Deletion of Hypoxia-Inducible Factor-1α in Adipocytes Enhances Glucagon-Like Peptide-1 Secretion and Reduces Adipose Tissue Inflammation

It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.     

Adipose Tissue-specific Inhibition of Hypoxia-inducible Factor 1α Induces Obesity and Glucose Intolerance by Impeding Energy Expenditure in Mice

Hypoxia in adipose tissue has been postulated as a possible contributor to obesity-related chronic inflammation, insulin resistance, and metabolic dysfunction. HIF1α (hypoxia-inducible factor 1α), a master signal mediator of hypoxia response, is elevated in obese adipose tissue. However, the role of HIF1α in obesity-related pathologies remains to be determined. Here we show that transgenic mice with adipose tissue-selective expression of a dominant negative version of HIF1α developed more severe obesity and were more susceptible to high fat diet-induced glucose intolerance and insulin resistance compared with their wild type littermates. Obesity in the transgenic mice was attributed to impaired energy expenditure and reduced thermogenesis. Histological examination of interscapular brown adipose tissue (BAT) in the transgenic mice demonstrated a markedly increased size of lipid droplets and decreased mitochondrial density in adipocytes, a phenotype similar to that in white adipose tissue. These changes in BAT of the transgenic mice were accompanied by decreased mitochondrial biogenesis and reduced expression of key thermogenic genes. In the transgenic mice, angiogenesis in BAT was decreased but was little affected in white adipose tissue. These findings support an indispensable role of HIF1α in maintaining the thermogenic functions of BAT, possibly through promoting angiogenesis and mitochondrial biogenesis in this tissue.     

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

Aims

Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.

Methods and Results

EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.

Conclusions

EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.     

Involvement of Inducible 6-Phosphofructo-2-kinase in the Anti-diabetic Effect of Peroxisome Proliferator-activated Receptor γ Activation in Mice

PFKFB3 is the gene that codes for the inducible isoform of 6-phosphofructo-2-kinase (iPFK2), a key regulatory enzyme of glycolysis. As one of the targets of peroxisome proliferator-activated receptor γ (PPARγ), PFKFB3/iPFK2 is up-regulated by thiazolidinediones. In the present study, using PFKFB3/iPFK2-disrupted mice, the role of PFKFB3/iPFK2 in the anti-diabetic effect of PPARγ activation was determined. In wild-type littermate mice, PPARγ activation (i.e. treatment with rosiglitazone) restored euglycemia and reversed high fat diet-induced insulin resistance and glucose intolerance. In contrast, PPARγ activation did not reduce high fat diet-induced hyperglycemia and failed to reverse insulin resistance and glucose intolerance in PFKFB3^+/− mice. The lack of anti-diabetic effect in PFKFB3^+/− mice was associated with the inability of PPARγ activation to suppress adipose tissue lipolysis and proinflammatory cytokine production, stimulate visceral fat accumulation, enhance adipose tissue insulin signaling, and appropriately regulate adipokine expression. Similarly, in cultured 3T3-L1 adipocytes, knockdown of PFKFB3/iPFK2 lessened the effect of PPARγ activation on stimulating lipid accumulation. Furthermore, PPARγ activation did not suppress inflammatory signaling in PFKFB3/iPFK2-knockdown adipocytes as it did in control adipocytes. Upon inhibition of excessive fatty acid oxidation in PFKFB3/iPFK2-knockdown adipocytes, PPARγ activation was able to significantly reverse inflammatory signaling and proinflammatory cytokine expression and restore insulin signaling. Together, these data demonstrate that PFKFB3/iPFK2 is critically involved in the anti-diabetic effect of PPARγ activation.     

Interleukin-1β Downregulates RBP4 Secretion in Human Adipocytes

Aims/hypothesis

The excessive accumulation of adipose tissue in the obese state is linked to an altered secretion profile of adipocytes, chronic low-grade inflammation and metabolic complications. RBP4 has been implicated in these alterations, especially insulin resistance. The aim of the present study was to determine if a local inflammatory micro-environment in adipose tissue regulates RBP4 expression and secretion.

Methods

Human SGBS and primary adipocytes cultured with conditioned media from human THP-1 macrophages were used as an in vitro model for adipose inflammation. Adipocytes were exposed to recombinant TNF-α, IL-1β, IL-6 or IL-8. In addition, coexpression of IL-1β and RBP4 was measured in adipose tissue samples from 18 healthy females. RBP4 expression was studied by quantitative PCR and ELISA.

