Adipocyte Insulin Sensitivity Research Articles

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.

Overexpression of PID1 reduces high density lipoprotein level and functionality in swine.

Phosphotyrosine interaction domain 1 (PID1), a protein with a phosphotyrosine-binding (PTB) domain, interacts with the lipoprotein receptor-related protein 1 (LRP1) to reduce the insulin sensitivity of adipocyte. Considering the role of LRP1 in lipid metabolism, we investigated the effect of PID1 on the content and biological activities of serum lipoproteins in pigs. PID1-transgenic pigs were genetated by sperm and magnetic nanoparticles-mediated method. The levels of PID1 in PID1-transgenic pig's liver were higher than that in the wild-type pig's liver. We found that serum levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I) were significantly reduced in PID1-transgenic pigs. On the other hand, PID1-transgenic pigs displayed increased non-HDL-C levels. Serum levels of total cholesterol and triglycerides were comparable between the PID1-transgenic and the wild-type pigs. Further, the HDL isolated from PID1-transgenic pigs showed a significant reduction in cholesterol efflux ability. In addition, serum superoxide dismutase activity of PID1-transgenic pigs was also obviously lowered compared with that of wild type pigs. In conclusion, these results suggest that PID1 might be able to adjust HDL-C levels in serum and HDL cholesterol efflux ability.

Procyanidin C1, a Component of Cinnamon Extracts, Is a Potential Insulin Sensitizer That Targets Adipocytes.

Natural products are one of the main sources for discovering new lead compounds. We previously reported that cinnamon extract has a promising effect in regulating lipid tissue volume and insulin sensitivity in vivo. However, its effective component and the underlying mechanism are not known. In the present study, we analyzed the effect of different components of cinnamon on regulating insulin sensitivity in 3T3-L1 adipocytes. Functional assay revealed that, of the six major components of cinnamon extracts, the B-type procyanidin, procyanidin C1, improves the differentiation of 3T3-L1 cells (TG content: 1.10 ± 0.09 mM at a dosage of 25 μM vs 0.67 ± 0.02 mM in vehicle group, p < 0.001) and promotes insulin-induced glucose uptake (8.58 ± 1.43 at a dosage of 25 μM vs 3.05 ± 1.24 in vehicle group, p < 0.001). Mechanism studies further suggested that procyanidin C1 activates the AKT-eNOS pathway, thus up-regulating glucose uptake and enhancing insulin sensitivity in mature adipocytes. Taken together, our study identified B-type procyanidinC1, a component of cinnamon extract, that stimulates preadipocyte differentiation and acts as a potential insulin action enhancer through the AKT-eNOS pathway in mature adipocytes.

Abstract 2844: In vitro recapitulation of in vivo obesity-promoted colorectal cancer growth using a patient-derived xenograft engineered tumor model

