Metabolic syndrome in mice induced by expressing a transcriptional activator in adipose tissue

Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11β-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11β-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.     

Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

Introduction

Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages.

Methods and results

Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes.

Conclusion

Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key source of inflammatory mediators in OSA.     

TNF-α and Temporal Changes in Sleep Architecture in Mice Exposed to Sleep Fragmentation

TNF-α plays critical roles in host-defense, sleep-wake regulation, and the pathogenesis of various disorders. Increases in the concentration of circulating TNF-α after either sleep deprivation or sleep fragmentation (SF) appear to underlie excessive daytime sleepiness in patients with sleep apnea (OSA). Following baseline recordings, mice were subjected to 15 days of SF (daily for 12 h/day from 07.00 h to 19.00 h), and sleep parameters were recorded on days1, 7 and 15. Sleep architecture and sleep propensity were assessed in both C57BL/6J and in TNF-α double receptor KO mice (TNFR KO). To further confirm the role of TNF-α, we also assessed the effect of treatment with a TNF- α neutralizing antibody in C57BL/6J mice. SF was not associated with major changes in global sleep architecture in C57BL/6J and TNFR KO mice. TNFR KO mice showed higher baseline SWS delta power. Further, following 15 days of SF, mice injected with TNF-α neutralizing antibody and TNFR KO mice showed increased EEG SWS activity. However, SWS latency, indicative of increased propensity to sleep, was only decreased in C57BL/6J, and was unaffected in TNFR KO mice as well as in C57BL/6J mice exposed to SF but treated with TNF-α neutralizing antibody. Taken together, our findings show that the excessive sleepiness incurred by recurrent arousals during sleep may be due to activation of TNF-alpha-dependent inflammatory pathways, despite the presence of preserved sleep duration and global sleep architecture.     

Islet-1: a potentially important role for an islet cell gene in visceral fat

Objective: To examine differences in gene expression between visceral (VF) and subcutaneous fat (SF) to identity genes of potential importance in regulation of VF. Methods and Procedures: We compared gene expression (by DNA array and quantitative PCR (qPCR)) in paired VF and SF adipose biopsies from 36 subjects (age 54 ± 15 years, 15 men/21 women) with varying degrees of adiposity and insulin resistance, in chow and fat fed mice (± rosiglitazone treatment) and in c‐Cbl^?/? mice. Gene expression was also examined in 3T3‐L1 preadipocytes during differentiation. Results: A twofold difference or more was found between VF and SF in 1,343 probe sets, especially for genes related to development, cell differentiation, signal transduction, and receptor activity. Islet‐1 (ISL1), a LIM‐homeobox gene with important developmental and regulatory function in islet, neural, and cardiac tissue, not previously recognized in adipose tissue was virtually absent in SF but substantially expressed in VF. ISL1 expression correlated negatively with BMI (r = ?0.37, P = 0.03), abdominal fat (by dual energy X‐ray absorptiometry, r = ?0.44, P = 0.02), and positively with circulating adiponectin (r = 0.33, P = 0.04). In diet‐induced obese mice, expression was reduced in the presence or absence of rosiglitazone. Correspondingly, expression was increased in the c‐Cbl^?/? mouse, which is lean and insulin sensitive (IS). ISL1 expression was increased sevenfold in 3T3‐L1 preadipocytes during early (day 1) differentiation and was reduced by day 2 differentiation. Discussion: An important developmental and regulatory gene ISL1 is uniquely expressed in VF, probably in the preadipocyte. Our data suggest that ISL1 may be regulated by adiposity and its role in metabolic regulation merits further study.     

