Insulin Signaling In Adipose Tissue Research Articles

Altered extracellular matrix dynamics is associated with insulin resistance in adolescent children with obesity.

The objective of this study was to examine the hypothesis that abdominal and gluteal adipocyte turnover, lipid dynamics, and fibrogenesis are dysregulated among insulin-resistant (IR) compared with insulin-sensitive (IS) adolescents with obesity. Seven IS and seven IR adolescents with obesity participated in a 3-h oral glucose tolerance test and a multi-section magnetic resonance imaging scan of the abdominal region to examine body fat distribution patterns and liver fat content. An 8-week 70% deuterated water (2 H2 O) labeling protocol examined adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from biopsied abdominal and gluteal fat. Abdominal and gluteal subcutaneous adipose tissue (SAT) turnover rates of lipid components were similar among IS and IR adolescents with obesity. However, the insoluble collagen (type I, subunit α2) isoform measured from abdominal, but not gluteal, SAT was elevated in IR compared with IS individuals. In addition, abdominal insoluble collagen Iα2 was associated with ratios of visceral-to-total (visceral adipose tissue + SAT) abdominal fat and whole-body and adipose tissue insulin signaling, and it trended toward a positive association with liver fat content. Altered extracellular matrix dynamics, but not expandability, potentially decreases abdominal SAT lipid storage capacity, contributing to the pathophysiological pathways linking adipose tissue and whole-body IR with altered ectopic storage of lipids within the liver among IR adolescents with obesity.

Inhibiting Phosphatidylcholine Remodeling in Adipose Tissue Increases Insulin Sensitivity.

Cell membrane phosphatidylcholine composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT); changes in membrane phosphatidylcholine saturation are implicated in metabolic disorders. Here, we identified LPCAT3 as the major isoform of LPCAT in adipose tissues and created adipocyte-specific Lpcat3-knockout mice to study adipose tissue lipid metabolism. Transcriptome sequencing and plasma adipokine profiling were used to investigate how LPCAT3 regulates adipose tissue insulin signaling. LPCAT3 deficiency reduced polyunsaturated phosphatidylcholines in adipocyte plasma membranes, increasing insulin sensitivity. LPCAT3 deficiency influenced membrane lipid rafts, which activated insulin receptors and AKT in adipose tissue, and attenuated diet-induced insulin resistance. Conversely, higher LPCAT3 activity in adipose tissues from ob/ob, db/db, and high-fat diet-fed mice reduced insulin signaling. Adding polyunsaturated phosphatidylcholines to mature human or mouse adipocytes in vitro worsened insulin signaling. We suggest that targeting LPCAT3 in adipose tissues to manipulate membrane phospholipid saturation is a new strategy to treat insulin resistance.

730-P: Effects of Obstructive Sleep Apnea Treatment on Whole-Body and Adipose Tissue Insulin Sensitivity—Role of Senescence and Mitigation by Adjuvant Metformin Therapy

Introduction: Obstructive sleep apnea (OSA) is a prevalent sleep disorder linked to high type 2 diabetes risk. Positive airway pressure (PAP) therapy is the standard of care for OSA, but it inconsistently improves glucose homeostasis. Senescent cells are common in adipose tissue (AT) of patients with OSA and may impair insulin signaling. However, the effects of PAP therapy on AT senescence are not known. Addition of metformin, an insulin-sensitizer with anti-senescent effects, may be a good adjuvant therapeutic strategy to improve glycemic control in patients with OSA. Methods: We randomly assigned 16 nondiabetic participants with OSA (Mean± SD; age: 50.9 ± 6.7 y, BMI: 36.5 ± 2.9 kg/m2) in a 1:1 ratio to receive either 2g metformin or placebo daily along with PAP therapy for 3 months in a double-blind pilot study. Whole body and AT insulin sensitivity was determined by 2h oral glucose tolerance test (OGTT). AT biopsy was obtained to estimate tissue insulin signaling and senescence. Results: The metformin and placebo groups had comparable age, BMI and OSA severity at baseline. PAP compliance was 38% and 75% in the metformin and placebo groups, respectively. Matsuda Index, glucose area under the curve (2h AUC), and free-fatty acid suppression at baseline and follow-up were not different in metformin or placebo treated groups. Compared to baseline, insulin AUC and insulin to glucose AUC ratio during OGTT was unchanged in the metformin but increased in the placebo group. Metformin, but not placebo, was associated with increased insulin mediated AKT phosphorylation and decreased senescence biomarkers (p21 and MCP1) in AT during the follow-up visit. Conclusion: Metformin, as adjunct to PAP therapy, attenuated insulin rises during OGTT, improved AT insulin signaling, and lowered senescence biomarkers in OSA patients. Larger trials of longer treatment duration are needed to determine if metformin can prevent diabetes in OSA patients. Disclosure S. Rodrigues: None. W.S. Dantas: None. R.A. Beyl: None. R.C. Hebert: None. I. Griffith: None. M. Tanksley: None. E.C. Mader: None. J.P. Kirwan: None. C.L. Axelrod: None. E. Ravussin: Research Support; Eli Lilly and Company, Novartis AG. Advisory Panel; Novartis. P. Singh: None. Funding National Institutes of Health (P30DK072476, U54GM104940); CAPES (88887.470405/2019-00 to S.R.)