Abstract Strong epidemiological evidence links obesity to an increased risk of colorectal cancer (CRC). However, the precise molecular mechanisms underlying such an association have not been fully elucidated, partly due to the lack of physiologically relevant models. Here, we established a novel PEG-fibrinogen-based engineered tumor model using patient-derived xenograft (PDX) CRC co-cultured with 3T3-L1 adipocytes and an orthotopically implanted PDX CRC model of obesity. The PDX CRC cells were isolated from PDXs propagated subcutaneously in NOD-SCID mice and encapsulated within a biomimetic polymer, PEG-fibrinogen, to create 3D engineered PDX CRC tumors (3DePCCTs) or cultured in a standard 2D manner. We compared cell viability, colony area, and cell subpopulations within the 3DePCCTs and stiffness of the 3DePCCTs to the in vivo propagated tumors. A stage IV CRC tumor was employed in vivo and in vitro to study obesity-promoted tumor growth. Responsiveness of the 3DePCCTs to the growth promoting effects of obesity was investigated through continuous co-culture with insulin sensitive (IS) and resistant (IR) (treated with TNFα and 1% hypoxia) 3T3-L1 adipocytes (adipocytes replaced every 72 hrs). The responsiveness of the stage IV CRC PDX line was validated using an orthotopically implanted CRC model of obesity in which Rag1tm1Mom mice were fed a high fat Western diet + 4% sugar water (HFWD+S) to induce obesity or a chow diet to maintain a lean phenotype. The cells within the 3DePCCTs remained viable and the PDX-line dependent differential growth of tumor colony area within the 3DePPCTs recapitulated line-dependent difference in in vivo tumor growth. Based on flow cytometry, human (70±3%) and Cytokeratin 20+ (31±1) cell subpopulations within the 3DePCCTs were similar to the original in vivo tumor tissue and maintained over time, whereas supporting mouse stromal cells took over 2D cultured cells (n=3 batches). Stiffness of the 3DePCCTs was within the range of the in vivo tumor tissue stiffness (0.3 to3.6 KPa, n=3 tissues). In vitro adipocytes maintained IR for at least 72 hrs based on significantly higher MCP1 (at least 10 folds) and lower GLUT4 (maximally 0.1 fold) (n=3 batches). The in vitro coculture model revealed a significantly (p &amp;lt; 0.05) greater number of cancer cell colonies within 3DePCCTs after 8 days of co-culture with IR adipocytes compared to IS adipocytes and this difference increased through day 29. In the in vivo model, a significant (p &amp;lt; 0.05) greater than 2-fold increase in weight of PDX CRC tumors grown in mice fed the HFWD+S diet was observed. We have established the ability to maintain PDX CRC cells in 3D culture long-term (29 days) and generated a novel in vitro obesity-mimetic engineered tumor model that recapitulated the growth promoting effects of the in vivo orthotopic, IR tumor microenvironment and could be used to examine mechanistic questions and therapeutic targets. Citation Format: Iman Hassani, Benjamin Anbiah, Bulbul Ahmed, Nicole L. Habbit, Michael W. Greene, Elizabeth A. Lipke. In vitro recapitulation of in vivo obesity-promoted colorectal cancer growth using a patient-derived xenograft engineered tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2844.

Cocoa Shell Phenolic Compounds Preserve Mitochondrial Function and Insulin Sensitivity in Adipocytes by Attenuating Their Inflammatory Interplay with Macrophages (FS15-06-19)

ObjectivesThe objective was to evaluate the effect of an aqueous phenolic extract from cocoa shell (CAE) and its main phenolic compounds to prevent the adipogenesis-induced loss of mitochondrial function and insulin sensitivity, targeting the inflammatory interplay macrophages-adipocytes in vitro. MethodsCAE and its main phenolic compounds (protocatechuic acid, procyanidin B2, and epicatechin) were tested in cell culture of RAW264.7 macrophages and 3T3-L1 mature adipocytes. Macrophages were stimulated with lipopolysaccharide (LPS, 1 μg/mL). Adipocytes were treated with the conditioned media (CM) from LPS-stimulated macrophages. Biomarkers for inflammation, mitochondrial function, and insulin sensitivity were determined after 24 h co-treatment with CAE and LPS/CM using chemical and immunochemical techniques. ResultsLPS-stimulated macrophages showed reduced inducible nitric oxide synthase and cyclooxygenase-2 expression and lowered NO and pro-inflammatory cytokines release when treated with CAE and pure phenolics. Inflammatory crosstalk created by stimulating adipocytes with CM was arrested; CAE diminished tumor necrosis factor-α (67%, p < 0.05) and highly promoted adiponectin secretion (12.3-fold, p < 0.05). Mitochondrial function, measured by reactive oxygen species production, mitochondrial content, and activity, was preserved in CM-treated adipocytes through up-regulating peroxisome proliferator-activated receptor gamma coactivator 1-α expression (46%, p < 0.05). Significant (p < 0.05) increases in insulin receptor (9-fold, p < 0.05), phosphoinositide 3-kinase (3-fold, p < 0.05), protein kinase B (4-fold, p < 0.05) phosphorylation and a decrease in insulin receptor substrate 1 serine phosphorylation induced increased glucose uptake (34%, p < 0.05) and glucose transporter 4 translocation (14-fold, p < 0.05) in CM-induced adipocytes. ConclusionsCocoa shell extract polyphenols reduced macrophages-adipocytes inflammatory interaction preventing mitochondrial dysfunction and insulin resistance. For the first time, cocoa shell aqueous extract showed a positive effect on adipogenesis and inflammation-related disorders. Funding SourcesUniversidad Autónoma de Madrid-Banco Santander, United States Department of Agriculture-National Institute of Food and Agriculture-HATCH, and the Spanish Ministry of Science and Innovation.

Fat-Specific Protein 27 Regulation of Vascular Function in Human Obesity.

