Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.     

Macrophage α1 AMP-activated Protein Kinase (α1AMPK) Antagonizes Fatty Acid-induced Inflammation through SIRT1

In this study, we aim to determine cellular mechanisms linking nutrient metabolism to the regulation of inflammation and insulin resistance. The nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 show striking similarities in nutrient sensing and regulation of metabolic pathways. We find that the expression, activity, and signaling of the major isoform α1AMPK in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli and in nutrient-rich conditions, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFAs), and diet-induced obesity. Activating AMPK signaling in macrophages by 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside or constitutively active α1AMPK (CA-α1) significantly inhibits; although inhibiting α1AMPK by short hairpin RNA knock-down or dominant-negative α1AMPK (DN-α1) increases LPS- and FFA-induced tumor necrosis factor α expression. Chromatin immunoprecipitation and luciferase reporter assays show that activation of AMPK by CA-α1 in macrophages significantly inhibits LPS- or FFA-induced NF-κB signaling. More importantly, in a macrophage-adipocyte co-culture system, we find that inactivation of macrophage AMPK signaling inhibits adipocyte insulin signaling and glucose uptake. Activation of AMPK by CA-α1 increases the SIRT1 activator NAD⁺ content and SIRT1 expression in macrophages. Furthermore, α1AMPK activation mimics the effect of SIRT1 on deacetylating NF-κB, and the full capacity of AMPK to deacetylate NF-κB and inhibit its signaling requires SIRT1. In conclusion, AMPK negatively regulates lipid-induced inflammation, which acts through SIRT1, thereby contributing to the protection against obesity, inflammation, and insulin resistance. Our study defines a novel role for AMPK in bridging the signaling between nutrient metabolism and inflammation.     

Interferon �� Attenuates Insulin Signaling, Lipid Storage, and Differentiation in Human Adipocytes via Activation of the JAK/STAT Pathway

Recent reports demonstrate T-cell infiltration of adipose tissue in early obesity. We hypothesized that interferon (IFN) γ, a major T-cell inflammatory cytokine, would attenuate human adipocyte functions and sought to establish signaling mechanisms. Differentiated human adipocytes were treated with IFNγ ± pharmacological inhibitors prior to insulin stimulation. [³H]Glucose uptake and AKT phosphorylation were assessed as markers of insulin sensitivity. IFNγ induced sustained loss of insulin-stimulated glucose uptake in human adipocytes, coincident with reduced Akt phosphorylation and down-regulation of the insulin receptor, insulin receptor substrate-1, and GLUT4. Loss of adipocyte triglyceride storage was observed with IFNγ co-incident with reduced expression of peroxisome proliferator-activated receptor γ, adiponectin, perilipin, fatty acid synthase, and lipoprotein lipase. Treatment with IFNγ also blocked differentiation of pre-adipocytes to the mature phenotype. IFNγ-induced robust STAT1 phosphorylation and SOCS1 mRNA expression, with modest, transient STAT3 phosphorylation and SOCS3 induction. Preincubation with a non-selective JAK inhibitor restored glucose uptake and Akt phosphorylation while completely reversing IFNγ suppression of adipogenic mRNAs and adipocyte differentiation. Specific inhibition of JAK2 or JAK3 failed to block IFNγ effects suggesting a predominant role for JAK1-STAT1. We demonstrate that IFNγ attenuates insulin sensitivity and suppresses differentiation in human adipocytes, an effect most likely mediated via sustained JAK-STAT1 pathway activation.     

Glucagon-Like Peptide-1 Induced Signaling and Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent Pancreatic β-Cells

Background

Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic β-cells, in particular cAMP, Ca²⁺ and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors.

Methodology/Prinicipal Findings

GLP-1 or Ex-4 at high glucose caused release (∼20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on β-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca²⁺]_i and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered.

Conclusions/Significance

The results indicate that GLP-1 barely affects β-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the β-cell, and that the β-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a “push” (fuel substrate driven) process, rather than a “pull” mechanism secondary to enhanced insulin release as well as to Ca²⁺, cAMP and PKB signaling.     

