COBL, MKX and MYOC Are Potential Regulators of Brown Adipose Tissue Development Associated with Obesity-Related Metabolic Dysfunction in Children.

Obesity is already accompanied by adipose tissue (AT) dysfunction and metabolic disease in children and increases the risk of premature death. Due to its energy-dissipating function, brown AT (BAT) has been discussed as being protective against obesity and related metabolic dysfunction. To analyze the molecular processes associated with BAT development, we investigated genome-wide expression profiles in brown and white subcutaneous and perirenal AT samples of children. We identified 39 upregulated and 26 downregulated genes in uncoupling protein 1 (UCP1)-positive compared to UCP1-negative AT samples. We prioritized for genes that had not been characterized regarding a role in BAT biology before and selected cordon-bleu WH2 repeat protein (COBL), mohawk homeobox (MKX) and myocilin (MYOC) for further functional characterization. The siRNA-mediated knockdown of Cobl and Mkx during brown adipocyte differentiation in vitro resulted in decreased Ucp1 expression, while the inhibition of Myoc led to increased Ucp1 expression. Furthermore, COBL, MKX and MYOC expression in the subcutaneous AT of children is related to obesity and parameters of AT dysfunction and metabolic disease, such as adipocyte size, leptin levels and HOMA-IR. In conclusion, we identify COBL, MKX and MYOC as potential regulators of BAT development and show an association of these genes with early metabolic dysfunction in children.

The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional relevance.

BACKGROUND: G protein-coupled receptors (GPCR) are well-characterized regulators of a plethora of physiological functions among them the modulation of adipogenesis and adipocyte function. The class of Adhesion GPCR (aGPCR) and their role in adipose tissue, however, is poorly studied. With respect to the demand for novel targets in obesity treatment, we present a comprehensive study on the expression and function of this enigmatic GPCR class during adipogenesis and in mature adipocytes. METHODS: The expression of all aGPCR representatives was determined by reanalyzing RNA-Seq data and by performing qPCR in different mouse and human adipose tissues under low- and high-fat conditions. The impact of aGPCR expression on adipocyte differentiation and lipid accumulation was studied by siRNA-mediated knockdown of all expressed members of this receptor class. The biological characteristics and function of mature adipocytes lacking selected aGPCR were analyzed by mass spectrometry and biochemical methods (lipolysis, glucose uptake, adiponectin secretion). RESULTS: More than ten aGPCR are significantly expressed in visceral and subcutaneous adipose tissues and several aGPCR are differentially regulated under high-caloric conditions in human and mouse. Receptor knockdown of six receptors resulted in an impaired adipogenesis indicating their expression is essential for proper adipogenesis. The altered lipid composition was studied in more detail for two representatives, ADGRG2/GPR64 and ADGRG6/GPR126. While GPR126 is mainly involved in adipocyte differentiation, GPR64 has an additional role in mature adipocytes by regulating metabolic processes. CONCLUSIONS: Adhesion GPCR are significantly involved in qualitative and quantitative adipocyte lipid accumulation and can control lipolysis. Factors driving adipocyte formation and function are governed by signaling pathways induced by aGPCR yielding these receptors potential targets for treating obesity.

Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high fat diet-induced obesity by counteracting adipose tissue inflammation in vivo.

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 -/-) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 -/- mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 -/-. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.

Molecular Mechanisms of Vaspin Action - From Adipose Tissue to Skin and Bone, from Blood Vessels to the Brain.

Visceral adipose tissue-derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that were specifically expressed or overexpressed in the intra-abdominal or visceral adipose tissue (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral obesity, insulin resistance, hyperinsulinemia and -glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type 2 diabetes.The follow-up study reporting the cloning, expression and functional characterization of vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.

Genomic redistribution of GR monomers and dimers mediates transcriptional response to exogenous glucocorticoid in vivo.

Glucocorticoids (GCs) are commonly prescribed drugs, but their anti-inflammatory benefits are mitigated by metabolic side effects. Their transcriptional effects, including tissue-specific gene activation and repression, are mediated by the glucocorticoid receptor (GR), which is known to bind as a homodimer to a palindromic DNA sequence. Using ChIP-exo in mouse liver under endogenous corticosterone exposure, we report here that monomeric GR interaction with a half-site motif is more prevalent than homodimer binding. Monomers colocalize with lineage-determining transcription factors in both liver and primary macrophages, and the GR half-site motif drives transcription, suggesting that monomeric binding is fundamental to GR's tissue-specific functions. In response to exogenous GC in vivo, GR dimers assemble on chromatin near ligand-activated genes, concomitant with monomer evacuation of sites near repressed genes. Thus, pharmacological GCs mediate gene expression by favoring GR homodimer occupancy at classic palindromic sites at the expense of monomeric binding. The findings have important implications for improving therapies that target GR.