Abstract 317: Adipose Tissue Phosphatidylcholine Remodeling Modulates Insulin Signaling

Phosphatidylcholines (PCs) are major components of cell membranes. Previous studies indicated that membrane PC composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT) and an alteration in cell membrane saturation has been implicated in a variety of metabolic disorders, including obesity, insulin resistance, and cardiovascular diseases. LPCAT3 is the major isoform of LPCAT in adipose tissues. To study the impact of LPCAT3 on adipose tissue lipid metabolism and its metabolic consequences, we prepared adipocyte-specific Lpcat3 gene knockout (KO) mice. Based on the analysis of bulk RNA sequencing, we gained insights to investigate the role of LPCAT3 in regulating adipose tissue insulin signaling. We revealed that LPCAT3 deficiency significantly reduced polyunsaturated PCs in the plasma membrane of adipocytes, resulting in increased insulin sensitivity. Mechanistically, we demonstrated that inhibiting LPCAT3 in adipose tissue promoted insulin receptor and AKT activation by influencing membrane lipid rafts and attenuated diet-induced insulin resistance. Conversely, adipose tissues from ob/ob, db/db, and high fat diet-fed mice had significantly higher LPCAT3 activity which blunts insulin signaling. Moreover, the treatment of polyunsaturated PCs on human or mouse mature adipocytes significantly worsened cellular insulin signaling. Our findings suggest that targeting LPCAT3 in adipose tissues to manipulate membrane phospholipid saturation would be a new strategy to treat insulin resistance.

PSUN123 Increased Expression of Insulin Signaling Intermediates After Six-Months Of Medical Care In Women With Obesity And Polycystic Ovary Syndrome

Abstract Background Recently an androgen-induced obesity-independent mechanism of insulin resistance in a mouse model mimicking lean polycystic ovary syndrome (PCOS) was discovered. To date there is little to no data on the mechanism of insulin resistance or molecular insulin signaling in adipose tissue of women with PCOS undergoing Vertical Sleeve Gastrectomy (VSG). Does the mechanism of androgen receptor (AR)-mediated regulation of insulin signaling seen in the lean PCOS mouse model occur in adipose tissue of women with obesity and PCOS? Objective We hypothesize that in women with obesity and PCOS, those with higher levels of free androgen index will display more dysregulation in the insulin signaling pathway in adipose tissue. More specifically, we believe that visceral more than subcutaneous adipose tissue in the women in the study will display more dysregulation; and lastly that VSG will alleviate insulin signaling dysregulation. Briefly, insulin activates insulin receptor (IR) which leads to activation of phospho-AKT which increases phosphorylated forkhead box 01 (p-FOXO1, gluconeogenesis regulation) and transcriptionally regulates acetyl co-A carboxylase (ACC, de novo lipogenesis (DNL) regulation). Methods This is a nested mechanistic study within a randomised controlled clinical trial. We performed molecular analyses (Western blots for protein and qPRC for mRNA expression, respectively, n = 3-4/group) exploring intermediates in the insulin signaling pathway (IR, p-AKT, p-FOXO1, and ACC) in (i) visceral and subcutaneous (SubQ) adipose tissue from women with obesity and PCOS before VSG bariatric surgery (n = 3/group); and (ii) SubQ WAT from women with obesity and PCOS at baseline and six-months after medical intervention with metformin (MTF) and lifestyle modification. Results Subcutaneous (SubQ) white adipose tissue (WAT) displayed similar levels of p-ACC compared to visceral WAT from women pre-VSG. Six months of medical care increased p-ACC in SubQ WAT by 5-fold compared to baseline. IR displayed similar expression in the visceral compared to SubQ WAT in women pre-VSG. IR expression increased by 20-fold at 6-months of medical care compared to baseline in SubQ WAT. p-FOXO1 displayed similar expression in the visceral compared to SubQ WAT. However, p-FOXO1 was decreased at 6-months of medical care compared to baseline. p-AKT levels were similar in visceral compared to SubQ WAT pre-VSG. However, p-AKT levels were significantly increased by 4-fold at 6-months of medical care compared to baseline. Conclusion None of the insulin signaling intermediates measured were different in visceral compared to SubQ in women with obesity and PCOS. Additionally, the findings indicated lowered DNL, lowered regulation of gluconeogenic genes and thus lowered gluconeogenesis, and increased insulin action in SubQ WAT in women with obesity and PCOS after 6-months of medical care. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Effects of SGLT2 inhibition on lipid transport in adipose tissue in type 2 diabetes.

