Insulin-induced Phosphorylation Research Articles

Knockdown of ACOT4 alleviates gluconeogenesis and lipid accumulation in hepatocytes

Acyl-CoA thioesterase 4 (ACOT4) has been reported to be related to acetyl-CoA carboxylase activity regulation; However, its exact functions in liver lipid and glucose metabolism are still unclear. Here, we discovered explored the regulatory roles of ACOT4 in hepatic lipid and glucose metabolism in vitro. We found that the expression level of ACOT4 was significantly increased in the hepatic of db/db and ob/ob mice as well as obese mice fed a high fat diet. Adenovirus-mediated overexpression of ACOT4 promoted gluconeogenesis and high-glucose/high-insulin-induced lipid accumulation and impaired insulin sensitivity in primary mouse hepatocytes, whereas ACOT4 knockdown notably suppressed gluconeogenesis and decreased the triglycerides accumulation in hepatocytes. Furthermore, ACOT4 knockdown increased insulin-induced phosphorylation of AKT and GSK-3β in primary mouse hepatocytes. Mechanistically, we found that upregulation of ACOT4 expression inhibited AMP-activated protein kinase (AMPK) activity, and its knockdown had the opposite effect. However, activator A769662 and inhibitor compound C of AMPK suppressed the impact of the change in ACOT4 expression on AMPK activity. Our data indicated that ACOT4 is related to hepatic glucose and lipid metabolism, primarily via the regulation of AMPK activity. In conclusion, ACOT4 is a potential target for the therapy of non-alcoholic fatty liver (NAFLD) and type 2 diabetes.

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to wild-type mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

SAT047 Regulation Of The Insulin Receptor And The Insulin-like Growth Factor 1 Receptor By miR-322-5p, A miR-16 Family Member, In Hypothalamic Neuronal Models

Abstract Disclosure: W. He: None. N. Loganathan: None. K.W. Mak: None. E. McIlwraith: None. D.D. Belsham: None. Insulin signals through the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R) in hypothalamic neurons to control food intake and peripheral metabolism. A contributor to obesity, and subsequent comorbidities such type 2 diabetes and heart disease, is the development of cellular insulin resistance in hypothalamic neurons. However, the molecular changes to neuronal insulin signaling remain to be fully elucidated. MicroRNAs (miRNAs) inhibit translation of specific mRNAs; thus, this study aimed to understand the involvement of miRNAs in the regulation of insulin signaling and resistance in hypothalamic neurons. To profile miRNAs expressed in hypothalamic neurons, RNA from the whole hypothalami of 14-week-old CD1 male mice (n = 4), as well as the immortalized hypothalamic neuronal cell lines mHypoE-46 (n = 3) and mHypoA-59 (n = 3), each co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP), were assessed using the Affymetrix GeneChip miRNA 4.0 Array. Notably, miR-16 family members, including miR-16-5p, miR-15b-5p, and miR-322-5p, were among the most highly expressed miRNAs in each case. In the mHypoA-59 neurons, overexpression of miR-322-5p for 24 hours decreased the mRNA levels of Igf1r (-26.1%; p = 0.0007; n = 4) and the protein level of INSR-beta (-31.8%; p = 0.0023). These results suggest that the miR-16 family plays an inhibitory role in insulin signaling in hypothalamic neurons. To study the involvement of miRNAs in hyperinsulinemia-induced neuronal insulin resistance, the mHypoE-46 neurons were treated with 100 nM insulin for 24 hours to induce cellular insulin resistance, as characterized by decreased INSR-beta protein (-96.3%; p < 0.0001; n = 4) and a resulting decrease in insulin-induced phosphorylation of protein kinase B (AKT) (-67.6%; p = 0.01; n = 4). GeneChip miRNA array analysis showed that insulin overexposure disrupted the expression of 48 miRNAs (p < 0.05, n = 3), including the upregulation of miR-18a-3p, mir-322, miR-494-3p, and miR-671-3p. Upon RT-qPCR validation (n = 3), we established that prolonged (24 hours), but not acute (1-6 hours), insulin exposure upregulated miR-18a-3p (+62%; p = 0.0104), miR-671-3p (+111%; p = 0.0079), and miR-1983 (+97%; p = 0.0181). miR-671-3p, miR-494-3p, and miR-18a-3p have been shown to decrease phosphatase and tensin homolog (PTEN) levels, which can promote neuronal insulin resistance based on bioinformatic analysis. Overall, these results suggest hyperinsulinemia disrupts the expression of specific miRNAs in hypothalamic neurons to promote cellular insulin resistance. Knowledge derived from these studies will provide insight into hypothalamus-derived miRNAs that could be targeted for miRNA-based diagnostics and therapeutics for early central insulin resistance in humans.(Supported by the CRC, CIHR, NSERC, EndoSoc, and BBDC) Presentation: Saturday, June 17, 2023

