in both normal and in borderline subjects and chronic intake of such a dose of ABA improves blood glucose, lipids and morphometric parameters (waist circumference and body mass index) in borderline subjects for prediabetes and the metabolic syndrome. This review summarizes the most recent results obtained in vivo with microgram amounts of ABA, the role of the receptor LANCL2 in the hormone’s action and the significance of the endowment by mammals of two different hormones controlling the metabolic response to glucose availability. Finally, open issues in need of further investigation and perspectives for the clinical use of nutraceutical ABA are discussed.

Adipose tissue‐specific disruption of the BBSome cause insulin resistance

The BBSome is a protein complex composed of 8 Bardet‐Biedl syndrome (BBS) proteins including BBS1. Patients carrying mutations in any of the BBS genes display several features including obesity and type 2 diabetes mellitus. We previously demonstrated that the BBSome is required for membrane localization of the insulin receptor. We also showed that mice lacking several Bbs genes developed obesity associated with increased blood glucose, impaired glucose clearance and insulin resistance. However, the role and contribution of Bbs genes in insulin‐sensitive tissues such as adipose tissue for the development of obesity and diabetes remains unclear. To address this, we used a new conditional knockout mouse where exon 3 of the Bbs1 gene is floxed (Bbs1flox). Cre‐mediated recombination causes a frame shift resulting in a premature stop. We assessed whether deletion of the Bbs1 gene in adipocytes (by breeding Bbs1flox mice with AdiponectinCre mice) affects glucose homeostasis. Interestingly, no obesity phenotype was observed in both male and female AdipoCre/Bbs1flox mice fed either normal chow or high fat diet (45%), suggesting that development of obesity in BBS is not due to defects in adipose tissue. White fat adipose (WAT) cell size was similar between AdipoCre/Bbs1flox mice and control animals. However, mRNA and protein expression of PPARg was significantly decreased in WAT of AdipoCre/Bbs1flox mice compared to control animals (48%± 5% for mRNA and 47%± 14% for protein, respectively). In addition, fat acid synthetase (FAS) mRNA was reduced in WAT of AdipoCre/Bbs1flox mice relative to controls (34%± 10% vs control. Insulin‐induced activation of AKT was significantly decreased in the WAT, but not in the liver and skeletal muscle of AdipoCre/Bbs1flox mice versus control animals. Finally, fasting glucose level was similar between AdipoCre/Bbs1flox mice and control animals, and glucose tolerance test revealed no difference between AdipoCre/Bbs1flox mice and controls. However, insulin tolerance test uncovered impaired insulin‐induced glucose clearance in AdipoCre/Bbs1flox mice. These findings demonstrate the importance of the adipocyte BBSome for insulin sensitivity and glucose homeostasis.

Metabolic syndrome diminishes insulin-induced Akt activation and causes a redistribution of Akt-interacting proteins in cardiomyocytes.

Metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors, with insulin resistance as a critical component for its development. Insulin signaling in the heart leads to Akt (also known as PKB) activation, a serine/threonine protein kinase, which regulates cardiac glucose metabolism and growth. Cardiac metabolic inflexibility, characterized by impaired insulin-induced glucose uptake and oxidation, has been reported as an early and consistent change in the heart of different models of MetS and diabetes; however, the evaluation of Akt activation has yielded variable results. Here we report in cardiomyocytes of MetS rats, diminished insulin-induced glucose uptake and Akt activation, evaluated by its impaired mobilization towards the plasma membrane and phosphorylation, and reflected in a re-distribution of its interacting proteins, assessed by label-free mass spectrometry (data are available via ProteomeXchange with identifier PXD013260). We report 45 proteins with diminished abundance in Akt complex of MetS cardiomyocytes, mainly represented by energy metabolism-related proteins, and also, 31 Akt-interacting proteins with increased abundance, which were mainly related to contraction, endoplasmic reticulum stress, and Akt negative regulation. These results emphasize the relevance of Akt in the regulation of energy metabolism in the heart and highlight Akt-interacting proteins that could be involved in the detrimental effects of MetS in the heart.