Obesity and malnutrition similarly alter the renin–angiotensin system and inflammation in mice and human adipose

The main goal of the present study was to evaluate the metabolic profile, inflammatory markers and the gene expression of the renin–angiotensin system (RAS) components in the visceral adipose tissue of eutrophic, obese and malnourished individuals and mice models of obesity and food restriction. Male Swiss mice were divided into eight groups and fed different levels of food restriction (20%, 40%, or 60%) using standard or high-fat diet. Metabolic profile and adipose tissues were assessed. The expression of AGT (Angiotensinogen), ACE (Angiotensin-converting enzyme), ACE2 (Angiotensin-converting enzyme 2), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the mice epididymal adipose tissue and the human visceral adipose tissue was assessed. The main findings showed reduced body weight, improved metabolism, decreased adipose tissues weight and reduced adipocyte area in mice submitted to food restriction. Diminished expression of IL-6, TNF-α, AGT, AT1 and ACE was detected in the 20% and 40% food restriction animal groups, although they were increased in the 60% malnourished group. Increased expression of IL-6, TNF-α, AGT and ACE in obese and malnourished individuals was observed. Adipocytes size was increased in obese individuals and reduced in malnutrition. In conclusion, we found that food restriction of 20% and 40% improved the metabolic profile, ameliorated the inflammatory status and down-regulated the RAS in mice. Severe 60% food restriction (malnutrition), however, stimulated a proinflammatory state and increased AGT and ACE expression in the adipose tissue of mice. A similar profile was observed in the adipose tissue of obese and malnourished humans, supporting the critical role of inflammation and RAS as mediators of metabolic disorders.     

Gene expression profile of mouse white adipose tissue during inflammatory stress: age‐dependent upregulation of major procoagulant factors

Tolerance to physiological stress resulting from inflammatory disease decreases significantly with age. High mortality rates, increased cytokine production, and pronounced thrombosis are characteristic complications of aged mice with acute systemic inflammation induced by injection with lipopolysaccharide (LPS). As adipose tissue is now recognized as an important source of cytokines, we determined the effects of aging on visceral white adipose tissue gene expression during LPS‐induced inflammation in male C57BL/6 mice. Microarray analysis revealed that the expression of 6025 genes was significantly changed by LPS; of those, the expression of 667 showed an age‐associated difference. Age‐associated differences were found in many genes belonging to the inflammatory response and blood clotting pathways. Genes for several procoagulant factors were upregulated by LPS; among these, tissue factor, thrombospondin‐1, and plasminogen activator inhibitors‐1 and ‐2, exhibited age‐associated increases in expression which could potentially contribute to augmented thrombosis. Further analysis by qRT–PCR, histological examination, and cell fraction separation revealed that most inflammatory and coagulant‐related gene expression changes occur in resident stromal cells rather than adipocytes or infiltrating cells. In addition, basal expression levels of 303 genes were altered by aging, including increased expression of component of Sp100‐rs (Csprs). This study indicates that adipose tissue is a major organ expressing genes for multiple inflammatory and coagulant factors and that the expression of many of these is significantly altered by aging during acute inflammation. Data presented here provide a framework for future studies aimed at elucidating the impact of adipose tissue on age‐associated complications during sepsis and systemic inflammation.     

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.     

转基因和基因敲除内脂素对小鼠脂肪积累的对比研究

目的内脂素（visfatin）也被叫做尼克酰胺磷酸核糖基转移酶,是一种脂肪因子,研究表明其与肥胖有关,但是与脂肪积累的关系仍然不明确,本研究是以内脂素转基因和内脂素基因敲除杂合子小鼠为对象,研究内脂素与脂肪积累的关系。方法 Western blot法对比分析转基因、基因敲除杂合子和野生型小鼠脂肪组织中内脂素表达水平。从2月龄开始对3种雌性小鼠饲喂高脂饲料,分别在2、4、6、8、9月龄测定其体重变化,并在9月龄时利用磁共振成像定性观测小鼠脂肪积累及分布,称量皮下和腹腔脂肪总重量并对腹腔脂肪组织进行组织学观察。结果内脂素转基因小鼠脂肪组织中内脂素的表达量比野生小鼠增加37%,基因敲除杂合子小鼠比野生小鼠降低了55%。饲喂7个月高脂饲料后,转基因小鼠体重平均27.8±0.8 g,野生小鼠体重平均33.6±1.1 g,基因敲除杂合子小鼠体重平均37.6±1.9 g。皮下和腹腔脂肪总重量测定结果显示转基因小鼠的脂肪总重量比野生小鼠降低了40%,基因敲除杂合子小鼠的脂肪总重量比野生小鼠增加了37%,组织学染色显示,内脂素转基因小鼠的平均单个脂肪细胞面积最小,而基因敲除杂合子小鼠面积最大。结果证实,内脂素表达量与体重、皮下和内脏脂肪总重量及脂肪细胞大小呈负相关。结论在饲喂高脂饲料的情况下,内脂素可以抑制脂肪的积累。     

Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes

Recently, vaspin was identified as an adipokine with insulin-sensitizing effects, which is predominantly secreted from visceral adipose tissue in a rat model of type 2 diabetes. In this study, we examined whether vaspin mRNA expression is a marker of visceral obesity and correlates with anthropometric and metabolic parameters in paired samples of visceral and subcutaneous adipose tissue from 196 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance. Vaspin mRNA expression was only detectable in 23% of the visceral and in 15% of the subcutaneous (SC) adipose tissue samples. Vaspin mRNA expression was not detectable in lean subjects (BMI<25) and was more frequently detected in patients with type 2 diabetes. No significant correlations were found between visceral vaspin gene expression and visceral fat area or SC vaspin expression. However, visceral vaspin expression significantly correlates with BMI, % body fat, and 2 h OGTT plasma glucose. Subcutaneous vaspin mRNA expression is significantly correlated with WHR, fasting plasma insulin concentration, and glucose infusion rate during steady state of an euglycemic-hyperinsulinemic clamp. Multivariate linear regression analysis revealed % body fat as strongest predictor of visceral vaspin and insulin sensitivity as strongest determinant of SC vaspin mRNA expression. In conclusion, our data indicate that induction of human vaspin mRNA expression in adipose tissue is regulated in a fat depot-specific manner and could be associated with parameters of obesity, insulin resistance, and glucose metabolism.     

Bofu-Tsu-Shosan,an Oriental Herbal Medicine,Exerts a Combinatorial Favorable Metabolic Modulation Including Antihypertensive Effect on a Mouse Model of Human Metabolic Disorders with Visceral Obesity

Accumulating evidence indicates that metabolic dysfunction with visceral obesity is a major medical problem associated with the development of hypertension, type 2 diabetes (T2DM) and dyslipidemia, and ultimately severe cardiovascular and renal disease. Therefore, an effective anti-obesity treatment with a concomitant improvement in metabolic profile is important for the treatment of metabolic dysfunction with visceral obesity. Bofu-tsu-shosan (BOF) is one of oriental herbal medicine and is clinically available to treat obesity in Japan. Although BOF is a candidate as a novel therapeutic strategy to improve metabolic dysfunction with obesity, the mechanism of its beneficial effect is not fully elucidated. Here, we investigated mechanism of therapeutic effects of BOF on KKAy mice, a model of human metabolic disorders with obesity. Chronic treatment of KKAy mice with BOF persistently decreased food intake, body weight gain, low-density lipoprotein cholesterol and systolic blood pressure. In addition, both tissue weight and cell size of white adipose tissue (WAT) were decreased, with concomitant increases in the expression of adiponectin and peroxisome proliferator-activated receptors genes in WAT as well as the circulating adiponectin level by BOF treatment. Furthermore, gene expression of uncoupling protein-1, a thermogenesis factor, in brown adipose tissue and rectal temperature were both elevated by BOF. Intriguingly, plasma acylated-ghrelin, an active form of orexigenic hormone, and short-term food intake were significantly decreased by single bolus administration of BOF. These results indicate that BOF exerts a combinatorial favorable metabolic modulation including antihypertensive effect, at least partially, via its beneficial effect on adipose tissue function and its appetite-inhibitory property through suppression on the ghrelin system.     

High-fat diet induces changes in adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner

Protein carbonylation is the covalent modification of proteins by α,β-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC–MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.     