The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

Brown adipocyte mineralocorticoid receptor deficiency impairs metabolic regulation in diet-induced obese mice

Activation of brown adipose tissue (BAT) contributes to energy dissipation and metabolic health. Although mineralocorticoid receptor (MR) antagonists have been demonstrated to improve metabolism under obesity, the underlying mechanisms remain incompletely understood. We aimed to evaluate the role of BAT MR in metabolic regulation. After 8 weeks of high-fat diet (HFD) feeding, BAT MR KO (BMRKO) mice manifested significantly increased bodyweight, fat mass, serum fasting glucose, and impaired glucose homeostasis compared with littermate control (LC) mice, although insulin resistance and fasting serum insulin were not significantly changed. Metabolic cage experiments showed no change in O2 consumption, CO2 production, or energy expenditure in obese BMRKO mice. RNA sequencing analysis revealed downregulation of genes related to fatty acid metabolism in BAT of BMRKO-HFD mice compared with LC-HFD mice. Moreover, H&E and immunohistochemical staining demonstrated that BMRKO exacerbated HFD-induced macrophage infiltration and proinflammatory genes in epididymal white adipose tissue (eWAT). BMRKO-HFD mice also manifested significantly increased liver weights and hepatic lipid accumulation, an increasing trend of genes related to lipogenesis and lipid uptake, and significantly decreased genes related to lipolytic and fatty acid oxidation in the liver. Finally, the level of insulin-induced AKT phosphorylation was substantially blunted in eWAT but not liver or skeletal muscle of BMRKO-HFD mice compared with LC-HFD mice. These data suggest that BAT MR is required to maintain metabolic homeostasis, likely through its regulation of fatty acid metabolism in BAT and impacts on eWAT and liver.

Role of Dgat2 in Glucose Uptake and Fatty Acid Metabolism in C2C12 Skeletal Myotubes.

Acyl-coenzyme A (CoA):diacylglycerol acyltransferase 2 (DGAT2) catalyzes the last stage of triacylglycerol (TAG) synthesis, a process that forms ester bonds with diacylglycerols (DAG) and fatty acyl-CoA substrates. The enzymatic role of Dgat2 has been studied in various biological species. Still, the full description of how Dgat2 channels fatty acids in skeletal myocytes and the consequence thereof in glucose uptake have yet to be well established. Therefore, this study explored the mediating role of Dgat2 in glucose uptake and fatty acid partitioning under short interfering ribonucleic acid (siRNA)-mediated Dgat2 knockdown conditions. Cells transfected with Dgat2 siRNA downregulated glucose transporter type 4 (Glut4) messenger RNA (mRNA) expression and decreased the cellular uptake of [1-14C]-labeled 2-deoxyglucose up to 24.3% (p < 0.05). Suppression of Dgat2 deteriorated insulininduced Akt phosphorylation. Dgat2 siRNA reduced [1-14C]-labeled oleic acid incorporation into TAG, but increased the level of [1-14C]-labeled free fatty acids at 3 h after initial fatty acid loading. In an experiment of chasing radioisotope-labeled fatty acids, Dgat2 suppression augmented the level of cellular free fatty acids. It decreased the level of re-esterification of free fatty acids to TAG by 67.6% during the chase period, and the remaining pulses of phospholipids and cholesteryl esters were decreased by 34.5% and 61%, respectively. Incorporating labeled fatty acids into beta-oxidation products increased in Dgat2 siRNA transfected cells without gene expression involving fatty acid oxidation. These results indicate that Dgat2 has regulatory function in glucose uptake, possibly through the reaction of TAG with endogenously released or recycled fatty acids.