Resveratrol bidirectionally regulates insulin effects in skeletal muscle through alternation of intracellular redox homeostasis

AimsReactive oxygen species (ROS) bidirectionally regulate insulin sensitivity in skeletal muscle. Insulin-induced ROS generation elevates insulin-regulated metabolic effects; however, chronic oxidative stress causes severe insulin resistance in skeletal muscle. Resveratrol (RV), as a natural antioxidant, eliminates intracellular ROS. It's unclear that whether it has different roles in insulin signaling pathway in skeletal muscle. Main methodsC57BL/6J mice and C2C12 myotubes were used to assess metabolic regulation effects of RV. Protein activation was detected using Immunofluorescence and Western Blot analysis. ROS were analyzed using confocal microscope and flow cytometry sorting (FACS). Intracellular reducing molecules were detected using an enzymatic method. Glucose uptake was measured using a fluorescent deoxyglucose analog (2-NBDG). Key findingsWe found that RV attenuated insulin-stimulated AKT phosphorylation via elimination of insulin-induced ROS generation in skeletal muscle, suggesting that RV decreased activation of the insulin-induced AKT signaling. In skeletal muscle of insulin resistance, RV reduced oxidative stress, restored intracellular glutathione (GSH) level, and enhanced insulin-induced AKT activation and glucose absorption. These results suggested that RV ameliorated insulin resistance by change of redox levels in skeletal muscle. SignificanceThis study revealed bidirectional regulation effects of RV on insulin-stimulated metabolism in skeletal muscle through alternation of intracellular redox homeostasis, which might provide a guidance role for treatment of metabolic diseases.

Intraflagellar‐transport A dysfunction causes hyperphagia‐induced systemic insulin resistance in a pre‐obese state

Deletion of murine Thm1, an intraflagellar transport A (IFT-A) component that mediates ciliary protein trafficking, causes hyperphagia, obesity, and metabolic syndrome. The role of Thm1 or IFT-A in adipogenesis and insulin sensitivity is unknown. Here, we report that Thm1 knockdown in 3T3-L1 pre-adipocytes promotes adipogenesis and enhances insulin sensitivity in vitro. Yet, pre-obese Thm1 conditional knockout mice show systemic insulin resistance. While insulin-induced AKT activation in Thm1 mutant adipose depots and skeletal muscle are similar to those of control littermates, an attenuated insulin response arises in the mutant liver. Insulin treatment of control and Thm1 mutant primary hepatocytes results in similar AKT activation. Moreover, pair-feeding Thm1 conditional knockout mice produces a normal insulin response, both in the liver and systemically. Thus, hyperphagia caused by a cilia defect, induces hepatic insulin resistance via a non-cell autonomous mechanism. In turn, hepatic insulin resistance drives systemic insulin resistance prior to an obese phenotype. These data demonstrate that insulin signaling across cell types is regulated differentially, and that the liver is particularly susceptible to hyperphagia-induced insulin resistance and a critical determinant of systemic insulin resistance.

Integration of TGF-\u03b2-induced Smad signaling in the insulin-induced transcriptional response in endothelial cells

Insulin signaling governs many processes including glucose homeostasis and metabolism, and is therapeutically used to treat hyperglycemia in diabetes. We demonstrated that insulin-induced Akt activation enhances the sensitivity to TGF-β by directing an increase in cell surface TGF-β receptors from a pool of intracellular TGF-β receptors. Consequently, increased autocrine TGF-β signaling in response to insulin participates in insulin-induced angiogenic responses of endothelial cells. With TGF-β signaling controlling many cell responses, including differentiation and extracellular matrix deposition, and pathologically promoting fibrosis and cancer cell dissemination, we addressed to which extent autocrine TGF-β signaling participates in insulin-induced gene responses of human endothelial cells. Transcriptome analyses of the insulin response, in the absence or presence of a TGF-β receptor kinase inhibitor, revealed substantial positive and negative contributions of autocrine TGF-β signaling in insulin-responsive gene responses. Furthermore, insulin-induced responses of many genes depended on or resulted from autocrine TGF-β signaling. Our analyses also highlight extensive contributions of autocrine TGF-β signaling to basal gene expression in the absence of insulin, and identified many novel TGF-β-responsive genes. This data resource may aid in the appreciation of the roles of autocrine TGF-β signaling in normal physiological responses to insulin, and implications of therapeutic insulin usage.

Free fatty acid-induced H2O2 activates TRPM2 to aggravate endothelial insulin resistance via Ca2+-dependent PERK/ATF4/TRB3 cascade in obese mice