Depletion of Jmjd1c impairs adipogenesis in murine 3T3-L1 cells.

Differentiation of adipocytes is a highly regulated process modulated by multiple transcriptional co-activators and co-repressors. JMJD1C belongs to the family of jumonji C (jmjC) domain-containing histone demethylases and was originally described as a ligand-dependent co-activator of thyroid hormone and androgen receptors. Here, we explored the potential role of Jmjd1c in white adipocyte differentiation. To investigate the relevance of Jmjd1c in adipogenesis, murine 3T3-L1 preadipocyte cells with transient knock-down of Jmjd1c (3T3_Jmjd1c) were generated. Depletion of Jmjd1c led to the formation of smaller lipid droplets, reduced accumulation of triglycerides and maintenance of a more fibroblast-like morphology after adipocyte differentiation. Concomitantly, insulin stimulated uptake of glucose and fatty acids was significantly reduced in 3T3_Jmjd1c adipocytes. In line with these observations we detected lower expression of key genes associated with lipid droplet formation (Plin1, Plin4, Cidea) and uptake of glucose and fatty acids (Glut4, Fatp1, Fatp4, Aqp7) respectively. Finally, we demonstrate that depletion of Jmjd1c interferes with mitotic clonal expansion (MCE), increases levels of H3K9me2 (dimethylation of lysine 9 of histone H3) at promotor regions of adipogenic transcription factors (C/EBPs and PPARγ) and leads to reduced induction of these key regulators. In conclusion, we have identified Jmjd1c as a modulator of adipogenesis. Our data suggest that Jmjd1c may participate in MCE and the activation of the adipogenic transcription program during the induction phase of adipocyte differentiation in 3T3-L1 cells.

Inhibition of AHCY impedes proliferation and differentiation of mouse and human adipocyte progenitor cells.

S-adenosyl-homocysteine-hydrolase (AHCY) plays an important role in the methionine cycle regulating cellular methylation levels. AHCY has been reported to influence proliferation and differentiation processes in different cell types, e.g. in cancer cells and mouse embryonic stem cells. In the development of adipose tissue, both the proliferation and differentiation of adipocyte progenitor cells (APCs) are important processes, which in the context of obesity are often dysregulated. To assess whether AHCY might also be involved in cell proliferation and differentiation of APCs, we investigated the effect of reduced AHCY activity on human and mouse APCs in vitro. We show that the inhibition of AHCY using adenosine dialdehyde (AdOx) and the knockdown of AHCY using gene-specific siRNAs reduced APC proliferation and number. Inhibition of AHCY further reduced APC differentiation into mature adipocytes and the expression of adipogenic differentiation markers. Global DNA methylation profiling in human APCs revealed that inhibition of AHCY is associated with alterations in CpG methylation levels of genes involved in fat cell differentiation and pathways related to cellular growth. Our findings suggest that AHCY is necessary for the maintenance of APC proliferation and differentiation and inhibition of AHCY alters DNA methylation processes leading to a dysregulation of the expression of genes involved in the regulation of these processes.

Differential expression of immunoregulatory cytokines in adipose tissue and liver in response to high fat and high sugar diets in female mice.

A comprehensive understanding of how dietary components impact immunoregulatory gene expression in adipose tissue (AT) and liver, and their respective contributions to metabolic health in mice, remains limited. The current study aimed to investigate the metabolic consequences of a high-sucrose diet (HSD) and a high-fat diet (HFD) in female mice with a focus on differential lipid- and sucrose-induced changes in immunoregulatory gene expression in AT and liver. Female C57BL/6 J mice were fed a purified and macronutrient matched high fat, high sugar, or control diets for 12 weeks. Mice were extensively phenotyped, including glucose and insulin tolerance tests, adipose and liver gene and protein expression analysis by qPCR and Western blot, tissue lipid analyses, as well as histological analyses. Compared to the control diet, HSD- and HFD-fed mice had significantly higher body weights, with pronounced obesity along with glucose intolerance and insulin resistance only in HFD-fed mice. HSD-fed mice exhibited an intermediate phenotype, with mild metabolic deterioration at the end of the study. AT lipid composition was significantly altered by both diets, and inflammatory gene expression was only significantly induced in HFD-fed mice. In the liver however, histological analysis revealed that both HSD- and HFD-fed mice had pronounced ectopic lipid deposition indicating hepatic steatosis, but more pronounced in HSD-fed mice. This was in line with significant induction of pro-inflammatory gene expression specifically in livers of HSD-fed mice. Overall, our findings suggest that HFD consumption in female mice induces more profound inflammation in AT with pronounced deterioration of metabolic health, whereas HSD induced more pronounced hepatic steatosis and inflammation without yet affecting glucose metabolism.