BackgroundPathophysiological mechanisms that connect obesity to cardiovascular disease are incompletely understood. FSP27 (Fat‐specific protein 27) is a lipid droplet‐associated protein that regulates lipolysis and insulin sensitivity in adipocytes. We unexpectedly discovered extensive FSP27 expression in human endothelial cells that is downregulated in association with visceral obesity. We sought to examine the functional role of FSP27 in the control of vascular phenotype.Methods and ResultsWe biopsied paired subcutaneous and visceral fat depots from 61 obese individuals (body mass index 44±8 kg/m2, age 48±4 years) during planned bariatric surgery. We characterized depot‐specific FSP27 expression in relation to adipose tissue microvascular insulin resistance, endothelial function and angiogenesis, and examined differential effects of FSP27 modification on vascular function. We observed markedly reduced vasodilator and angiogenic capacity of microvessels isolated from the visceral compared with subcutaneous adipose depots. Recombinant FSP27 and/or adenoviral FSP27 overexpression in human tissue increased endothelial nitric oxide synthase phosphorylation and nitric oxide production, and rescued vasomotor and angiogenic dysfunction (P<0.05), while siRNA‐mediated FSP27 knockdown had opposite effects. Mechanistically, we observed that FSP27 interacts with vascular endothelial growth factor‐A and exerts robust regulatory control over its expression. Lastly, in a subset of subjects followed longitudinally for 12±3 months after their bariatric surgery, 30% weight loss improved metabolic parameters and increased angiogenic capacity that correlated positively with increased FSP27 expression (r=0.79, P<0.05).ConclusionsOur data strongly support a key role and functional significance of FSP27 as a critical endogenous modulator of human microvascular function that has not been previously described. FSP27 may serve as a previously unrecognized regulator of arteriolar vasomotor capacity and angiogenesis which are pivotal in the pathogenesis of cardiometabolic diseases linked to obesity.

Effects of a High Fat Meal Challenge with Different Doses of Spice Blend on Postprandial Fatty Acid Suppression (P08-094-19)

ObjectivesHigh fat meals can increase inflammation and plasma triglycerides. Poor suppression of postprandial adipocyte lipolysis results in higher levels of non-esterified fatty acids (NEFA) in insulin resistant subjects. This impaired suppression of NEFA may exacerbate hypertriglyceridemia following a high fat meal by increasing available fatty acids. We have shown myristic acid (MA) and stearic acid (SA) are less suppressed than other fatty acids 1 hour following a glucose challenge, and this pattern may indicate optimal adipocyte insulin sensitivity. It is unknown if the same pattern occurs in other contexts, such as high fat meal. MethodsPlasma samples were collected from 12 obese male subjects at baseline and 4 time points following a high fat meal (1076 kcal, 39% kcal from saturated fat) on three visits. Meals contained either 0, 2, or 6 grams of a spice blend. Plasma was analyzed by GC-MS to measure multiple fatty acids, including MA, SA, palmitic acid (PA), oleic acid (OA), and linoleic acid (LA). ResultsAt 1 hour following the high fat meal, MA and SA were less suppressed than OA, PA, and LA (P < 0.0001). Total NEFA concentrations were most suppressed (61%; CI: 46%, 76%) at 2 hours following the meal and remained suppressed until 4 hours. PA (59%, P < 0.0001), SA (43%, P < 0.001), OA (68%, P < 0.0001), and LA (71%, P < 0.0001) also achieved maximal suppression at 2 hours and remained suppressed. MA was suppressed (34%, P = 0.0002) at 1 hour, then returned to baseline. Compared to baseline, all saturated fatty acids increased as % of total NEFA at 2 hours (MA: 0 hr, 1.8%, 2 hr, 3.7%, P < 0.0001; PA: 0 hr, 24%, 2 hr, 26%, P = 0.009; SA: 0 hr, 8%, 2 hr, 12%, P < 0.0001), while the unsaturated fatty acids decreased (OA: 0 hr, 33%, 2 hr, 25%, P < 0.0001; LA: 0 hr, 24%, 2 hr, 19%, P < 0.0001). There was no significant effect of spice. ConclusionsNEFA was most suppressed at 2 hours following a high fat meal challenge, but MA and SA were suppressed less than all other fatty acids. Saturated fatty acids increased as a % of total NEFA following the meal challenge, while unsaturated fatty acids decreased. These data support our previous findings that MA and SA are less suppressed by insulin than other fatty acids. Funding SourcesMcCormick Science Institute; Penn State Department of Nutritional Sciences; National Center for Advancing Translational Sciences, NIH, (UL1 TR002014).