Results

RBP4 mRNA expression and secretion was significantly reduced upon incubation with macrophage-conditioned media in SGBS adipocytes and human primary adipocytes. Out of several factors studied we identified IL-1β as a new factor regulating RBP4. IL-1β significantly downregulated RBP4 mRNA and secretion in a time- and dose-dependent manner. IL-1β mediated its inhibitory effects on RBP4 expression via IL-1 receptor and NF-κB, as incubation with the IL-1 receptor blocking antibody and the NF-κB inhibitors CAPE and SC-514 reversed its effect. Most interestingly, RBP4 mRNA was negatively correlated with IL-1β mRNA in subcutaneous adipose tissue.

Conclusions

Adipose tissue inflammation as found in the obese state might lead to a downregulation in local RBP4 levels. IL-1β was identified as a major factor contributing to the decrease in RBP4. The increase in circulating RBP4 that often precedes the development of systemic insulin resistance is most likely unrelated to inflammatory processes in adipose tissue.     

Momordica charantia (Bitter Melon) Reduces Obesity-Associated Macrophage and Mast Cell Infiltration as well as Inflammatory Cytokine Expression in Adipose Tissues

Obesity is a world-wide epidemic disease that correlates closely with type 2 diabetes and cardiovascular diseases. Obesity-induced chronic adipose tissue inflammation is now considered as a critical contributor to the above complications. Momordica charantia (bitter melon, BM) is a traditional Chinese food and well known for its function of reducing body weight gain and insulin resistance. However, it is unclear whether BM could alleviate adipose tissue inflammation caused by obesity. In this study, C57BL/6 mice were fed high fat diet (HFD) with or without BM for 12 weeks. BM-contained diets ameliorated HFD-induced obesity and insulin resistance. Histological and real-time PCR analysis demonstrated BM not only reduced macrophage infiltration into epididymal adipose tissues (EAT) and brown adipose tissues (BAT). Flow cytometry show that BM could modify the M1/M2 phenotype ratio of macrophages in EAT. Further study showed that BM lowered mast cell recruitments in EAT, and depressed pro-inflammatory cytokine monocyte chemotactic protein-1 (MCP-1) expression in EAT and BAT as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in EAT. Finally, ELISA analysis showed BM-contained diets also normalized serum levels of the cytokines. In summary, in concert with ameliorated insulin resistance and fat deposition, BM reduced adipose tissue inflammation in diet-induced obese (DIO) mice.     

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.     

Angiotensin II Receptor Blocker Ameliorates Stress-Induced Adipose Tissue Inflammation and Insulin Resistance

A strong causal link exists between psychological stress and insulin resistance as well with hypertension. Meanwhile, stress-related responses play critical roles in glucose metabolism in hypertensive patients. As clinical trials suggest that angiotensin-receptor blocker delays the onset of diabetes in hypertensive patients, we investigated the effects of irbesartan on stress-induced adipose tissue inflammation and insulin resistance. C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle, 3 and 10 mg/kg/day irbesartan. The plasma concentrations of lipid and proinflammatory cytokines [Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor-α, and interleukin-6] were assessed with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was observed with CD11b-positive cell counts and mRNA expressions of CD68 and F4/80 using immunohistochemistry and RT-PCR methods respectively. The mRNA levels of angiotensinogen, proinflammatory cytokines shown above, and adiponectin in WAT were also assessed with RT-PCR method. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and mRNA expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. Restraint stress increased monocyte accumulation, plasma free fatty acids, expression of angiotensinogen and proinflammatory cytokines including MCP-1, and reduced adiponectin. Irbesartan reduced stress-induced monocyte accumulation in WAT in a dose dependent manner. Irbesartan treatment also suppressed induction of adipose angiotensinogen and proinflammatory cytokines in WAT and blood, and reversed changes in adiponectin expression. Notably, irbesartan suppressed stress-induced reduction in adipose tissue weight and free fatty acid release, and improved insulin tolerance with restoration of IRS-1 and GLUT4 mRNA expressions in WAT. The results indicate that irbesartan improves stress-induced adipose tissue inflammation and insulin resistance. Our results suggests that irbesartan treatment exerts additive benefits for glucose metabolism in hypertensive patients with mental stress.     