Thyroid hormones regulate Zfp423 expression in regionally distinct adipose depots through direct and cell-autonomous action.

The hypothalamic pituitary thyroid axis is a major regulator of many differentiation processes, including adipose tissue. However, it remains unclear whether and how thyroid hormone (TH) signaling contributes to preadipocyte commitment and differentiation into mature adipocytes. Here, we show a cell-autonomous effect of TH on the transcriptional regulation of zinc finger protein 423 (Zfp423), an early adipogenic determination factor, in murine adipose depots. Mechanistically, binding of the unliganded TH receptor to a negative TH responsive element within the Zfp423 promoter activates transcriptional activity that is reversed upon TH binding. Zfp423 upregulation is associated with increased GFP+ preadipocyte recruitment in stromal vascular fraction isolated from white fat of hypothyroid Zfp423GFP reporter mice. RNA sequencing identified Zfp423-driven gene programs that are modulated in response to TH during adipogenic differentiation. Collectively, we identified Zfp423 as a key molecule that integrates TH signaling into the regulation of adipose tissue plasticity.

LRP1 is the cell-surface endocytosis receptor for vaspin in adipocytes.

Vaspin is a serine protease inhibitor that protects against adipose tissue inflammation and insulin resistance, two key drivers of adipocyte dysfunction and metabolic disorders in obesity. Inhibition of target proteases such as KLK7 has been shown to reduce adipose tissue inflammation in obesity, while vaspin binding to cell surface GRP78 has been linked to reduced obesity-induced ER stress and insulin resistance in the liver. However, the molecular mechanisms by which vaspin directly affects cellular processes in adipocytes remain unknown. Using fluorescently labeled vaspin, we found that vaspin is rapidly internalized by mouse and human adipocytes, but less efficiently by endothelial, kidney, liver, and neuronal cells. Internalization occurs by active, clathrin-mediated endocytosis, which is dependent on vaspin binding to the LRP1 receptor, rather than GRP78 as previously thought. This was demonstrated by competition experiments and RNAi-mediated knock-down in adipocytes and by rescuing vaspin internalization in LRP1-deficient Pea13 cells after transfection with a functional LRP1 minireceptor. Vaspin internalization is further increased in mature adipocytes after insulin-stimulated translocation of LRP1. Although vaspin has nanomolar affinity for LRP1 clusters II-IV, binding to cell surface heparan sulfates is required for efficient LRP1-mediated internalization. Native, but not cleaved vaspin, and also vaspin polymers are efficiently endocytosed, and ultimately targeted for lysosomal degradation. Our study provides mechanistic insight into the uptake and degradation of vaspin in adipocytes, thereby broadening our understanding of its functional repertoire. We hypothesize the vaspin-LRP1 axis to be an important mediator of vaspin effects not only in adipose tissue but also in other LRP1-expressing cells.

Overexpressing high levels of human vaspin limits high fat diet-induced obesity and enhances energy expenditure in a transgenic mouse.

Adipose tissue inflammation and insulin resistance are hallmarks in the development of metabolic diseases resulting from overweight and obesity, such as type 2 diabetes and non-alcoholic fatty liver disease. In obesity, adipocytes predominantly secrete proinflammatory adipokines that further promote adipose tissue dysfunction with negative effects on local and systemic insulin sensitivity. Expression of the serpin vaspin (SERPINA12) is also increased in obesity and type 2 diabetes, but exhibits compensatory roles in inflammation and insulin resistance. This has in part been demonstrated using vaspin-transgenic mice. We here report a new mouse line (h-vaspinTG) with transgenic expression of human vaspin in adipose tissue that reaches vaspin concentrations three orders of magnitude higher than wild type controls (>200 ng/ml). Phenotyping under chow and high-fat diet conditions included glucose-tolerance tests, measurements of energy expenditure and circulating parameters, adipose tissue and liver histology. Also, ex vivo glucose uptake in isolated adipocytes and skeletal muscle was analyzed in h-vaspinTG and littermate controls. The results confirmed previous findings, revealing a strong reduction in diet-induced weight gain, fat mass, hyperinsulinemia, -glycemia and -cholesterolemia as well as fatty liver. Insulin sensitivity in adipose tissue and muscle was not altered. The h-vaspinTG mice showed increased energy expenditure under high fat diet conditions, that may explain reduced weight gain and overall metabolic improvements. In conclusion, this novel human vaspin-transgenic mouse line will be a valuable research tool to delineate whole-body, tissue- and cell-specific effects of vaspin in health and disease.