The Roles of Adipokines,Proinflammatory Cytokines,and Adipose Tissue Macrophages in Obesity-Associated Insulin Resistance in Modest Obesity and Early Metabolic Dysfunction

The roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in obesity-associated insulin resistance have been explored in both animal and human studies. However, our current understanding of obesity-associated insulin resistance relies on studies of artificial metabolic extremes. The purpose of this study was to explore the roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in human patients with modest obesity and early metabolic dysfunction. We obtained omental adipose tissue and fasting blood samples from 51 females undergoing gynecologic surgery. We investigated serum concentrations of proinflammatory cytokines and adipokines as well as the mRNA expression of proinflammatory and macrophage phenotype markers in visceral adipose tissue using ELISA and quantitative RT-PCR. We measured adipose tissue inflammation and macrophage infiltration using immunohistochemical analysis. Serum levels of adiponectin and leptin were significantly correlated with HOMA-IR and body mass index. The levels of expression of MCP-1 and TNF-α in visceral adipose tissue were also higher in the obese group (body mass index ≥ 25). The expression of mRNA MCP-1 in visceral adipose tissue was positively correlated with body mass index (r = 0.428, p = 0.037) but not with HOMA-IR, whereas TNF-α in visceral adipose tissue was correlated with HOMA-IR (r = 0.462, p = 0.035) but not with body mass index. There was no obvious change in macrophage phenotype or macrophage infiltration in patients with modest obesity or early metabolic dysfunction. Expression of mRNA CD163/CD68 was significantly related to mitochondrial-associated genes and serum inflammatory cytokine levels of resistin and leptin. These results suggest that changes in the production of inflammatory biomolecules precede increased immune cell infiltration and induction of a macrophage phenotype switch in visceral adipose tissue. Furthermore, serum resistin and leptin have specific roles in the regulation of adipose tissue macrophages in patients with modest obesity or early metabolic dysfunction.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Hepatic metabolic adaptation and adipose tissue expansion are altered in mice with steatohepatitis induced by high-fat high sucrose diet

Background: Obesity is a chronic progressive disease with several metabolic alterations. Nonalcoholic fatty liver disease (NAFLD) is an important comorbidity of obesity that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis or hepatocarcinoma. This study aimed at clarifying the molecular mechanisms underlying the metabolic alterations in hepatic and adipose tissue during high-fat high-sucrose diet-induced NAFLD development in mice. Methods: Twenty-four male mice (C57BL/6J) were randomly allocated into 3 groups (n = 8 mice per group) to receive a chow diet, a high-fat diet (HFD), or a high-fat high-sucrose diet (HF-HSD) for 20 weeks. At sacrifice, liver and adipose tissue were obtained for histopathological, metabolomic, and protein expression analyses. Results: HF-HSD (but not HFD) was associated with NASH and increased oxidative stress. These animals presented an inhibition of hepatic autophagy and alterations in AMP-activated protein kinase/mammalian target of rapamycin activity. We also observed that the ability of metabolic adaptation was adversely affected by the increase of damaged mitochondria. NASH development was associated with changes in adipose tissue dynamics and increased amounts of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in visceral adipose tissue. Conclusion: HF-HSD led to a metabolic blockage and impaired hepatic mitochondria turnover. In addition, the continuous accumulation of fatty acids produced adipose tissue dysfunction and hepatic fat accumulation that favored the progression to NASH.     

Coordinated regulation of nutrient and inflammatory responses by STAMP2 is essential for metabolic homeostasis

Metabolic and inflammatory pathways crosstalk at many levels, and, while required for homeostasis, interaction between these pathways can also lead to metabolic dysregulation under conditions of chronic stress. Thus, we hypothesized that mechanisms might exist to prevent overt inflammatory responses during physiological fluctuations in nutrients or under nutrient-rich conditions, and we identified the six-transmembrane protein STAMP2 as a critical modulator of this integrated response system of inflammation and metabolism in adipocytes. Lack of STAMP2 in adipocytes results in aberrant inflammatory responses to both nutrients and acute inflammatory stimuli. Similarly, in whole animals, visceral adipose tissue of STAMP2(-/-) mice exhibits overt inflammation, and these mice develop spontaneous metabolic disease on a regular diet, manifesting insulin resistance, glucose intolerance, mild hyperglycemia, dyslipidemia, and fatty liver disease. We conclude that STAMP2 participates in integrating inflammatory and metabolic responses and thus plays a key role in systemic metabolic homeostasis.