Tissue-specific role of glycogen synthase kinase 3beta in glucose homeostasis and insulin action

Dysregulation of the protein kinase glycogen synthase kinase 3 (GSK-3) has been implicated in the development of type 2 diabetes mellitus. GSK-3 protein expression and kinase activity are elevated in diabetes, while selective GSK-3 inhibitors have shown promise as modulators of glucose metabolism and insulin sensitivity. There are two GSK-3 isoforms in mammals, GSK-3α and GSK-3β. Mice engineered to lack GSK-3β die in late embryogenesis from liver apoptosis, whereas mice engineered to lack GSK-3α are viable and exhibit improved insulin sensitivity and hepatic glucose homeostasis. To assess the potential role of GSK-3β in insulin function, a conditional gene-targeting approach whereby mice in which expression of GSK-3β was specifically ablated within insulin-sensitive tissues were generated was undertaken. Liver-specific GSK-3β knockout mice are viable and glucose and insulin tolerant and display “normal” metabolic characteristics and insulin signaling. Mice lacking expression of GSK-3β in skeletal muscle are also viable but, in contrast to the liver-deleted animals, display improved glucose tolerance that is coupled with enhanced insulin-stimulated glycogen synthase regulation and glycogen deposition. These data indicate that there are not only distinct roles for GSK-3α and GSK-3β within the adult but also tissue-specific phenotypes associated with each of these isoforms.     

Lipocalin 2 Expression and Secretion Is Highly Regulated by Metabolic Stress,Cytokines, and Nutrients in Adipocytes

Lipocalin 2 (Lcn2) has been recently characterized as a new adipokine having a role in innate immunity and energy metabolism. Nonetheless, the metabolic regulation of Lcn2 production in adipocytes has not been comprehensively studied. To better understand the Lcn2 biology, we investigated the regulation of Lcn2 expression in adipose tissue in response to metabolic stress in mice as well as the control of Lcn2 expression and secretion by cytokines and nutrients in 3T3-L1 adipocytes. Our results showed that the mRNA expression of Lcn2 was upregulated in white and brown adipose tissues as well as liver during fasting and cold stress in mice. Among pro-inflammatory cytokines TNFα, IL-1β, and IL-6, IL-1β showed most profound effect on Lcn2 expression and secretion in 3T3-L1 adipocytes. Insulin stimulated Lcn2 expression and secretion in a dose-dependent manner; this insulin effect was significantly abolished in the presence of low concentration of glucose. Moreover, insulin-stimulated Lcn2 expression and secretion was also attenuated when glucose was replaced by 3-O-methyl-d-glucose or by blocking NFκB pathway activation. Additionally, we showed that palmitate and oleate induced Lcn2 expression and secretion more significantly than EPA, while phytanic acid reduced Lcn2 production. Our results demonstrated that Lcn2 production in adipocytes is highly responsive to metabolic stress, cytokines, and nutrient signals, suggesting an important role of Lcn2 in adipocyte metabolism and inflammation.     

A Novel Peroxisome Proliferator-activated Receptor (PPAR)α Agonist and PPARγ Antagonist,Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice

A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia.     

Regulation of adipogenic differentiation and adipose tissue inflammation by interferon regulatory factor 3

Dysfunction of adipocytes and adipose tissue is a primary defect in obesity and obesity-associated metabolic diseases. Interferon regulatory factor 3 (IRF3) has been implicated in adipogenesis. However, the role of IRF3 in obesity and obesity-associated disorders remains unclear. Here, we show that IRF3 expression in human adipose tissues is positively associated with insulin sensitivity and negatively associated with type 2 diabetes. In mouse pre-adipocytes, deficiency of IRF3 results in increased expression of PPARγ and PPARγ-mediated adipogenic genes, leading to increased adipogenesis and altered adipocyte functionality. The IRF3 knockout (KO) mice develop obesity, insulin resistance, glucose intolerance, and eventually type 2 diabetes with aging, which is associated with the development of white adipose tissue (WAT) inflammation. Increased macrophage accumulation with M1 phenotype which is due to the loss of IFNβ-mediated IL-10 expression is observed in WAT of the KO mice compared to that in wild-type mice. Bone-marrow reconstitution experiments demonstrate that the nonhematopoietic cells are the primary contributors to the development of obesity and both hematopoietic and nonhematopoietic cells contribute to the development of obesity-related complications in IRF3 KO mice. This study demonstrates that IRF3 regulates the biology of multiple cell types including adipocytes and macrophages to prevent the development of obesity and obesity-related complications and hence, could be a potential target for therapeutic interventions for the prevention and treatment of obesity-associated metabolic disorders.Subject terms: Interferons, Preclinical research     