Chemical Inducers of Obesity-Associated Metabolic Stress Activate Inflammation and Reduce Insulin Sensitivity in 3T3-L1 Adipocytes.

Obesity is accompanied by dyslipidemia, hypoxia, endoplasmic reticulum (ER) stress, and inflammation, representing the major risk factor for the development of insulin resistance (IR) and type 2 diabetes. We modeled these conditions in cultured 3T3-L1 adipocytes and studied their effect on insulin signaling, glucose uptake, and inflammatory response via activation of stress-dependent JNK1/2 kinases. Decreased insulin-induced phosphorylation of the insulin cascade components IRS, Akt, and AS160 was observed under all tested conditions (lipid overloading of cells by palmitate, acute inflammation induced by bacterial lipopolysaccharide, hypoxia induced by Co2+, and ER stress induced by brefeldin A). In all the cases, except the acute inflammation, glucose uptake by adipocytes was reduced, and the kinetics of JNK1/2 activation was bi-phasic exhibiting sustained activation for 24 h. By contrast, in acute inflammation, JNK1/2 phosphorylation increased transiently and returned to the basal level within 2-3 h of stimulation. These results suggest a critical role of sustained (latent) vs. transient (acute) inflammation in the induction of IR and impairment of glucose utilization by adipose tissue. The components of the inflammatory signaling can be promising targets in the development of new therapeutic approaches for preventing IR and type 2 diabetes.

SAT-002 Measurement of Visceral Fat Using Computed Tomography in Two Subtypes of Primary Aldosteronism

Context: A strong association between excess visceral fat and the presence of metabolic factors, hypertension and type 2 diabetes have been demonstrated. Patients with primary aldosteronism (PA) are complicated by metabolic syndrome more frequently than those without PA. Aldosterone-induced mineralocorticoid receptor activation has been reported to impair insulin sensitivity in adipocytes and skeletal muscle. An excess of visceral fat has been hypothesized to cause an elevation of aldosterone secretion, and to cause insulin resistance in patients with PA. Objectives: To clarify the role of visceral fat in the pathophysiology of PA, we investigated the correlation of visceral fat parameters with plasma aldosterone concentration (PAC), and the correlation of visceral fat parameters with homeostasis model assessment insulin resistance (HOMA-R) in patients with PA. Materials and methods: This retrospective observational study comprised 131 patients diagnosed with PA between 2007 and 2017 at Sapporo City General Hospital. We divided participants into two subtypes, aldosterone producing adenoma (APA, n = 47) and idiopathic hyperaldosteronism (IHA, n = 84), utilizing adrenal venous sampling. We excluded patients with suspected autonomous cortisol secretion, defined as serum cortisol levels ≧ 3 μg/dL after a 1-mg dexamethasone suppression test. We analyzed the correlations of visceral fat percentage (VF%), visceral fat area (VFA) evaluated by computed tomography studies and PAC, and the correlations of VF% and VFA with HOMA-R in each subtype group. Results: Age and sex distribution was not different between patients with APA and patients with IHA (p = 0.85, p = 0.18, respectively). PAC was significantly higher in patients with APA than patients with IHA (p < 0.001). Serum potassium levels were significantly lower in patients with APA than patients with IHA (p < 0.001). Body mass index was not different between these two subtypes (p = 0.87). VF% was significantly higher in patients with IHA than in those with APA (p = 0.02). The number of patients treated as type 2 diabetes mellitus with oral medicine, HbA1c, and HOMA-R were not different between these two subtypes (p =0.76, p =0.22, p = 0.12, respectively). Patients with IHA showed a positive correlation of VF% with PAC (r = 0.377, p < 0.001), and VFA with PAC (r = 0.443, p < 0.001). In contrast, patients with APA showed no significant correlation of VF% and VFA with PAC. Patients with APA and patients with IHA showed a positive correlation of VF% and VFA with HOMA-R. Conclusions: Between two subtypes of PA, the association of visceral fat on insulin resistance was not different. The association of visceral fat on PAC was different in two subtypes of PA. Only in patients with IHA, visceral fat may play a role in regulation of aldosterone secretion.