SGLT2 inhibition induces an insulin-independent reduction in plasma glucose causing increased lipolysis and subsequent lipid oxidation by energy-consuming tissues. However, it is unknown whether SGLT2 inhibition also affects lipid storage in adipose tissue. Therefore, we aimed to determine the effects of SGLT2 inhibition on lipid storage and lipolysis in adipose tissue. We performed a randomized, double-blinded, placebo-controlled crossover design of 4 weeks of empagliflozin 25 mg and placebo once-daily in 13 individuals with type 2 diabetes treated with metformin. Adipose tissue fatty acid uptake, lipolysis rate and clearance were measured by 11C-palmitate PET/CT. Adipose tissue glucose uptake was measured by 18F-FDG PET/CT. Protein and gene expression of pathways involved in lipid storage and lipolysis were measured in biopsies of abdominal s.c. adipose tissue. Subjects were weight stable, which allowed us to quantify the weight loss-independent effects of SGLT2 inhibition. We found that SGLT2 inhibition did not affect free fatty acids (FFA) uptake in abdominal s.c. adipose tissue but increased FFA uptake in visceral adipose tissue by 27% (P < 0.05). In addition, SGLT2 inhibition reduced GLUT4 protein (P = 0.03) and mRNA content (P = 0.01) in abdominal s.c. adipose tissue but without affecting glucose uptake. In addition, SGLT2 inhibition decreased the expression of genes involved in insulin signaling in adipose tissue. We conclude that SGLT2 inhibition reduces GLUT4 gene and protein expression in abdominal s.c. adipose tissue, which could indicate a rebalancing of substrate utilization away from glucose oxidation and lipid storage capacity through reduced glycerol formation.

Reversible insulin resistance in muscle and fat unrelated to the metabolic syndrome in patients with acromegaly.

SummaryBackgroundPatients with active acromegaly exhibit insulin resistance despite a lean phenotype whereas controlled disease improves insulin sensitivity and increases fat mass. The mechanisms underlying this paradox remain elusive, but growth hormone (GH)-induced lipolysis plays a central role. The aim of the study was to investigative the molecular mechanisms of insulin resistance dissociated from obesity in patients with acromegaly.MethodsIn a prospective study, twenty-one patients with newly diagnosed acromegaly were studied at diagnosis and after disease control obtained by either surgery alone (n=10) or somatostatin analogue (SA) treatment (n=11) with assessment of body composition (DXA scan), whole body and tissue-specific insulin sensitivity and GH and insulin signalling in adipose tissue and skeletal muscle.FindingsDisease control of acromegaly significantly reduced lean body mass (p<0.001) and increased fat mass (p<0.001). At diagnosis, GH signalling (pSTAT5) was constitutively activated in fat and enhanced expression of GH-regulated genes (CISH and IGF-I) were detected in muscle and fat. Insulin sensitivity in skeletal muscle, liver and adipose tissue increased after disease control regardless of treatment modality. This was associated with enhanced insulin signalling in both muscle and fat including downregulation of phosphatase and tensin homolog (PTEN) together with reduced signalling of GH and lipolytic activators in fat.InterpretationIn conclusion, the study support that uncontrolled lipolysis is a major feature of insulin resistance in active acromegaly, and is characterized by upregulation of PTEN and suppression of insulin signalling in both muscle and fat.FundingThis work was supported by a grant from the Independent Research Fund, Denmark (7016-00303A) and from the Alfred Benzon Foundation, Denmark.