Metabolomics Analysis Reveals that AICAR Affects Glycerolipid,Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells

AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using ¹³C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.     

Direct Angiotensin II Type 2 Receptor Stimulation Ameliorates Insulin Resistance in Type 2 Diabetes Mice with PPARγ Activation

Objectives

The role of angiotensin II type 2 (AT₂) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT₂ receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue.

Methods

T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[³H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined.

Results

Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored β cell damage in diabetic pancreatic tissue.

Conclusion

The present study demonstrated that direct AT₂ receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic β cells.     

Effects of a high-salt diet on adipocyte glucocorticoid receptor and 11-β hydroxysteroid dehydrogenase 1 in salt-sensitive hypertensive rats

High-salt diets decrease insulin sensitivity in salt-sensitive hypertensive rats, and glucocorticoids promote adipocyte growth and may have pathophysiological roles in the metabolic syndrome. The aim of this study was to clarify the relationship between high-salt diet and the adipocyte glucocorticoid hormones in salt-sensitive hypertensive rats. Six-week-old Dahl salt-sensitive (DS) hypertensive rats and salt-resistant (DR) rats were fed a high-salt diet or a normal-salt diet for 4 weeks. Fasting blood glucose (FBG), serum adiponectin, plasma insulin, and corticosterone in plasma and in visceral adipose tissues, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activities in adipose tissues and glucose uptake in isolated muscle were measured. Animals underwent an oral glucose tolerance test (OGTT). The expression of mRNA for glucocorticoid receptor (GR), 11β-HSD1 and tumor necrosis factor-α (TNF-α) in adipose tissues were measured using a real-time PCR. A high-salt diet did not influence FBG; however, decreased 2-deoxy glucose uptake and plasma insulin during OGTT in DS rats. The high-salt diet increased significantly adipose tissue corticosterone concentration and 11β-HSD1 activities, gene expression for GR, 11β-HSD1 and TNF-α in adipose tissues in DS rats compared with DR rats (p < 0.05). The high-salt diet did not influence plasma corticosterone and serum adiponectin concentration in DS and DR rats. These results suggest that changes in GR and 11β-HSD1 in adipose tissue may contribute to insulin sensitivity in salt-sensitive hypertensive rats.     

The Gustatory Signaling Pathway and Bitter Taste Receptors Affect the Development of Obesity and Adipocyte Metabolism in Mice

Intestinal chemosensory signaling pathways involving the gustatory G-protein, gustducin, and bitter taste receptors (TAS2R) have been implicated in gut hormone release. Alterations in gut hormone profiles may contribute to the success of bariatric surgery. This study investigated the involvement of the gustatory signaling pathway in the development of diet-induced obesity and the therapeutic potential of targeting TAS2Rs to induce body weight loss. α-gustducin-deficient (α-gust^-/-) mice became less obese than wild type (WT) mice when fed a high-fat diet (HFD). White adipose tissue (WAT) mass was lower in α-gust^-/- mice due to increased heat production as a result of increases in brown adipose tissue (BAT) thermogenic activity, involving increased protein expression of uncoupling protein 1. Intra-gastric treatment of obese WT and α-gust^-/- mice with the bitter agonists denatonium benzoate (DB) or quinine (Q) during 4 weeks resulted in an α-gustducin-dependent decrease in body weight gain associated with a decrease in food intake (DB), but not involving major changes in gut peptide release. Both WAT and 3T3-F442A pre-adipocytes express TAS2Rs. Treatment of pre-adipocytes with DB or Q decreased differentiation into mature adipocytes. In conclusion, interfering with the gustatory signaling pathway protects against the development of HFD-induced obesity presumably through promoting BAT activity. Intra-gastric bitter treatment inhibits weight gain, possibly by directly affecting adipocyte metabolism.     