Adipocyte alterations in endometriosis: reduced numbers of stem cells and microRNA induced alterations in adipocyte metabolic gene expression

BackgroundEndometriosis is an estrogen dependent, inflammatory disorder occurring in 5–10% of reproductive-aged women. Women with endometriosis have a lower body mass index (BMI) and decreased body fat compared to those without the disease, yet few studies have focused on the metabolic abnormalities in adipose tissue in women with endometriosis. Previously, we identified microRNAs that are differentially expressed in endometriosis and altered in the serum of women with the disease. Here we explore the effect of endometriosis on fat tissue and identified a role for endometriosis-related microRNAs in fat metabolism and a reduction in adipocyte stem cell number.MethodsPrimary adipocyte cells cultured from 20 patients with and without endometriosis were transfected with mimics and inhibitors of microRNAs 342-3p or Let 7b-5p to model the status of these microRNAs in endometriosis. RNA was extracted for gene expression analysis by qRT-PCR. PCNA expression was used as a marker of adipocyte proliferation. Endometriosis was induced experimentally in 9-week old female C57BL/6 mice and after 10 months fat tissue was harvested from both the subcutaneous (inguinal) and visceral (mesenteric) tissue. Adipose-derived mesenchymal stem cells in fat tissue were characterized in both endometriosis and non-endometriosis mice by FACS analysis.ResultsGene expression analysis showed that endometriosis altered the expression of Cebpa, Cebpb, Ppar-γ, leptin, adiponectin, IL-6, and HSL, which are involved in driving brown adipocyte differentiation, appetite, insulin sensitivity and fat metabolism. Each gene was regulated by an alteration in microRNA expression known to occur in endometriosis. Analysis of the stem cell content of adipose tissue in a mouse model of endometriosis demonstrated a reduced number of adipocyte stem cells.ConclusionsWe demonstrate that microRNAs Let-7b and miR-342-3p affected metabolic gene expression significantly in adipocytes of women with endometriosis. Similarly, there is a reduction in the adipose stem cell population in a mouse model of endometriosis. Taken together these data suggest that endometriosis alters BMI in part through an effect on adipocytes and fat metabolism.

Relationship of phenolic composition of selected purple maize (Zea mays L.) genotypes with their anti-inflammatory, anti-adipogenic and anti-diabetic potential

This study aimed to investigate the associations between phenolic composition of selected purple maize genotypes and their anti-inflammatory, anti-adipogenic and anti-diabetic properties in vitro. Anthocyanin-rich water extracts (PMWs) from 20 purple maize genotypes were evaluated in RAW 264.7 macrophages and 3T3-L1 adipocytes under different conditions. Cyanidin-3-O-glucoside (C3G), pelargonidin-3-O-glucoside (Pr3G), peonidin-3-O-glucoside (P3G) and corresponding acylated forms were major anthocyanins in PMW, accompanied by ten tentatively identified non-anthocyanin phenolics. Correlation studies showed that C3G, P3G, and derivatives, but not Pr3G and its acylated form contributed to the biological properties of PMW. Besides anthocyanins, quercetin, luteolin, and rutin were the dominant anti-inflammatory and anti-diabetic components, in terms of down-regulating pro-inflammatory mediator production in inflamed macrophages and adipocytes, modulating diabetes-related key enzymes and improving insulin sensitivity in insulin-resistant adipocytes. Quercetin and phenolic acids, especially vanillic acid and protocatechuic acid, were closely associated with anti-adipogenic properties of PMW via inhibition of the preadipocyte-adipocyte transition.

Two nights of recovery sleep restores the dynamic lipemic response, but not the reduction of insulin sensitivity, induced by five nights of sleep restriction.