Myoglobin-mediated lipid shuttling increases adrenergic activation of brown and white adipocyte metabolism and is as a marker of thermogenic adipocytes in humans.

BACKGROUND: Recruitment and activation of brown adipose tissue (BAT) results in increased energy expenditure (EE) via thermogenesis and represents an intriguing therapeutic approach to combat obesity and treat associated diseases. Thermogenesis requires an increased and efficient supply of energy substrates and oxygen to the BAT. The hemoprotein myoglobin (MB) is primarily expressed in heart and skeletal muscle fibres, where it facilitates oxygen storage and flux to the mitochondria during exercise. In the last years, further contributions of MB have been assigned to the scavenging of reactive oxygen species (ROS), the regulation of cellular nitric oxide (NO) levels and also lipid binding. There is a substantial expression of MB in BAT, which is induced during brown adipocyte differentiation and BAT activation. This suggests MB as a previously unrecognized player in BAT contributing to thermogenesis. METHODS AND RESULTS: This study analyzed the consequences of MB expression in BAT on mitochondrial function and thermogenesis in vitro and in vivo. Using MB overexpressing, knockdown or knockout adipocytes, we show that expression levels of MB control brown adipocyte mitochondrial respiratory capacity and acute response to adrenergic stimulation, signalling and lipolysis. Overexpression in white adipocytes also increases their metabolic activity. Mutation of lipid interacting residues in MB abolished these beneficial effects of MB. In vivo, whole-body MB knockout resulted in impaired thermoregulation and cold- as well as drug-induced BAT activation in mice. In humans, MB is differentially expressed in subcutaneous (SC) and visceral (VIS) adipose tissue (AT) depots, differentially regulated by the state of obesity and higher expressed in AT samples that exhibit higher thermogenic potential. CONCLUSIONS: These data demonstrate for the first time a functional relevance of MBs lipid binding properties and establish MB as an important regulatory element of thermogenic capacity in brown and likely beige adipocytes.

Functional changes of the gastric bypass microbiota reactivate thermogenic adipose tissue and systemic glucose control via intestinal FXR-TGR5 crosstalk in diet-induced obesity.

BACKGROUND: Bariatric surgery remains the most effective therapy for adiposity reduction and remission of type 2 diabetes. Although different bariatric procedures associate with pronounced shifts in the gut microbiota, their functional role in the regulation of energetic and metabolic benefits achieved with the surgery are not clear. METHODS: To evaluate the causal as well as the inherent therapeutic character of the surgery-altered gut microbiome in improved energy and metabolic control in diet-induced obesity, an antibiotic cocktail was used to eliminate the gut microbiota in diet-induced obese rats after gastric bypass surgery, and gastric bypass-shaped gut microbiota was transplanted into obese littermates. Thorough metabolic profiling was combined with omics technologies on samples collected from cecum and plasma to identify adaptions in gut microbiota-host signaling, which control improved energy balance and metabolic profile after surgery. RESULTS: In this study, we first demonstrate that depletion of the gut microbiota largely reversed the beneficial effects of gastric bypass surgery on negative energy balance and improved glucolipid metabolism. Further, we show that the gastric bypass-shaped gut microbiota reduces adiposity in diet-induced obese recipients by re-activating energy expenditure from metabolic active brown adipose tissue. These beneficial effects were linked to improved glucose homeostasis, lipid control, and improved fatty liver disease. Mechanistically, these effects were triggered by modulation of taurine metabolism by the gastric bypass gut microbiota, fostering an increased abundance of intestinal and circulating taurine-conjugated bile acid species. In turn, these bile acids activated gut-restricted FXR and systemic TGR5 signaling to stimulate adaptive thermogenesis. CONCLUSION: Our results establish the role of the gut microbiome in the weight loss and metabolic success of gastric bypass surgery. We here identify a signaling cascade that entails altered bile acid receptor signaling resulting from a collective, hitherto undescribed change in the metabolic activity of a cluster of bacteria, thereby readjusting energy imbalance and metabolic disease in the obese host. These findings strengthen the rationale for microbiota-targeted strategies to improve and refine current therapies of obesity and metabolic syndrome. Video Abstract Bariatric Surgery (i.e. RYGB) or the repeated fecal microbiota transfer (FMT) from RYGB donors into DIO (diet-induced obesity) animals induces shifts in the intestinal microbiome, an effect that can be impaired by oral application of antibiotics (ABx). Our current study shows that RYGB-dependent alterations in the intestinal microbiome result in an increase in the luminal and systemic pool of Taurine-conjugated Bile acids (TCBAs) by various cellular mechanisms acting in the intestine and the liver. TCBAs induce signaling via two different receptors, farnesoid X receptor (FXR, specifically in the intestines) and the G-protein-coupled bile acid receptor TGR5 (systemically), finally resulting in metabolic improvement and advanced weight management. BSH, bile salt hydrolase; BAT brown adipose tissue.