Phosphorylation of insulin receptor substrates (IRS-1 and IRS-2) is attenuated following cecal ligation and puncture in mice

BackgroundSepsis is characterized as an insulin resistant state. However, the effects of sepsis on insulin’s signal transduction pathway are unknown. The molecular activity driving insulin signaling is controlled by tyrosine phosphorylation of the insulin receptor β-subunit (IRβ) and of insulin receptor substrate molecules (IRS) -1 and IRS-2.HypothesisCecal ligation and puncture (CLP) attenuates IRβ, IRS-1 and IRS-2 phosphorylation.MethodsIACUC-approved studies conformed to ARRIVE guidelines. CLP was performed on C57BL/6 mice; separate cohorts received intraperitoneal insulin at baseline (T0) or at 23 or 47 h. post-CLP, 1 h before mice were euthanized. We measured levels of (1) glucose and insulin in serum, (2) IRβ, IRS-1 and IRS-2 in skeletal muscle and liver homogenate and (3) phospho-Irβ (pIRβ) in liver and skeletal muscle, phospho-IRS-1 (pIRS-1) in skeletal muscle and pIRS-2 in liver. Statistical significance was determined using ANOVA with Sidak’s post-hoc correction.ResultsCLP did not affect the concentrations of IRβ, IRS-1or IRS-2 in muscle or liver homogenate or of IRS-1 in liver. Muscle IRS-1 concentration at 48 h. post-CLP was higher than at T0. Post-CLP pIRS-1 levels in muscle and pIRβ and pIRS-2 levels in liver were indistinguishable from T0 levels. At 48 h. post-CLP pIRβ levels in muscle were higher than at T0. Following insulin administration, the relative abundance of pIRβ in muscle and liver at T0 and at both post-CLP time points was significantly higher than abundance in untreated controls. In T0 controls, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was higher than in untreated mice. However, at both post-CLP time points, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was not distinguishable from the abundance in untreated mice at the same time point. Serum glucose concentration was significantly lower than T0 at 24 h., but not 48 h., post-CLP. Glucose concentration was lower following insulin administration to T0 mice but not in post-CLP animals. Serum insulin levels were significantly higher than baseline at both post-CLP time points.ConclusionsCLP impaired insulin-induced tyrosine phosphorylation of both IRS-1 in muscle and IRS-2 in liver. These findings suggest that the molecular mechanism underlying CLP-induced insulin resistance involves impaired IRS-1/IRS-2 phosphorylation.

1561-P: Dietary Proteins Impact Glucose Tolerance and Hepatic Insulin Resistance in Mice through Modulation of the Urea Cycle

We recently reported that dietary proteins (DP) modulate diet-induced obesity and insulin resistance in mice (Nat Commun 12: 3377, 2021). We posit that these metabolic alterations are caused by differential activation of the hepatic urea cycle in response to different amino acid (AA) compositions and total nitrogen load. Objective: To test whether DP sources alter hepatic metabolism and insulin resistance through the urea cycle. Methods: Metabolic phenotypes, hepatic urea cycle and insulin resistance were measured in mice fed low-fat, low-sucrose (LFLS) or high-fat, high-sucrose (HFHS) diets containing either pork (PP), soy (SP) or casein (CP) protein for 12 weeks. Based on their AA composition, PP had the highest nitrogen load, followed by SP and CP. Results: When compared to SP and CP, feeding PP to HFHS mice induced more glucose intolerance which was correlated with higher post-prandial AA-derived nitrogen load in the portal vein. PP feeding in HFHS mice induced hepatic insulin resistance, revealed by impaired insulin-induced Akt phosphorylation on Ser473. Mice fed the LFLS diet were, however, protected from PP-induced glucose intolerance and insulin resistance, and this was associated with robust increase in the protein levels of urea cycle enzymes (CPS1, OTC) in the liver. In starked contrast, induction of urea cycle enzymes was blunted in the liver of PP-fed HFHS mice. PP feeding also increased the expression of inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) as showed by DHE staining of liver sections. Conclusion: Dietary PP increases total nitrogen load activating the urea cycle, which may protect against metabolic dysfunction and hepatic insulin resistance in LFLS mice. This protective mechanism is lost in PP-fed HFHS mice, likely by downregulation of urea cycle activity, when the liver is overloaded with dietary fat. iNOS induction could be acting as an alternative nitrogen handling pathway, causing a build-up of mitochondrial ROS. Disclosure F.Bégin: None. A.Caron: None. A.Marette: Advisory Panel; Valbiotis, Amancia, Plexus, Acasti.