Chronic inadequate sleep is associated with increased risk of cardiometabolic diseases. The mechanisms involved are poorly understood but involve changes in insulin sensitivity, including within adipose tissue. The aim of this study was to assess the effects of sleep restriction on nonesterified fatty acid (NEFA) suppression profiles in response to an intravenous glucose tolerance test (IVGTT) and to assess whether 2 nights of recovery sleep (a "weekend") is sufficient to restore metabolic health. We hypothesized that sleep restriction impairs both glucose and lipid metabolism, specifically adipocyte insulin sensitivity, and the dynamic lipemic response of adipocyte NEFA release during an IVGTT. Fifteen healthy men completed an inpatient study of 3 baseline nights (10 h of time in bed/night), followed by 5 nights of 5 h of time in bed/night and 2 recovery nights (10 h of time in bed/night). IVGTTs were performed on the final day of each condition. Reductions in insulin sensitivity without a compensatory change in acute insulin response to glucose were consistent with prior studies (insulin sensitivity P = 0.002; acute insulin response to glucose P = 0.23). The disposition index was suppressed by sleep restriction and did not recover after recovery sleep (P < 0.0001 and P = 0.01, respectively). Fasting NEFAs were not different from baseline in either the restriction or recovery conditions. NEFA rebound was significantly suppressed by sleep restriction (P = 0.01) but returned to baseline values after recovery sleep. Our study indicates that sleep restriction impacts NEFA metabolism and demonstrates that 2 nights of recovery sleep may not be adequate to restore glycemic health.

Early Changes in Adipose Tissue Morphology, Gene Expression, and Metabolism After RYGB in Patients With Obesity and T2D.

Roux-en-Y gastric bypass (RYGB) surgery effectively prevents or treats type 2 diabetes (T2D). Adipose tissue (AT) mechanisms may be of importance. To assess the relationship between early changes in whole-body and AT metabolism in surgically treated patients with T2D. A randomized single-center study. Nineteen patients with T2D with body mass index 30 to 45 kg/m2. Thirteen patients were assessed at baseline and 4 and 24 weeks after RYGB (preceded by a 4-week low-calorie diet) and compared with 6 control patients continuing standard medical treatment: oral glucose tolerance test, subcutaneous AT biopsies for gene expression, adipocyte size, glucose uptake, lipolysis, and insulin action. At 4 and 24 weeks post-RYGB, all patients but one had stopped diabetes medication. Fasting glucose, HbA1c, and insulin levels decreased and the Matsuda index increased compared with baseline (P < 0.01 for all), indicating improved whole-body insulin sensitivity. Mean adipocyte size significantly reduced, more at 4 than at 24 weeks; at 4 weeks, glucose uptake per adipocyte was lowered, and isoproterenol-stimulated lipolysis tended to increase, whereas the fold insulin effects on glucose uptake and lipolysis were unchanged. Expression of genes involved in fatty acid oxidation, CPT1b and adiponectin, was increased at 4 weeks, whereas leptin and E2F1 (involved in cell proliferation) were reduced (P < 0.05 for all). Glycemic control and in vivo insulin sensitivity improved 4 weeks after RYGB, but adipocyte insulin sensitivity did not change despite a marked reduction in adipocyte size. Thus, mechanisms for a rapid improvement of T2D after RYGB may occur mainly in other tissues than adipose.

Combating Insulin Resistance Using Medicinal Plants as a Supplementary Therapy to Metformin in 3T3-L1 Adipocytes: Improving Early Intervention-Based Diabetes Treatment

One in eleven people will have a form of diabetes during their lifetime (1). In 2012, diabetes and its life-threatening side effects cost 245 billion dollars in the United States (2). Averting type 2 diabetes (T2D) will significantly reduce health care costs and engender a healthier population. A primary cause of diabetes is insulin resistance, which is caused by disruption of insulin signal transduction. The objective was to maximize insulin sensitivity by creating a more effective, early intervention-based treatment to avert severe T2D. This treatment combined metformin, “the insulin sensitizer”, and medicinal plants, curcumin, fenugreek, and nettle. To conduct this study, insulin resistance was induced using free fatty acids (FFAs). Insulin sensitivity in adipocytes (propagated and differentiated from 3T3-L1 fibroblasts) was measured using assays specific for leptin concentration, glucose uptake, and Akt phosphorylation. These three proteins/pathways served as quantitative measurements for insulin sensitivity, since they are essential to insulin signaling. It was hypothesized that a dual therapy would maximize insulin sensitivity. This hypothesis was supported as each plant-enhanced combination treatment attenuated the effects of FFAs, increasing and reversing each biological marker of functional insulin signaling to normal function. This indicates that each treatment has the potential to combat obesity, augment glucose uptake, and heighten Akt/PI3K pathway function. This study, therefore, offers an improved multifunctional early intervention-based diabetes treatment that maximizes insulin sensitivity.

Niacin induces miR-502-3p expression which impairs insulin sensitivity in human adipocytes.