Proline oxidase–adipose triglyceride lipase pathway restrains adipose cell death and tissue inflammation

The nutrient-sensing lipolytic enzyme adipose triglyceride lipase (ATGL) has a key role in adipose tissue function, and alterations in its activity have been implicated in many age-related metabolic disorders. In adipose tissue reduced blood vessel density is related to hypoxia state, cell death and inflammation. Here we demonstrate that adipocytes of poorly vascularized enlarged visceral adipose tissue (i.e. adipose tissue of old mice) suffer from limited nutrient delivery. In particular, nutrient starvation elicits increased activity of mitochondrial proline oxidase/dehydrogenase (POX/PRODH) that is causal in triggering a ROS-dependent induction of ATGL. We demonstrate that ATGL promotes the expression of genes related to mitochondrial oxidative metabolism (peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptor-γ coactivator-1α), thus setting a metabolic switch towards fat utilization that supplies energy to starved adipocytes and prevents cell death, as well as adipose tissue inflammation. Taken together, these results identify ATGL as a stress resistance mediator in adipocytes, restraining visceral adipose tissue dysfunction typical of age-related metabolic disorders.     

Losartan Attenuates Insulin Resistance and Regulates Browning Phenomenon of White Adipose Tissue in ob/ob Mice

Insulin resistance (IR) is a villain role to the pathology of fatty liver diseases implicated in adipose tissue dysfunction, which is characterized by lipid droplets (LDs) accumulation and hypoxia-inducible factor 1α (HIF1α) related macrophage infiltration. HIF1α is required for its lipogenic actions in adipocytes, while and it regulates M1 and M2 polarization features of macrophages. Losartan has been shown to be an insulin sensitizer in obese states, actions involving in HIF1α signaling. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, GTT, ITT, and HOMA-IR were identified losartan alleviated IR signaling in obese mice. This alleviation may through inhibits HIF1α by suppressing STAT3-NF-κB signaling, which, in turn, revealed HIF1α-dependent decreases the angiogenesis pathway in adipose tissue, including regulation of VEGF and TGFβR2 levels. In white adipose tissue, a set of lipogenesis-related genes, Srebp1, Fas, and Scd-1 were markedly downregulated after losartan intervention, as well as reduced LDs size and LD-associated proteins, perilipin family proteins (PLINs) compared with obese mice. Losartan abolished macrophage infiltration with upregulation of M2 and inhibition of M1 macrophage markers in obese mice. Our data suggest that losartan attenuated obese-induced fatty liver, linked to alleviating inflammation in adipose tissues and a shift in M1/M2 macrophage balance. Furthermore, losartan might improve mitochondria biogenesis by upregulating SIRT1, PGC1α, UCP1, and mRNA of Tfam, Cd137, Tmem26, Ucp1 expression in white adipose tissue compared with the obese group. Taken together, losartan may improve IR and adipose dysfunction by inhibiting lipotoxicity and HIF1α pathways.     

Adipolin/C1qdc2/CTRP12 protein functions as an adipokine that improves glucose metabolism

Obesity is a major risk factor for the development of insulin resistance and type 2 diabetes. Adipose tissue secretes various bioactive molecules, referred to as adipokines, whose dysregulation can mediate changes in glucose homeostasis and inflammatory responses. Here, we identify C1qdc2/CTRP12 as an insulin-sensitizing adipokine that is abundantly expressed by fat tissues and designate this adipokine as adipolin (adipose-derived insulin-sensitizing factor). Adipolin expression in adipose tissue and plasma was reduced in rodent models of obesity. Adipolin expression was also decreased in cultured 3T3-L1 adipocytes by treatment with inducers of endoplasmic reticulum stress and inflammation. Systemic administration of adipolin ameliorated glucose intolerance and insulin resistance in diet-induced obese mice. Adipolin administration also reduced macrophage accumulation and proinflammatory gene expression in the adipose tissue of obese mice. Conditioned medium from adipolin-expressing cells diminished the expression of proinflammatory cytokines in response to stimulation with LPS or TNFα in cultured macrophages. These data suggest that adipolin functions as an anti-inflammatory adipokine that exerts beneficial actions on glucose metabolism. Therefore, adipolin represents a new target molecule for the treatment of insulin resistance and diabetes.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Increasing Adipocyte Lipoprotein Lipase Improves Glucose Metabolism in High Fat Diet-induced Obesity