The effects of sucrose and arsenic on muscular insulin signaling pathway differ between gastrocnemius and quadriceps muscles

Background: Muscle insulin resistance can originate from a sedentary lifestyle, hypercaloric diets or, the exposure to endocrine-disrupting pollutants such as arsenic. In skeletal muscle, insulin stimulates glucose uptake by translocating GLUT4 to the sarcolemma. This study aimed to evaluate the alterations induced by sucrose and arsenic exposure in vivo on the pathways involved in insulin-stimulated GLUT4 translocation in the quadriceps and gastrocnemius muscles. Methods: Male Wistar rats were treated with 20 % sucrose (S), 50 mg/L sodium arsenite (A), or both (A+S) in drinking water for 8 weeks. The intraperitoneal insulin tolerance (ITT) test was on the seventh week of treatment. Quadriceps and gastrocnemius muscles were obtained after overnight fasting or after 30 min of intraperitoneal insulin injection. We assessed changes in GLUT4 translocation to the sarcolemma by cell fractionation and abundance of the proteins involved in GLUT4 translocation by Western blot. Results: Male rats consuming S and A+S gained more weight than control and A-treated animals. Rats consuming S, A, and A+S developed insulin resistance assessed through ITT. Neither treatments nor insulin stimulation in the quadriceps produced changes in GLUT4 levels in sarcolemma and Akt phosphorylation. Conversely, A and A+S decreased protein expression of Tether containing Ubx domain for GLUT4 (TUG), and A alone increased calpain-10 expression. All treatments reduced this muscle's protein levels of VAMP2. Conversely, S and A-treatment increased basal GLUT4 levels in the sarcolemma of the gastrocnemius, while all treatments inhibited insulin-induced GLUT4 translocation. These effects correlated with lower basal levels of TUG and impaired insulin-stimulated TUG proteolysis. Moreover, animals treated with S had reduced capn10 protein levels in this muscle, while A and A+S inhibited insulin-induced Akt phosphorylation. Conclusion: Arsenic and sucrose act through different pathways to impair GLUT4 trafficking, and the effects differ between the quadriceps and gastrocnemius. CONACYT-Fronteras #568492, DGAPA-PAPIIT-UNAM #IN20872 and DGAPA-PAPIIT-UNAM #IN205621 This is the full abstract presented at the American Physiology Summit 2023 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.

ANGPTL4 is a potential driver of HCV-induced peripheral insulin resistance

Chronic hepatitis C (CHC) is associated with the development of metabolic disorders, including both hepatic and extra-hepatic insulin resistance (IR). Here, we aimed at identifying liver-derived factor(s) potentially inducing peripheral IR and uncovering the mechanisms whereby HCV can regulate the action of these factors. We found ANGPTL4 (Angiopoietin Like 4) mRNA expression levels to positively correlate with HCV RNA (r = 0.46, p < 0.03) and HOMA-IR score (r = 0.51, p = 0.01) in liver biopsies of lean CHC patients. Moreover, we observed an upregulation of ANGPTL4 expression in two models recapitulating HCV-induced peripheral IR, i.e. mice expressing core protein of HCV genotype 3a (HCV-3a core) in hepatocytes and hepatoma cells transduced with HCV-3a core. Treatment of differentiated myocytes with recombinant ANGPTL4 reduced insulin-induced Akt-Ser473 phosphorylation. In contrast, conditioned medium from ANGPTL4-KO hepatoma cells prevented muscle cells from HCV-3a core induced IR. Treatment of HCV-3a core expressing HepG2 cells with PPARγ antagonist resulted in a decrease of HCV-core induced ANGPTL4 upregulation. Together, our data identified ANGPTL4 as a potential driver of HCV-induced IR and may provide working hypotheses aimed at understanding the pathogenesis of IR in the setting of other chronic liver disorders.

Heterocyclic amines reduce insulin-induced AKT phosphorylation and induce gluconeogenic gene expression in human hepatocytes.