Abstract 13123: Global Knockout of LDLRAP1 Regulates Atherosclerosis, Adipose Metabolism, and Insulin Resistance

Introduction: Atherosclerotic vascular syndromes account for 50% of all deaths in the United States, and are a considerable medical and socioeconomic problem contributing to mortality of several conditions, including myocardial infarction and stroke. It is accepted that atherosclerosis and symptoms of metabolic syndrome, such as obesity, insulin resistance, and hypercholesterolemia often coincide with and exacerbate one another, however the molecular events in common with these conditions are not yet fully understood. One event that may link these two disorders is lipid receptor trafficking and signaling of the LDL receptor (LDLR), which plays a key role in LDL uptake and regulation of plasma LDL concentration. Low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) is a cytosolic adaptor protein which interacts with the cytoplasmic tail of the LDLR, mediating internalization of the LDL-LDLR complex and connecting it to the cell’s endocytic machinery. The purpose of this study was to characterize the effects of LDLRAP1 global knockout on atherogenesis and metabolic function in order to gain a better understanding of the molecular mechanisms that interconnect atherosclerosis and metabolic syndrome. Results: LDLRAP1 global knockout mice fed a high-fat western diet (HFD) for 16 weeks displayed increased atherosclerotic plaque burden (p&lt;0.0001), serum cholesterol (p&lt;0.0001) and triglyceride levels (p&lt;0.01), and expression of lipid receptors SR-A1 and CD36 (p&lt;0.05), and gained significantly more weight compared to controls (p&lt;0.0001). LDLRAP1 -/- adipose also displayed decreased glucose uptake (p&lt;0.01) and pAKT and AKT protein levels (p&lt;0.05). Both before and after a HFD, LDLRAP1 -/- mice were also significantly insulin resistant (p&lt;0.01) with concomitant dysregulated insulin signaling in adipose tissue, and displayed metabolic differences as quantified by metabolic cage analysis (p&lt;0.05). Conclusions: These data suggest that in addition to a function in cholesterol internalization, LDLRAP1 participates in regulating adipose metabolism and insulin sensitivity. This implicates LDLRAP1 as a key molecule and potential therapeutic target in treatment of diseases which constitute metabolic syndrome.

Role of RAGE in obesity-induced adipose tissue inflammation and insulin resistance

AbatractObesity is known to be associated with adipose tissue inflammation and insulin resistance. Importantly, in obesity, the accumulation of proinflammatory macrophages in adipose tissue correlates with insulin resistance. We hypothesized that the receptor for advanced glycation end products (RAGE) and associated ligands are involved in adipose tissue insulin resistance, and that the activation of the AGE–RAGE axis plays an important role in obesity-associated inflammation. C57BL/6J mice (WT) and RAGE deficient (RAGE−/−) mice were fed a high fat diet (HFD) and subjected to glucose and insulin tolerance tests. Epdidymal adipose tissue (eAT) was collected and adipose stromal vascular cells isolated using flow cytometry. Visceral adipose tissue macrophage polarization was assessed by quantitative real time PCR. Immunoblotting was performed to evaluate the insulin signaling in adipose tissues. In additional studies, cell trafficking was assessed by injecting labeled blood monocytes into recipient mice. RAGE−/− mice displayed improved insulin sensitivity and glucose tolerance, accompanied by decreased body weight and eAT mass. Exogenous methylglyoxal (MGO) impaired insulin-stimulated AKT signaling in adipose tissues from WT mice fed a normal chow diet, but not in RAGE−/− mice. In contrast, in obese mice, treatment with MGO did not reduce insulin-induced phosphorylation of AKT in WT-HFD mice. Moreover, insulin-induced AKT phosphorylation was found to be impaired in adipose tissue from RAGE−/−-HFD mice. RAGE−/− mice displayed improved inflammatory profiles and evidence for increased adipose tissue browning. This observation is consistent with the finding of reduced plasma levels of FFA, glycerol, IL-6, and leptin in RAGE−/− mice compared to WT mice. Collectively the data demonstrate that RAGE-mediated adipose tissue inflammation and insulin-signaling are potentially important mechanisms that contribute to the development of obesity-associated insulin resistance.

MH-76, a Novel Non-Quinazoline α1-Adrenoceptor Antagonist, but Not Prazosin Reduces Inflammation and Improves Insulin Signaling in Adipose Tissue of Fructose-Fed Rats