MicroRNAs have been involved in insulin resistance (IR). As the mechanism whereby niacin, an anti-dyslipidemic agent, leads to IR remains elusive, we sought to identify differentially expressed microRNAs in adipose tissue (AT) of individuals receiving niacin and to explore the link between microRNAs, niacin and IR in human adipocytes.In a double-blind controlled study, 22 obese men received extended-release niacin or placebo over 8 weeks. Bioclinical data and subcutaneous AT biopsies were obtained before and after treatment. AT microRNA expression profiles were determined using RTqPCR for 758 human-specific microRNAs. hMADS adipocytes were treated with niacin, or acipimox (a niacin-like drug without effect on IR), or transfected with miR-502-3p. Glucose uptake and Western blotting were performed.In obese men, insulin sensitivity decreased after niacin treatment. In AT, the expression of 6 microRNAs including miR-502-3p was up-regulated. Treatment of hMADS adipocytes with niacin specifically increased miR-502-3p expression. Acipimox had no effect. Overexpression of miR-502-3p in adipocytes led to reduced insulin-induced glucose uptake and lower insulin-stimulated AKT phosphorylation.Long term niacin treatment altered microRNA expression levels in human AT. Increased miR-502-3p expression may play a role in the mediation of IR due to niacin in adipocytes.The study is registered in Clinical Trials NCT01083329 and EudraCT 2009-012124-85.

Chungkookjang with High Contents of Poly-γ-Glutamic Acid Improves Insulin Sensitizing Activity in Adipocytes and Neuronal Cells.

We hypothesized that soybeans fermented with Bacillus spp. for 48 h (chungkookjang) would be rich in poly-γ-glutamate (γ-PGA) and would have greater efficacy for improving insulin sensitivity and insulin secretion in 3T3-L1 adipocytes, min6 cells, and PC12 neuronal cells. We screened 20 different strains of B. subtillus and B. amyloliquefaciens spp. for γ-polyglutamate (PGA) production and their anti-diabetic and anti-dementia activities in cell-based studies. Chungkookjang made with two B. amyloliquefaciens spp. (BA730 and BA731) were selected to increase the isoflavonoid and γ-PGA. Insulin-stimulated glucose uptake was higher in 3T3-L1 adipocytes given both chungkookjang extracts than in the cells given vehicle (control). The ethanol extract of BA731 (BA731-E) increased the uptake the most. Triglyceride accumulation decreased in BA731-E and BA731-W and the accumulation increased in BA730-W and BA730-E. The mRNA expression of fatty acid synthetase and acetyl CoA carboxylase was much lower in BA731-E and BA731-W and it was higher in BA730-W than the control. BA730-E and BA730-W also increased peroxisome proliferator-activated receptor (PPAR)-γ activity. Glucose-stimulated insulin secretion increased with the high dosage of BA730-W and BA730-E in insulinoma cells, compared to the control. Insulin contents and cell survival in high glucose media were higher in cells with both BA731-E and BA730-E. Triglyceride deposition and TNF-α mRNA expression were lower in BA731 than the control. The high-dosage treatment of BA730-E and BA731-E increased differentiated neuronal cell survival after treating amyloid-β(25-35) compared to the control. Brain-derived neurotrophic factor and ciliary neurotrophic factor, indices of neuronal cell proliferation, were higher in BA730 and BA731 than in the control. Tau expression was also reduced in BA731 more than the control and it was a similar level of the normal-control. In conclusion, BA730 increased PPAR-γ activity and BA731 enhanced insulin sensitivity in the brain and periphery. BA730 and BA731 prevented and alleviated the symptoms of type 2 diabetes and Alzheimer’s disease with different pathways.

GLP-1 Improves Adipocyte Insulin Sensitivity Following Induction of Endoplasmic Reticulum Stress.

Glucagon-like peptide 1 (GLP-1) improves insulin resistance of adipose tissue in obese humans. However, the mechanism of this effect is unclear. Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin signaling. We hypothesized that GLP-1 could directly improve insulin signaling in ER-stressed adipocytes. Here, we examined the effects of GLP-1 on ER stress response in fat cells in an obese and insulin-resistant murine model. We found that GLP-1 analog liraglutide reduced ER stress related gene expression in visceral fat cells accompanied by improved systemic insulin tolerance. Consistently, GLP-1 decreased CHOP expression and increased insulin stimulated AKT phosphorylation (p-AKT) in thapsigargin, a ER stress inducer, treated white fat cells differentiated from visceral stromal vascular fraction. We further found blocking CHOP expression increased insulin stimulated p-AKT in ER-stressed fat cells. Of note, we found mTOR signaling pathway contributed to the expression of ATF4 and subsequently the CHOP expression in ER stress response, while GLP-1 inhibited mTOR activity as exemplified by elevated autophagosome formation and increased LC3II/LC3I ratio. These findings suggest that GLP-1 directly modulates the ER stress response partially via inhibiting mTOR signaling pathway, leading to increased insulin sensitivity in adipocytes.