Transient Receptor Potential Melastatin-2 (TRPM2) is a nonselective cation channel mediating Ca2+ influx in response to oxidative stress. Given that insulin resistance-related endothelial dysfunction in obesity attributes to fatty-acid-induced reactive oxygen species (ROS) overproduction, in this study, we addressed the possible role of TRPM2 in obesity-related endothelial insulin resistance and the underlying mechanisms. Whole-cell patch clamp technique, intracellular Ca2+ concentration measurement, western blot, vasorelaxation assay, and high-fat diet (HFD)-induced obese model were employed to assess the relationship between TRPM2 and endothelial insulin response. We found that both the expression and activity of TRPM2 were higher in endothelial cells of obese mice. Palmitate rose a cationic current in endothelial cells which was inhibited or enlarged by TRPM2 knockdown or overexpression. Silencing of TRPM2 remarkably improved insulin-induced endothelial Akt activation, nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production, while TRPM2 overexpression resulted in the opposite effects. Furthermore, TRPM2-mediated Ca2+ entry, CaMKII activation and the following activation of PERK/ATF4/TRB3 cascade were involved in the mechanism of obesity or palmitate-induced endothelial insulin resistance. Notably, in vivo study, knockdown of TRPM2 with adeno-associated virus harboring short-hairpin RNA (shRNA) against TRPM2 alleviated endothelial insulin resistance and ameliorated endothelium-dependent vasodilatation in obese mice. Thus, these results suggest that TRPM2-activated Ca2+ signaling is necessary to induce insulin resistance-related endothelial dysfunction in obesity. Downregulation or pharmacological inhibition of TRPM2 channels may lead to the development of effective drugs for treatment of endothelial dysfunction associated with oxidative stress state.

Methods to Investigate β-Arrestin Function in Metabolic Regulation.

Type II diabetes is one of the most serious worldwide public health problems, and its hallmark is insulin resistance, obesity associated with chronic inflammation, and defective islet β-cell function. β-Arrestins play important roles in diabetes pathogenesis through scaffolding insulin-induced AKT activation in the liver, suppressing peroxisome proliferator-activated receptor-γ-mediated adipogenesis and inflammatory responses in adipose tissue and through promoting GLP-1-induced insulin secretion in the islet. The current chapter provides detailed protocols for both in vitro and in vivo studies of the function of β-arrestins associated with type II diabetes.

Antagonistic interaction between Nodal and insulin modulates pancreatic \u03b2-cell proliferation and survival

BackgroundInsulin signaling pathway in β-cell is essential to promote β-cells proliferation and survival, while Nodal–ALK7–Smad3 signaling involves β-cells apoptosis. We attempted to address inter-relationship between Nodal and insulin in modulating β-cell proliferation and apoptosis.MethodsUsing INS-1 β-cells and isolated rat islets, we examined the effects of Nodal, insulin, or the two combined on β-cell proliferation and/or apoptosis.ResultsThe β-cells under high-glucose or palmitate conditions showed significant up-regulation of Nodal expression and activation of its downstream signaling pathway resulted in increased cleaved caspase-3. Insulin treatment led to significantly attenuated Nodal-induced cell apoptotic pathway. Similar results were found in directly Nodal-treated β-cell that insulin could partially block Nodal-induced up-regulation of ALK7–Smad3–caspase-3 signaling pathways with significantly attenuated β-cell apoptosis. Interestingly, we found that insulin-induced Akt activation and downstream molecules including GSK-3β, β-catenin and ERK1/2 was significantly attenuated by the co-treatment with Nodal, resulted in decreased cell proliferation. Furthermore, Nodal decreased glucose-evoked calcium influx and played a negative role during glucose-stimulated insulin secretion in the β-cells. Immunocytochemistry studies showed that Nodal treatment translocated Smad3 from cytosol mostly to the nucleus; however, co-treatment with insulin significantly decreased Smad3 nuclear localization. Co-immunoprecipitation experiments showed a directly interaction between Smad3 and Akt, and this interaction was enhanced by co-treatment with insulin.ConclusionsOur data suggest that the antagonistic interaction between Nodal and insulin has a role in the regulation of β-cell mass and secretion.

PO-193 Exercise training decreased lipid accumulation in murine skeletal muscle through Sestrin2-mediated SHIP2-JNK signaling pathway