Background: Quinazoline α1-adrenoceptors antagonists have been shown to exert moderately favorable effects on the metabolic profile in hypertensive patients. However, based on AntiHypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) results, they are no longer recommended as a first line therapy of hypertension. Recent studies have shown that quinazoline-based α1-adrenoceptors antagonists (prazosin, doxazosin) induce the apoptosis and necrosis, which may be responsible for ALLHAT outcomes; however, these effects were proven to be independent of α1-adrenoceptor blockade and were associated with the presence of quinazoline moiety. MH-76 (1-[3-(2,6-dimethylphenoxy)propyl]-4-(2-methoxyphenyl)piperazine hydrochloride)) is a non-quinazoline α1-adrenoceptor antagonist which, in fructose-fed rats, exerted antihypertensive effect, and, contrary to prazosin, reduced insulin resistance and abdominal adiposity. In this study we aimed to further investigate and compare the effects of MH-76 and prazosin on inflammation in adipose tissue of fructose-fed rats. Methods: Abdominal adipose tissue was collected from four groups of fructose-fed rats (Control, Fructose, Fructose + MH-76 and Fructose + Prazosin) and subjected to biochemical, histopathological and immunohistochemical studies. Moreover, selected tissue distribution studies were performed. Results: Treatment with MH-76 but not with prazosin improved endothelial integrity, reduced adipose tissue inflammation and infiltration by immune cells, resulting in lowering leptin, MCP-1, IL-6, TNF-α and PAI-1 levels. In adipose tissue from Fructose + MH-76 animals, a higher amount of eosinophils accompanied with higher IL-4 concentration was observed. Treatment with MH-76 but not with prazosin markedly reduced phosphorylation of IRS-1 at Ser307. Conclusion: MH-76 may improve insulin signaling in adipose tissue by reducing the pro-inflammatory cytokine production and inhibiting the inflammatory cells recruitment. In contrast, in adipose tissue from animals treated with prazosin, the inflammatory effect was clearly enhanced.

Hepatocyte‐specific Overexpression of Fatty Acid Desaturase 1 Improves Insulin Signaling and ameliorates hypertriglyceridemia in Male Rat

Objective Decreased fatty acid desaturase 1 (Fads1) activity results in the accumulation of saturated fat, which contributes to the development of nonalcoholic steatohepatitis (NASH). The aim of this study was to determine whether hepatic overexpression of Fads1 attenuates a dietary high-fat high-fructose (HFHF)-induced metabolic phenotype. Methods Hepatocyte-specific overexpression of Fads1 was achieved via the tail vein injection of recombinant adeno-associated virus (AAV) serotype 8 containing an albumin promoter with rat Fads1 sequence. Blank vectors served as controls. Male weanling Sprague-Dawley rats were assigned to five groups and fed with chow diet, low-fat high-fructose diet with AAV8 blank control (LFHF-AAV8-Blank), LFHF-AAV8-Fads1, high-fat high-fructose diet with AAV8 blank control (HFHF-AAV8-Blank), or HFHF-AAV8-Fads1 (n=8 rats/group) for 8 weeks. Metabolic phenotypes were characterized, including body weight, level of hepatic steatosis, glucose and insulin tolerance tests (GTT, ITT). Liver, skeletal muscle and adipose tissue insulin signaling were examined by western blot. Results Hepatocyte-specific overexpression of Fads1 significantly decreased body weight gain and improved glucose tolerance, but not hepatic steatosis, in LFHF-fed rats. These effects were not evident in HFHF-fed rats. On the contrary, white adipose tissue weight was markedly reduced in HFHF-AAV8-Fads1 rats compared to HFHF-AAV8-blank control rats, and to a lesser extent in LFHF rats. Both LFHF-AAV8-Fads1 and HFHF-AAV8-Fads1 rats displayed decreased fasting plasma triglycerides and increased plasma free fatty acids compared to their AAV8-blank control rats, and this was associated with upregulation of adipose tissue hormone sensitive lipase (HSL). While phosphorylated Akt (p-Akt) expression was downregulated, phosphorylated glycogen synthase kinase 3β (GSK-3β) was upregulated by both LFHF and HFHF diet in skeletal muscle, these alterations were not evident in the liver and adipose tissue. Moreover, decreased expression of p-Akt in skeletal muscle was partially restored in HFHF-AAV8-Fads1 rats, but not in LFHF-AAV8-Fads1 rats. Conclusion Our data demonstrated that hepatocyte-specific overexpression of Fads1 leads to the improvement of metabolic phenotypes in a diet- and tissue-specific manner, and this was associated with improved insulin signaling in skeletal muscle. The underlying mechanism(s) need further investigation.

Advanced lipodystrophy reverses fatty liver in mice lacking adipocyte hormone-sensitive lipase

Modulation of adipocyte lipolysis represents an attractive approach to treat metabolic diseases. Lipolysis mainly depends on two enzymes: adipose triglyceride lipase and hormone-sensitive lipase (HSL). Here, we investigated the short- and long-term impact of adipocyte HSL on energy homeostasis using adipocyte-specific HSL knockout (AHKO) mice. AHKO mice fed high-fat-diet (HFD) progressively developed lipodystrophy accompanied by excessive hepatic lipid accumulation. The increased hepatic triglyceride deposition was due to induced de novo lipogenesis driven by increased fatty acid release from adipose tissue during refeeding related to defective insulin signaling in adipose tissue. Remarkably, the fatty liver of HFD-fed AHKO mice reversed with advanced age. The reversal of fatty liver coincided with a pronounced lipodystrophic phenotype leading to blunted lipolytic activity in adipose tissue. Overall, we demonstrate that impaired adipocyte HSL-mediated lipolysis affects systemic energy homeostasis in AHKO mice, whereby with older age, these mice reverse their fatty liver despite advanced lipodystrophy.