Heterocyclic amines (HCAs) are well-known for their mutagenic properties. One of the major routes of human exposure is through consumption of cooked meat, as certain cooking methods favor formation of HCAs. Recent epidemiological studies reported significant associations between dietary HCA exposure and insulin resistance and type II diabetes. However, no previous studies have examined if HCAs, independent of meat consumption, contributes to pathogenesis of insulin resistance or metabolic disease. In the present study, we have assessed the effect of three HCAs commonly found in cooked meat (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [MeIQ], 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [PhIP]) on insulin signaling and glucose production. HepG2 or cryopreserved human hepatocytes were treated with 0-50μM of MeIQ, MeIQx, or PhIP for 3days. Treatment of HepG2 cells and hepatocytes with MeIQ and MeIQx resulted in a significant reduction in insulin-induced AKT phosphorylation, suggesting that HCA exposure decreases hepatic insulin signaling. HCA treatment also led to significant increases in expression of gluconeogenic genes, G6PC and PCK1, in both HepG2 and cryopreserved human hepatocytes. Additionally, the level of phosphorylated FOXO1, a transcriptional regulator of gluconeogenesis, was significantly reduced by HCA treatment in hepatocytes. Importantly, HCA treatment of human hepatocytes led to increases in extracellular glucose level in the presence of gluconeogenic substrates, suggesting that HCAs induce hepatic glucose production. The current findings suggest that HCAs induce insulin resistance and promote hepatic glucose production in human hepatocytes. This implicates that exposure to HCAs may lead to the development of type II diabetes or metabolic syndrome.

H2S- and Redox-State-Mediated PTP1B S-Sulfhydration in Insulin Signaling.

Because hydrogen sulfide (H2S) is classified as a gaseous signaling molecule, protein S-sulfhydration is known to be one of the mechanisms by which H2S signals are conducted. PTP1B, a negative regulator in insulin signaling, has been found to be S-sulfhydrated at Cys215-SH to form Cys215-SSH in response to endoplasmic reticulum (ER) stress. Therefore, we aimed to understand the change in PTP1B S-sulfhydration and cellular redox homeostasis in response to insulin stimulation. We demonstrated a feasible PEG-switch method to determine the levels of PTP1B S-sulfhydration. According to the results obtained from HEK293T and MDA-MB-231 cells, insulin induced a change in PTP1B S-sulfhydration that was similar to the change in Insulin receptor substrate 1 (IRS1) phosphorylation in both cell lines. However, insulin-induced PTP1B S-sulfhydration and IRS1 phosphorylation were only significantly affected by metformin in HEK293T cells. Insulin also induced an increase in reactive oxygen species (ROS) in both cell lines. However, the level of H2S, GSH, and GSSG was only significantly affected by insulin and metformin in HEK293T cells. HEK293T cells maintained high levels of H2S and cysteine, but low levels of GSSG and GSH in general compared to MDA-MB-231 cells. From these findings, we suggest that PTP1B activity is modulated by H2S and redox-regulated S-sulfhydration during insulin signaling.

Selective somatostatin receptor 5 inhibition improves hepatic insulin sensitivity.

Diabetes is a metabolic disorder with an increasing global prevalence. Somatostatin (SST), a peptide hormone, regulates hormone secretion via five SST receptor (SSTR) subtypes (SSTR1-5) in a tissue-specific manner. As SSTR5 is expressed in pancreatic β-cells and intestinal L-cells, studies have suggested that SSTR5 regulates glucose tolerance through insulin and incretin secretion, thereby having a prominent role in diabetes. Moreover, SSTR5 knockout (KO) mice display enhanced insulin sensitivity; however, the underlying mechanism has not been clarified. Therefore, in this study, we investigate the effect of SSTR5 blockade on insulin resistance and the target organ using SSTR5 KO mice and a selective SSTR5 antagonist (compound-1). High-fat diet (HFD)-fed SSTR5 KO mice exhibited significantly lower homeostasis model assessment of insulin resistance (HOMA-IR) than HFD-fed wild-type mice. Two-week oral administration of compound-1 dose-dependently and significantly reduced changes in the levels of glycosylated hemoglobin (GHb), plasma glucose, plasma insulin, and HOMA-IR in male KK-Ay /Ta Jcl mice (KK-Ay mice), a model of obese type 2 diabetes with severe insulin resistance. Additionally, compound-1 significantly increased the glucose infusion rate while decreasing hepatic glucose production in male KK-Ay mice, as evidenced by hyperinsulinemic-euglycemic clamp analyses. In addition, compound-1 ameliorated the insulin-induced Akt phosphorylation suppression by octreotide in the liver of male C57BL/6J mice. Collectively, our results demonstrate that selective SSTR5 inhibition can improve insulin sensitivity by enhancing liver insulin action; thus, selective SSTR5 antagonists represent potentially novel therapeutic agents for type 2 diabetes.