Objective Obesity is becoming increasingly prevalent and is an important contributor to the worldwide burden of diseases. It is widely accepted that exercise training is beneficial for the prevention and treatment of obesity. However, the underlying mechanism by which exercise training improving skeletal muscle lipid metabolism is still not fully described.
 Sestrins (Sestrin1-3) are highly conserved stress-inducible protein. Concomitant ablation of Sestrin2 and Sestrin3 has been reported to provoke hepatic mTORC1/S6K1 activation and insulin resistance even without nutritional overload and obesity, implicating that Sestrin2 and Sestrin3 have an important homeostatic function in the control of mammalian glucose and lipid metabolism. Our previous results demonstrated that physical exercise increased Sestrin2 expression in murine skeletal muscle, while the role of Sestrin2 in regulating lipid metabolism remains unknown. 
 SH2 domain containing inositol 5-phosphatase (SHIP2) acts as a negative regulator of the insulin signaling both in vitro and in vivo. An increased expression of SHIP2 inhibits the insulin-induced Akt activation, glucose uptake, and glycogen synthesis in 3T3-L1 adipocytes, L6 myotubes and tissues of animal models. Alterations of SHIP2 expression and/or enzymatic function appear to have a profound impact on the development of insulin resistance. However, the regulatory function of SHIP2 in lipid metabolism after exercise remains unclear. It has been reported that SHIP2 modulated lipid metabolism through regulating the activity of c-Jun N-terminal kinase (JNK) and Sterol regulatory element-binding protein-1 (SREBP-1).
 JNK is a subclass of mitogen-activated protein kinase (MAPK) signaling pathway in mammalian cells and plays a crucial role in metabolic changes and inflammation associated with a high-fat diet. Inhibition of JNK reduces lipid deposition and proteins level of fatty acid de novo synthesis in liver cells. It has been reported that Sestrin2 regulated the phosphorylation of JNK, however the underlying mechanism remains unclear. SREBP-1 is important in regulating cholesterol biosynthesis and uptake and fatty acid biosynthesis, and SREBP-1 expression produces two different isoforms, SREBP-1a and SREBP-1c. SREBP-1c is responsible for regulating the genes required for de novo lipogenesis and its expression is regulated by insulin. SREBP-1a regulates genes related to lipid and cholesterol production and its activity is regulated by sterol levels in the cell.
 Altogether, the purpose of this study was to explore the effect and underlying mechanism of Sestrin2 on lipid accumulation after exercise training.
 Methods Male wild type and SESN2−/− mice were divided into normal chow (NC) and high-fat diet (HFD) groups to create insulin resistance mice model. After 8 weeks the IR model group was then divided into HFD sedentary control and HFD exercise groups (HE). Mice in HE group underwent 6-week treadmill exercise to reveal the effect of exercise training on lipid metabolism in insulin resistance model induced by HFD. We explored the mechanism through which Sestrin2 regulated lipid metabolism in vitro by supplying palmitate, overexpressing or inhibiting SESNs, SHIP2 and JNK in myotubes.
 Results We found that 6-week exercise training decreased body weight, BMI and fat mass in wild type and SESN2-/- mice after high-fat diet (HFD) feeding. And exercise training decreased the level of plasma glucose, serum insulin, triglycerides and free fatty acids in wild type but not in Sestrin2-/- mice. Lipid droplet in skeletal muscle was also decreased in wild type but did not in Sestrin2-/- mice. Moreover, exercise training increased the proteins expression involved in fatty acid oxidation and decreased the proteins which related to fatty acid de novo synthesis. The results of oil red staining and the change of proteins related to fatty acid de novo synthesis and beta oxidation in myotubes treated with palmitate, Ad-SESN2 and siRNA-Sestrin2 were consisted with the results in vivo, which suggested that Sestrin2 was a key regulator in lipid metabolism. Exercise training increased Sestrin2 expression and reversed up-regulation of SHIP2 and pJNK induced by HFD in wild type mice but not in Sestrin2-/- mice. In parallel, overexpression of Sestrin2 decreased the level of SHIP2 and pJNK induced by palmitate while Sestrin2 knock down by siRNA-Sestrin2 treatment did not change the expression of SHIP2 and pJNK, which suggested that Sestrin2 modulated SHIP2 and JNK in the state of abnormal lipid metabolism. Inhibition of SHIP2 reduced the activity of JNK, increased lipid accumulation and the proteins of fatty acid synthesis after palmitate treatment and over expression of Sestrin2, which suggest that Sestrin2 modulated lipid metabolism through SHIP2/JNK pathway.
 Conclusions Sestrin2 plays an important role in improving lipid metabolism after exercise training, and Sestrin2 regulates lipid metabolism by SHIP2-JNK pathway in skeletal muscle.

Hyper-insulinemia increases the glutamate-excitotoxicity in cortical neurons: A mechanistic study

Insulin resistance in type-2 diabetic condition increases the risk of stroke and cognitive deficits in which involvement of glutamate has been postulated. It has been hypothesized that hyper-insulinemia in cortical neurons increases the vulnerability towards glutamate-induced excitotoxicity. To mimic insulin resistance, cortical neurons were incubated with high insulin (1 µM) and high glucose (50 mM final concentration) in in-vitro condition for 24 h. Pre-treatment of cortical neurons with high insulin blocked acute insulin-induced activation of Akt and GSK-3β but not in the case of high glucose. Our results demonstrate that chronic high insulin exposure increases glutamate-induced excitotoxity, which was blocked by insulin receptor antagonist (S961) and GSK-3β inhibitor (SB216763). These inhibitors also ameliorated pAkt (Ser473) and pGSK-3β(Ser9) levels after chronic insulin exposure. Increase in glutamate-excitotoxicity in insulin-resistant cortical neurons was found to be associated with increased expression of PICK1. However, GluR2 did not get altered in hyper-insulinemia condition. This study demonstrates that hyper-insulinemia increases glutamate excitotoxicity which could be attributed to activation of GSK-3β and increased expression of PICK1.