Short-term intermittent hypoxia induces simultaneous systemic insulin resistance and higher cardiac contractility in lean mice.

BackgroundIntermittent hypoxia (IH) is the major feature of obstructive sleep apnea syndrome, well‐known to induce cardiometabolic complications. We previously demonstrated that IH induces hyperinsulinemia and associated altered insulin signaling in adipose tissue, liver, and skeletal muscle, but impact of IH on cardiac insulin signaling and functional/structural consequences remains unknown. Therefore, the aims of this study were to investigate in both lean and obese mice the effects of chronic IH on the following: (1) cardiac insulin signaling and (2) cardiac remodeling and function.MethodsC57BL/6 J male mice were fed low‐fat (LFD) or high‐fat (HFD) diet for 20 weeks, and exposed to IH (21–5% FiO2, 60 s cycle, 8 h/day) or normoxia (N) for the last 6 weeks. Systemic insulin sensitivity was evaluated by an insulin tolerance test. Cardiac remodeling and contractile function were assessed by cardiac ultrasonography. Ultimately, hearts were withdrawn for biochemical and histological analysis.ResultsIn LFD mice, IH‐induced hyperinsulinemia and systemic insulin resistance that were associated with increased phosphorylations of cardiac insulin receptor and Akt on Tyr1150 and Ser473 residues, respectively. In addition, IH significantly increased cardiac interstitial fibrosis and cardiac contractility. In the HFD group, IH did not exert any additional effect, nor on insulin/Akt signaling, nor on cardiac remodeling and function.ConclusionOur study suggests that, despite systemic insulin resistance, IH exposure mediates an adaptive cardiac response in lean but not in obese mice. Further studies are needed to investigate which specific mechanisms are involved and to determine the long‐term evolution of cardiac responses to IH.

Systemic Insulin Resistance and Metabolic Perturbations in Chow Fed Inducible Nitric Oxide Synthase Knockout Male Mice: Partial Reversal by Nitrite Supplementation.

iNOS, an important mediator of inflammation, has emerged as an important metabolic regulator. There are conflicting observations on the incidence of insulin resistance (IR) due to hyperglycemia/dyslipidemia in iNOS−/− mice. There are reports that high fat diet (HFD) fed mice exhibited no change, protection, or enhanced susceptibility to IR. Similar observations were also reported for low fat diet (LFD) fed KO mice. In the present study chow fed iNOS−/− mice were examined for the incidence of IR, and metabolic perturbations, and also for the effect of sodium nitrite supplementation (50 mg/L). In IR-iNOS−/− mice, we observed significantly higher body weight, BMI, adiposity, blood glucose, HOMA-IR, serum/tissue lipids, glucose intolerance, enhanced gluconeogenesis, and disrupted insulin signaling. Expression of genes involved in hepatic and adipose tissue lipid uptake, synthesis, oxidation, and gluconeogenesis was upregulated with concomitant downregulation of genes for hepatic lipid excretion. Nitrite supplementation restored NO levels, significantly improved systemic IR, glucose tolerance, and also reduced lipid accumulation by rescuing hepatic insulin sensitivity, glucose, and lipid homeostasis. Obesity, gluconeogenesis, and adipose tissue insulin signaling were only partially reversed in nitrite supplemented iNOS−/− mice. Our results thus demonstrate that nitrite supplementation to iNOS−/− mice improves insulin sensitivity and metabolic homeostasis, thus further highlighting the metabolic role of iNOS.

Dysregulation of PP2A-Akt interaction contributes to Sucrose non-fermenting related kinase (SNRK) deficiency induced insulin resistance in adipose tissue