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.

RAGE displays sex-specific differences in obesity-induced adipose tissue insulin resistance

BackgroundThe receptor for advanced glycation end products (RAGE) plays an important role in obesity-associated insulin sensitivity. We have also previously reported that RAGE deficiency improved insulin resistance in obesity-induced adipose tissue. The current study was aimed to elucidate the sex-specific mechanism of RAGE deficiency in adipose tissue metabolic regulation and systemic glucose homeostasis.MethodsRAGE-deficient (RAGE−/−) mice were fed a high-fat diet (HFD) and subjected to glucose and insulin tolerance tests. Subcutaneous adipose tissue (sAT) was collected, and macrophage polarization was assessed by quantitative real-time PCR. Immunoblotting was performed to evaluate the insulin signaling in adipose tissues.ResultsUnder HFD feeding conditions, body weight and adipocyte size of female RAGE deficient (RAGE−/−) were markedly lower than that of male mice. Female RAGE−/− mice showed significantly improved glucose and insulin tolerance compared to male RAGE−/− mice, accompanied with increased M2 macrophages polarization. Expressions of genes involved in anti-oxidant and browning were up-regulated in adipose tissues of female RAGE−/− mice. Moreover, insulin-induced AKT phosphorylation was significantly elevated in adipose tissue in female RAGE−/− mice compared to male RAGE−/− mice.ConclusionsOur findings suggest that RAGE-mediated adipose tissue insulin resistance is sex-specific, which is associated with different expression of genes involved in anti-oxidant and browning and insulin-induced AKT phosphorylation.

Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling

Insulin signaling is mediated via a network of protein phosphorylation. Dysregulation of this network is central to obesity, type 2 diabetes and metabolic syndrome. Here we investigate the role of phosphatase binding protein Alpha4 (α4) that is essential for the serine/threonine protein phosphatase 2A (PP2A) in insulin action/resistance in adipocytes. Unexpectedly, adipocyte-specific inactivation of α4 impairs insulin-induced Akt-mediated serine/threonine phosphorylation despite a decrease in the protein phosphatase 2A (PP2A) levels. Interestingly, loss of α4 also reduces insulin-induced insulin receptor tyrosine phosphorylation. This occurs through decreased association of α4 with Y-box protein 1, resulting in the enhancement of the tyrosine phosphatase protein tyrosine phosphatase 1B (PTP1B) expression. Moreover, adipocyte-specific knockout of α4 in male mice results in impaired adipogenesis and altered mitochondrial oxidation leading to increased inflammation, systemic insulin resistance, hepatosteatosis, islet hyperplasia, and impaired thermogenesis. Thus, the α4 /Y-box protein 1(YBX1)-mediated pathway of insulin receptor signaling is involved in maintaining insulin sensitivity, normal adipose tissue homeostasis and systemic metabolism.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.

The tyrosine phosphorylation of GRK2 is responsible for activated D2R-mediated insulin resistance

Dopamine D2 receptor (D2R) plays a key role in the regulation of glucose homeostasis by stimulating the secretion of many glucoregulatory hormones. Insulin resistance (IR) is associated with the pathogenesis of metabolic disorders which occurs when PI3K/Akt signaling pathway is downregulated. However, the potential involvement of D2R in insulin resistance remains unclear. In the present study, we investigated the regulation of glucose transport by D2-like receptors and discovered that activation of D2R, but not D3R or D4R, suppressed insulin-induced 2-DOG uptake and Glut4 membrane translocation in a GRK2- and Src-dependent manner. Further study revealed that activation of D2R inhibits insulin-induced phosphorylation of Akt at Thr308 and Ser473, which are hallmarks of its kinase activity, by increasing the interaction of tyrosine phosphorylated GRK2 with Akt and then preventing Akt from interacting with PDK1. Thus, this study demonstrates that Src mediated GRK2 tyrosine phosphorylation is an essential physiological event that mediates the roles of D2R in insulin resistance.