The IRE1\u03b1-XBP1s pathway promotes insulin-stimulated glucose uptake in adipocytes by increasing PPAR\u03b3 activity

The peroxisome proliferator-activated receptor-γ (PPARγ) improves whole-body insulin sensitivity by regulating the adipogenic and metabolic functions of mature adipocytes. We have previously demonstrated that an active splice variant of X-box binding protein 1 (XBP1s) enhances PPARγ expression during adipogenesis. In this study, we investigated the role of XBP1s, particularly with respect to PPARγ, in the mechanisms underlying insulin sensitivity in mature adipocytes. Insulin was able to stimulate XBP1s generation by activating inositol-requiring enzyme 1 (IRE1) α and was also able to increase its transcriptional activity by inducing nuclear translocation. XBP1s also upregulated the levels of phosphorylated IRS1 and AKT, demonstrating a positive feedback regulatory mechanism linking insulin and XBP1s. XBP1s enhanced the expression of fibroblast growth factor 21 and, in turn, increased PPARγ activity, translocation of GLUT4 to the cell surface, and glucose uptake rate in adipocytes. In addition, XBP1s abolished palmitate-induced insulin resistance in adipocytes by increasing adiponectin secretion, repressing the secretion of pro-inflammatory adipokines such as leptin, monocyte chemoattractant protein 1, and tumor necrosis factor α, and decreasing fatty acid release. These findings provide a novel mechanism by which XBP1s stimulate insulin sensitivity in adipocytes through fibroblast growth factor 21 induction and PPARγ activation.

Modulation of the Inflammatory Status of Macrophages and Their Paracrine Effect on the Sensitivity of Adipocytes to Insulin with Sirtuin and PPARγ Receptor Activators.

We studied the effect of SIRT1 deacetylase and PPARγ receptor activators on proinflammatory (M1), anti-inflammatory (M2) polarization of RAW264.7 macrophages and their modulating effects on insulin sensitivity of adipocytes. In M1 macrophages, the expression of TNFα and CXCL9, secretion of CXCL11, ROS generation, and content of dendritic-like cells were elevated. In M2 macrophages, expression of IGF-1 and ALOX15 factors was enhanced. SIRT1 activator (DCHC) and PPARγ receptor ligand (rosiglitazone) reduced expression of inflammatory markers TNFα and CXCL9 and increased expression of IGF-1 and ALOX15. SIRT1 inhibitor Ex527 increased the proportion of dendritic cells in macrophage populations. The paracrine effect of M1-macrophage-conditioned media attenuated insulin-dependent phosphorylation of threonine (Thr308) in Akt kinase and enhanced phosphorylation of serine (Ser473). This effect was attenuated by DCHC and rosiglitazone.

Re-highlighting the action of PPARγ in treating metabolic diseases.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor family and plays an important role in adipocyte differentiation, glucose homeostasis, and insulin sensitivity. Thiazolidinediones (TZDs), synthetic ligands of PPARγ, have been used for the treatment of diabetes mellitus for two decades. TZDs were expected to be amazing drugs not only for type 2 diabetes but also for metabolic syndrome and atherosclerotic vascular disease because they can reduce both insulin resistance and inflammation in experimental studies. However, serious unwanted effects pushed TZDs back to an optional second-tier drug for type 2 diabetes. Nevertheless, PPARγ is still one of the most important targets for the treatment of insulin resistance and diabetes mellitus, and novel strategies to modulate PPARγ activity to enhance its beneficial effects and reduce unwanted adverse effects are anticipated. Recent studies showed that post-translational modification (PTM) of PPARγ regulates PPARγ activity or stability and may be a novel way to optimize PPARγ activity with reduced adverse effects. In this review, we will focus on recent advances in PTM of PPARγ and the mechanisms regulating PPARγ function as well as in the development of PPARγ modulators or agonists.