Cleavage of the vaspin N-terminus releases cell-penetrating peptides that affect early stages of adipogenesis and inhibit lipolysis in mature adipocytes.

Vaspin expression and function is related to metabolic disorders and comorbidities of obesity. In various cellular and animal models of obesity, diabetes and atherosclerosis vaspin has shown beneficial, protective and/or compensatory action. While testing proteases for inhibition by vaspin, we noticed specific cleavage within the vaspin N-terminus and sequence analysis predicted cell-penetrating activity for the released peptides. These findings raised the question whether these proteolytic peptides exhibit biological activity.We synthesized various N-terminal vaspin peptides to investigate cell-penetrating activity and analyse uptake mechanisms. Focusing on adipocytes, we performed microarray analysis and functional assays to elucidate biological activities of the vaspin-derived peptide, which is released by KLK7 cleavage (vaspin residues 21-30; VaspinN). Our study provides first evidence that proteolytic processing of the vaspin N-terminus releases cell-penetrating and bioactive peptides with effects on adipocyte biology. The VaspinN peptide increased preadipocyte proliferation, interfered with clonal expansion during the early stage of adipogenesis and blunted adrenergic cAMP-signalling, downstream lipolysis as well as insulin signalling in mature adipocytes.Protease-mediated release of functional N-terminal peptides presents an additional facet of vaspin action. Future studies will address the mechanisms underlying the biological activities and clarify, if vaspin-derived peptides may have potential as therapeutic agents for the treatment of metabolic diseases.

Leptin counteracts hypothermia in hypothyroidism through its pyrexic effects and by stabilizing serum thyroid hormone levels.

OBJECTIVE: Thyroid hormones (TH) are essential for the homeostatic control of energy metabolism and the regulation of body temperature. The hypothalamic-pituitary-thyroid (HPT) axis is regulated by negative feedback mechanisms, ensuring that TH levels are maintained at a constant level. However, the feedback mechanisms underlying the resetting of the HPT axis regulation in the control of body temperature are still not fully understood. Here, we aimed to determine the thermoregulatory response in hypothyroid mice to different environmental temperatures and the underlying mechanisms. METHODS: Distinct thermogenic challenges were induced in hypothyroid female C57BL/6N and leptin-deficient ob/ob mice through housing at either room temperature or thermoneutrality. The thermogenic and metabolic effects were analyzed through metabolic chambers, 18F-FDG-PET/MRI, infrared thermography, metabolic profiling, histology, gene expression and Western blot analysis. RESULTS: In hypothyroid mice maintained at room temperature, high leptin serum levels induce a pyrexic effect leading to the stabilization of body temperature through brown adipose tissue thermogenesis and white adipose tissue browning. Housing at thermoneutrality leads to the normalization of leptin levels and a reduction of the central temperature set point, resulting in decreased thermogenesis in brown and white adipose tissue and skeletal muscle and a significant decline in body temperature. Furthermore, anapyrexia in hypothyroid leptin-deficient ob/ob mice indicates that besides its pyrexic actions, leptin exerts a stimulatory effect on the HPT axis to stabilize the remaining TH serum levels in hypothyroid mice. CONCLUSION: This study led to the identification of a previously unknown endocrine loop in which leptin acts in concert with the HPT axis to stabilize body temperature in hypothyroid mice.

Alternatives for the worse: Molecular insights into adverse effects of bisphenol a and substitutes during human adipocyte differentiation.