Identification and validation of the pathways and functions regulated by the orphan nuclear receptor,ROR alpha1, in skeletal muscle

The retinoic acid receptor-related orphan receptor (ROR) alpha has been demonstrated to regulate lipid metabolism. We were interested in the RORα1 dependent physiological functions in skeletal muscle. This major mass organ accounts for ∼40% of the total body mass and significant levels of lipid catabolism, glucose disposal and energy expenditure. We utilized the strategy of targeted muscle-specific expression of a truncated (dominant negative) RORα1ΔDE in transgenic mice to investigate RORα1 signaling in this tissue. Expression profiling and pathway analysis indicated that RORα influenced genes involved in: (i) lipid and carbohydrate metabolism, cardiovascular and metabolic disease; (ii) LXR nuclear receptor signaling and (iii) Akt and AMPK signaling. This analysis was validated by quantitative PCR analysis using TaqMan low-density arrays, coupled to statistical analysis (with Empirical Bayes and Benjamini–Hochberg). Moreover, westerns and metabolic profiling were utilized to validate the genes, proteins and pathways (lipogenic, Akt, AMPK and fatty acid oxidation) involved in the regulation of metabolism by RORα1. The identified genes and pathways were in concordance with the demonstration of hyperglycemia, glucose intolerance, attenuated insulin-stimulated phosphorylation of Akt and impaired glucose uptake in the transgenic heterozygous Tg-RORα1ΔDE animals. In conclusion, we propose that RORα1 is involved in regulating the Akt2-AMPK signaling pathways in the context of lipid homeostasis in skeletal muscle.     

Adipocytokine Orosomucoid Integrates Inflammatory and Metabolic Signals to Preserve Energy Homeostasis by Resolving Immoderate Inflammation

Orosomucoid (ORM), also called α-1 acid glycoprotein, is an abundant plasma protein that is an immunomodulator induced by stressful conditions such as infections. In this study, we reveal that Orm is induced selectively in the adipose tissue of obese mice to suppress excess inflammation that otherwise disturbs energy homeostasis. Adipose Orm levels were elevated by metabolic signals, including insulin, high glucose, and free fatty acid, as well as by the proinflammatory cytokine tumor necrosis factor-α, which is found in increased levels in the adipose tissue of morbid obese subjects. In both adipocytes and macrophages, ORM suppressed proinflammatory gene expression and pathways such as NF-κB and mitogen-activated protein kinase signalings and reactive oxygen species generation. Concomitantly, ORM relieved hyperglycemia-induced insulin resistance as well as tumor necrosis factor-α-mediated lipolysis in adipocytes. Accordingly, ORM improved glucose and insulin tolerance in obese and diabetic db/db mice. Taken together, our results suggest that ORM integrates inflammatory and metabolic signals to modulate immune responses to protect adipose tissue from excessive inflammation and thereby from metabolic dysfunction.     

Pancreatic Insulin Content Regulation by the Estrogen Receptor ERα

The function of pancreatic β-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ERα and ERβ, are important molecules involved in glucose metabolism, yet their role in pancreatic β-cell physiology is still greatly unknown. In this report we show that both ERα and ERβ are present in pancreatic β-cells. Long term exposure to physiological concentrations of 17β-estradiol (E2) increased β-cell insulin content, insulin gene expression and insulin release, yet pancreatic β-cell mass was unaltered. The up-regulation of pancreatic β-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ERα and ERβ agonists as well as ERαKO and ERβKO mice suggests that the estrogen receptor involved is ERα. The up-regulation of pancreatic insulin content by ERα activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.