ObjectiveWe previously identified Sucrose non-fermenting related kinase (SNRK) as a regulator of adipose inflammation and energy homeostasis. In this study, we aimed to investigate the role of SNRK in insulin signaling in white (WAT) and brown adipose tissue (BAT). MethodsAdipose tissue specific (SNRK deficiency in both WAT and BAT) and BAT specific knockout mouse models were employed. Phosphoproteomic studies were conducted to identify the novel SNRK pathway regulating insulin signaling in adipose tissue. ResultsSNRK ablation is sufficient to inhibit insulin-stimulated AKT phosphorylation and glucose uptake in both WAT and BAT. Phosphoproteomic study using SNRK deficient versus wild type BAT samples revealed 99% reduction of phosphorylation on Serine 80 of PPP2R5D, the regulatory subunit of Protein phosphatase 2A (PP2A). Drastic (142.5-fold) induction of phosphorylation on Serine 80 of PPP2R5D was observed in SNRK-deficient primary brown adipocytes overexpressing SNRK compared to control protein. In vitro phosphorylation reaction followed by targeted phosphoproteomic detection further confirms that human recombinant SNRK is able to phosphorylate human recombinant PPP2R5D. Dephosphorylated PPP2R5D promotes constitutive assembly of PP2A-AKT complex, therefore inhibits insulin-induced AKT phosphorylation and subsequent glucose uptake in both BAT and WAT. Knockdown of PPP2R5D in adipocytes can improve insulin sensitivity in adipocytes without SNRK expression. ConclusionsOur findings demonstrate that SNRK regulates insulin signaling through controlling PPP2R5D phosphorylation, which subsequently impacts PP2A activity and then AKT phosphorylation in both WAT and BAT. SNRK may represent a promising potential target for treating insulin resistance-related metabolic disorders.

1781-P: Adipocyte GI Signaling Regulates Whole-Body Glucose Homeostasis and Insulin Sensitivity

Adipocyte dysfunction links obesity to insulin resistance and type 2 diabetes (T2D). Receptor-mediated activation of heterotrimeric G proteins plays a pivotal role in regulating adipocyte function. Little is known about the potential in vivo metabolic roles of Gi-type G proteins expressed by adipocytes, primarily due to the lack of suitable animal models. To address this issue, we generated adipocre-ROSA 26PTXmice in which Gisignaling was selectively disrupted in adipocytes ('adipo-Gi KO mice'). Compared to control mice, adipo-Gi KO mice displayed increased lipolysis and decreased fat mass when maintained on regular chow or a high fat diet (HFD). Moreover, HFD adipo-Gi KO mice showed impaired glucose tolerance and reduced insulin sensitivity. In vitro studies with primary adipocytes prepared from adipo-Gi KO mice indicated that the lack of Gisignaling led to impaired insulin signaling and reduced insulin-stimulated glucose uptake. In parallel, we also used a chemo-genetic approach to generate a mouse line selectively expressing a CNO-sensitive, Gi-coupled DREADD (designer receptor exclusively activated by a designer drug) in adipocytes (adipo-GiD mice). CNO-induced activation of adipocyte Gi signaling in adipo-GiD mice significantly improved glucose tolerance and insulin sensitivity independent of the diet that the mice consumed (regular chow or HFD). In vivo 14C-2-deoxy-glucose uptake assays demonstrated that activation of adipocyte Gisignaling enhanced insulin-induced glucose uptake in adipose tissues. Stimulation of adipocyte Gisignaling activation also further enhanced insulin signaling in adipose tissues. Taken together, our data clearly demonstrate that adipocyte Gisignaling is a critical regulator of insulin signaling in adipocytes and is critical for the maintenance of glucose homeostasis. A better understanding of the mechanisms through which Gipromotes adipocyte insulin signaling should stimulate the development of novel antidiabetic drugs. Disclosure L. Wang: None. S. Pydi: None. L. Zhu: None. S. Jain: None. Y. Cui: None. O. Gavrilova: None. J. Wess: None. Funding National Institutes of Health

Prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows

BackgroundOverconditioned dairy cows are susceptible to excessive lipolysis and increased insulin resistance during the transition period. The associations among body fat reserve, insulin resistance, and lipolysis in adipose tissues (AT) remain to be elucidated. Therefore, this study aimed to investigate whether excessive fat reserves influence the insulin signaling pathway in AT postpartum.ResultsTwenty multiparous dairy cows were selected and assigned to one of two groups, according to prepartum body condition score (BCS): Control group (BCS = 3.0–3.5; n = 10) and Overconditioned group (BCS ≥ 4.0; n = 10). Blood samples were collected on days −14, −7, −4, −2, −1, 0, 1, 2, 4, 7, and 14 relative to parturition. Subcutaneous AT were collected on day 2 following parturition for quantitative real-time polymerase chain reaction and western blot analyses. No differences were observed between the two groups in serum glucose, non-esterified fatty acids, β-hydroxybutyric acid, tumor necrosis factor (TNF) α, insulin, or leptin concentrations during the experimental period. Compared with the control cows, the overconditioned cows had lower serum triglyceride levels and higher adiponectin concentrations. In the AT postpartum, insulin receptor mRNA and protein levels were lower in the overconditioned cows than in the control cows, and no differences were found in glucose transporter 4 mRNA. Compared with the control cows, the overconditioned cows had lower mRNA levels of TNFα and higher mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) in AT postpartum. The phosphorylated protein kinase B (AKT) content and phosphorylation rate of AKT were increased in the overconditioned cows compared with the control cows, which suggested that the downstream insulin signaling in AT was affected.ConclusionsIn the present study, transition dairy cows with higher BCS did not show more fat mobilization. The changes of insulin signaling pathway in AT postpartum of overconditioned cows may be partly related to the expression of PPARγ and TNFα, and the secretion of adiponectin.