Lipid accumulation in liver and skeletal muscle contributes to co-morbidities associated with diabetes and obesity. We made a transgenic mouse in which the adiponectin (Adipoq) promoter drives expression of lipoprotein lipase (LPL) in adipocytes to potentially increase adipose tissue lipid storage. These mice (Adipoq-LPL) have improved glucose and insulin tolerance as well as increased energy expenditure when challenged with a high fat diet (HFD). To identify the mechanism(s) involved, we determined whether the Adipoq-LPL mice diverted dietary lipid to adipose tissue to reduce peripheral lipotoxicity, but we found no evidence for this. Instead, characterization of the adipose tissue of the male mice after HFD challenge revealed that the mRNA levels of peroxisome proliferator-activated receptor-γ (PPARγ) and a number of PPARγ-regulated genes were higher in the epididymal fat pads of Adipoq-LPL mice than control mice. This included adiponectin, whose mRNA levels were increased, leading to increased adiponectin serum levels in the Adipoq-LPL mice. In many respects, the adipose phenotype of these animals resembles thiazolidinedione treatment except for one important difference, the Adipoq-LPL mice did not gain more fat mass on HFD than control mice and did not have increased expression of genes in adipose such as glycerol kinase, which are induced by high affinity PPAR agonists. Rather, there was selective induction of PPARγ-regulated genes such as adiponectin in the adipose of the Adipoq-LPL mice, suggesting that increasing adipose tissue LPL improves glucose metabolism in diet-induced obesity by improving the adipose tissue phenotype. Adipoq-LPL mice also have increased energy expenditure.     

GLUT4 Defects in Adipose Tissue Are Early Signs of Metabolic Alterations in Alms1GT/GT,a Mouse Model for Obesity and Insulin Resistance

Dysregulation of signaling pathways in adipose tissue leading to insulin resistance can contribute to the development of obesity-related metabolic disorders. Alström Syndrome, a recessive ciliopathy, caused by mutations in ALMS1, is characterized by progressive metabolic alterations such as childhood obesity, hyperinsulinemia, and type 2 diabetes. Here we investigated the role of Alms1 disruption in AT expansion and insulin responsiveness in a murine model for Alström Syndrome. A gene trap insertion in Alms1 on the insulin sensitive C57BL6/Ei genetic background leads to early hyperinsulinemia and a progressive increase in body weight. At 6 weeks of age, before the onset of the metabolic disease, the mutant mice had enlarged fat depots with hypertrophic adipocytes, but without signs of inflammation. Expression of lipogenic enzymes was increased. Pre-adipocytes isolated from mutant animals demonstrated normal adipogenic differentiation but gave rise to mature adipocytes with reduced insulin-stimulated glucose uptake. Assessment of whole body glucose homeostasis revealed glucose intolerance. Insulin stimulation resulted in proper AKT phosphorylation in adipose tissue. However, the total amount of glucose transporter 4 (SLC4A2) and its translocation to the plasma membrane were reduced in mutant adipose depots compared to wildtype littermates. Alterations in insulin stimulated trafficking of glucose transporter 4 are an early sign of metabolic dysfunction in Alström mutant mice, providing a possible explanation for the reduced glucose uptake and the compensatory hyperinsulinemia. The metabolic signaling deficits either reside downstream or are independent of AKT activation and suggest a role for ALMS1 in GLUT4 trafficking. Alström mutant mice represent an interesting model for the development of metabolic disease in which adipose tissue with a reduced glucose uptake can expand by de novo lipogenesis to an obese state.     

Adipose Tissue Promotes a Serum Cytokine Profile Related to Lower Insulin Sensitivity after Chronic Central Leptin Infusion

Obesity is an inflammatory state characterized by an augment in circulating inflammatory factors. Leptin may modulate the synthesis of these factors by white adipose tissue decreasing insulin sensitivity. We have examined the effect of chronic central administration of leptin on circulating levels of cytokines and the possible relationship with cytokine expression and protein content as well as with leptin and insulin signaling in subcutaneous and visceral adipose tissues. In addition, we analyzed the possible correlation between circulating levels of cytokines and peripheral insulin resistance. We studied 18 male Wistar rats divided into controls (C), those treated icv for 14 days with a daily dose of 12 μg of leptin (L) and a pair-fed group (PF) that received the same food amount consumed by the leptin group. Serum leptin and insulin were measured by ELISA, mRNA levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-10 and tumor necrosis factor-α (TNF-α) by real time PCR and serum and adipose tissue levels of these cytokines by multiplexed bead immunoassay. Serum leptin, IL-2, IL-4, IFN-γ and HOMA-IR were increased in L and TNF-α was decreased in PF and L. Serum leptin and IL-2 levels correlate positively with HOMA-IR index and negatively with serum glucose levels during an ip insulin tolerance test. In L, an increase in mRNA levels of IL-2 was found in both adipose depots and IFN-γ only in visceral tissue. Activation of leptin signaling was increased and insulin signaling decreased in subcutaneous fat of L. In conclusion, leptin mediates the production of inflammatory cytokines by adipose tissue independent of its effects on food intake, decreasing insulin sensitivity.