Esaxerenone, a selective mineralocorticoid receptor blocker, improves insulin sensitivity in mice consuming high-fat diet

BackgroundEsaxerenone is a novel, non-steroidal selective mineralocorticoid receptor (MR) blocker. MR activation plays a crucial role in the development of cardiovascular and metabolic diseases. In this study, we investigated the effects of esaxerenone on various metabolic parameters in mice. Materials and methodsEsaxerenone (3 mg/kg/day) was orally administered to high-fat diet (HFD)-fed male C57BL/6 mice. Mice fed a normal diet (ND) served as controls. Glucose and insulin tolerance, plasma lipid levels, and transaminase levels were assessed as metabolic parameters. Macrophage accumulation in the adipose tissue was evaluated using histological analysis. 3T3-L1 adipocytes, HepG2 cells, and C2C12 myotubes were used for in vitro experiments. Gene expression and insulin signaling were examined using quantitative RT-PCR and western blotting, respectively. ResultsHFD successfully induced insulin resistance compared with that in ND. Esaxerenone ameliorated insulin resistance (P < 0.05) without altering other metabolic parameters, such as the lipid profile. Esaxerenone administration tended to decrease plasma transaminase levels compared with those in the non-treated group. In the adipose tissue, esaxerenone decreased macrophage accumulation (P < 0.05) and increased the expression levels of adiponectin and PPARγ. Aldosterone significantly decreased the expression levels of PPARγ and adiponectin in 3T3-L1 adipocytes. Furthermore, aldosterone attenuated insulin-induced Akt phosphorylation in 3T3-L1 adipocytes, HepG2 cells, and C2C12 myotubes in a dose-dependent manner (P < 0.01). These effects were ameliorated by pretreatment with esaxerenone. ConclusionEsaxerenone ameliorated insulin resistance in HFD-fed mice. Reduction of inflammation and improvement in insulin signaling may underlie the beneficial effects of esaxerenone.

Metabolic Remodeling with Hepatosteatosis Induced Vascular Oxidative Stress in Hepatic ERK2 Deficiency Mice with High Fat Diets.

We previously demonstrated the marked hepatosteatosis and endothelial dysfunction in hepatocyte-specific ERK2 knockout mice (LE2KO) with a high-fat/high-sucrose diet (HFHSD), but detailed metabolic changes and the characteristics in insulin-sensitive organs were not tested. This study aimed to characterize metabolic remodeling with changes in insulin-sensitive organs, which could induce endothelial dysfunction in HFHSD-LE2KO. The serum glucose and fatty acid (FA) were modestly higher in HFHSD-LE2KO than HFHSD-Control. FA synthesis genes were up-regulated, which was associated with the decreased phosphorylation of AMPK and ACC, and with the up-regulation of SREBP-1 in the liver from HFHSD-LE2KO. In FA and amino acids fraction analysis, arachidonic acid/eicosapentaenoic acid ratio, L-ornithine/arginine ratio, asymmetric dimethylarginine and homocysteine levels were elevated in HFHSD-LE2KO. Insulin-induced phosphorylation of AKT was blunted in skeletal muscle. Serum leptin and IL-1β were elevated, and serum adiponectin was decreased with the enlargement of epididymal adipocytes. Finally, the enhanced superoxide levels in the aorta, which were blunted with CCCP, apocynin, and tempol, were observed in HFHSD-LE2KO. A pre-incubation of aortic rings with tempol improved endothelial dysfunction in HFHSD-LE2KO. HFHSD-LE2KO revealed an acceleration of FA synthesis in the liver leading to insulin resistance in skeletal muscle and the enlargement of visceral adipocytes. Global metabolic remodeling such as changes in arginine metabolism, ω3/ω6 ratio, and adipocytokines, could affect the vascular oxidative stress and endothelial dysfunction in HFHSD-LE2KO.