Statins Promote Interleukin-1β-Dependent Adipocyte Insulin Resistance Through Lower Prenylation, Not Cholesterol.

Statins lower cholesterol and adverse cardiovascular outcomes, but this drug class increases diabetes risk. Statins are generally anti-inflammatory. However, statins can promote inflammasome-mediated adipose tissue inflammation and insulin resistance through an unidentified immune effector. Statins lower mevalonate pathway intermediates beyond cholesterol, but it is unknown whether lower cholesterol underpins statin-mediated insulin resistance. We sought to define the mevalonate pathway metabolites and immune effectors that propagate statin-induced adipose insulin resistance. We found that LDL cholesterol lowering was dispensable, but statin-induced lowering of isoprenoids required for protein prenylation triggered NLRP3/caspase-1 inflammasome activation and interleukin-1β (IL-1β)-dependent insulin resistance in adipose tissue. Multiple statins impaired insulin action at the level of Akt/protein kinase B signaling in mouse adipose tissue. Providing geranylgeranyl isoprenoids or inhibiting caspase-1 prevented statin-induced defects in insulin signaling. Atorvastatin (Lipitor) impaired insulin signaling in adipose tissue from wild-type and IL-18-/- mice, but not IL-1β-/- mice. Atorvastatin decreased cell-autonomous insulin-stimulated lipogenesis but did not alter lipolysis or glucose uptake in 3T3-L1 adipocytes. Our results show that statin lowering of prenylation isoprenoids activates caspase-1/IL-1β inflammasome responses that impair endocrine control of adipocyte lipogenesis. This may allow the targeting of cholesterol-independent statin side effects on adipose lipid handling without compromising the blood lipid/cholesterol-lowering effects of statins.

Elucidation of a New Mechanism of Onset of Insulin Resistance: Effects of Statins and Tumor Necrosis Factor-α on Insulin Signal Transduction

Impaired insulin signaling in adipose tissue and skeletal muscle causes insulin resistance associated with the development of type 2 diabetes. However, the molecular mechanisms underlying insulin resistance remain to be elucidated. In this review, we describe the current understanding of the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) and tumor necrosis factor (TNF)-α on insulin signal transduction in adipocytes. First, we determined that atorvastatin inhibits the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 through a decrease in the RhoA-Rho-kinase pathway, resulting in the inhibition of glucose uptake. Second, we found that TNF-α induces IRS-1 phosphorylation at serine residues 636/639 and inhibits the tyrosine phosphorylation of IRS-1 through the increase in both extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation. Interestingly, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMP-activated protein kinase activator, suppresses TNF-α-induced IRS-1 serine phosphorylation at 636/639 and the phosphorylation of ERK by enhancing interactions between ERK and dual-specificity phosphatase-9. These results may be helpful in understanding the mechanisms underlying insulin resistance.

Functional Roles of Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) in Adipocytes

Adipose tissue fibrosis is recognized as a hallmark of adipose tissue dysfunction. We have previously identified PRELP as a novel regulator of adipogenesis by using lentiviral short hairpin RNA libraries targeting murine genomes. PRELP belongs to the family of leucine-rich repeat proteins, which are characterized by a series of adjacent leucine-rich motifs flanked by disulfide-bounded domains. Functionally, PRELP anchors basement membranes to connective tissues. However, roles of PRELP in adipocytes remain unclear. In the present study, we show that PRELP expression is up-regulated both in adipocytes of high-fat diet induced obesity mice and ob/ob mice. PRELP reduction in 3T3-L1 adipocytes leads to enhanced insulin signaling and insulin-stimulated glucose uptake. To determine functions of PRELP in the development of metabolic phenotypes, we generated adipocyte-specific PRELP transgenic mice. Adipose-specific overexpression of PRELP results in the development of significant adipose tissue fibrosis and insulin resistance on high fat diet without difference of body weight and adiposity. This phenotype is associated with increased expression of inflammation genes and decreased insulin signaling in adipose tissue of PRELP transgenic mice. Furthermore, we find that global Prelp deficient mice are protected from adipose tissue fibrosis and insulin resistance associated with high fat feeding. These data suggest that PRELP might play a key role in the development of adipose tissue remodeling in obesity. Disclosure J. Eguchi: None. J. Wada: None.