Bisphenol A (BPA), which is used in a variety of consumer-related plastic products, was reported to cause adverse effects, including disruption of adipocyte differentiation, interference with obesity mechanisms, and impairment of insulin- and glucose homeostasis. Substitute compounds are increasingly emerging but are not sufficiently investigated.We aimed to investigate the mode of action of BPA and four of its substitutes during the differentiation of human preadipocytes to adipocytes and their molecular interaction with peroxisome proliferator-activated receptor γ (PPARγ), a pivotal regulator of adipogenesis.Binding and effective biological activation of PPARγ were investigated by surface plasmon resonance and reporter gene assay, respectively. Human preadipocytes were continuously exposed to BPA, BPS, BPB, BPF, BPAF, and the PPARγ-antagonist GW9662. After 12 days of differentiation, lipid production was quantified via Oil Red O staining, and global protein profiles were assessed using LC-MS/MS-based proteomics. All tested bisphenols bound to human PPARγ with similar efficacy as the natural ligand 15d-PGJ2in vitroand provoked an antagonistic effect on PPARγ in the reporter gene assay at non-cytotoxic concentrations. During the differentiation of human preadipocytes, all bisphenols decreased lipid production. Global proteomics displayed a down-regulation of adipogenesis and metabolic pathways, similar to GW9662. Interestingly, pro-inflammatory pathways were up-regulated, MCP1 release was increased, and adiponectin decreased. pAKT/AKT ratios revealed significantly reduced insulin sensitivity by BPA, BPB, and BPS upon insulin stimulation.Thus, our results show that not only BPA but also its substitutes disrupt crucial metabolic functions and insulin signaling in adipocytes under low, environmentally relevant concentrations. This effect, mediated through inhibition of PPARγ, may promote hypertrophy of adipose tissue and increase the risk of developing metabolic syndrome, including insulin resistance.

Role of Kallikrein 7 in Body Weight and Fat Mass Regulation.

Increased plasma and adipose tissue protease activity is observed in patients with type 2 diabetes and obesity. It has been proposed that specific proteases contribute to the link between obesity, adipose tissue inflammation and metabolic diseases. We have recently shown that ablation of the serine protease kallikrein-related peptidase 7 (Klk7) specifically in adipose tissue preserves systemic insulin sensitivity and protects mice from obesity-related AT inflammation. Here, we investigated whether whole body Klk7 knockout (Klk7-/-) mice develop a phenotype distinct from that caused by reduced Klk7 expression in adipose tissue. Compared to littermate controls, Klk7-/- mice gain less body weight and fat mass both under chow and high fat diet (HFD) feeding, are hyper-responsive to exogenous insulin and exhibit preserved adipose tissue function due to adipocyte hyperplasia and lower inflammation. Klk7-/- mice exhibit increased adipose tissue thermogenesis, which is not related to altered thyroid function. These data strengthen our recently proposed role of Klk7 in the regulation of body weight, energy metabolism, and obesity-associated adipose tissue dysfunction. The protective effects of Klk7 deficiency in obesity are likely linked to a significant limitation of adipocyte hypertrophy. In conclusion, our data indicate potential application of specific KLK7 inhibitors to regulate KLK7 activity in the development of obesity and counteract obesity-associated inflammation and metabolic diseases.

Atg7 Knockdown Reduces Chemerin Secretion in Murine Adipocytes.

CONTEXT: In individuals with obesity, adipocyte endocrine function is affected by altered autophagy. Genetic variants in autophagy-related gene 7 (ATG7) correlated with serum chemerin (RARRES2) concentrations. OBJECTIVES: To investigate a functional interplay between chemerin and ATG7, how it may relate to autophagy-mediated adipocyte dysfunction in obesity, and the relevance of genetic ATG7 variants in chemerin physiology. DESIGN: Adipose ATG7 mRNA expression and adiposity measures were available in two human study cohorts. The effect of a high-calorie diet on adipose Rarres2 and Atg7 expression was investigated in mice. In 3T3-L1 adipocytes, the effect of Atg7 knockdown on chemerin expression and secretion was studied. The influence of single nucleotide polymorphisms on ATG7 transcription and chemerin physiology was investigated using a luciferase assay. SETTING: Mouse model, clinical trials, in vitro studies. PARTICIPANTS: Native American (n = 83) and white (n = 100) cohorts. MAIN OUTCOME MEASURE: Adipocyte chemerin expression and secretion. RESULTS: In mice fed a high-calorie diet, adipose Atg7 mRNA expression did not parallel an increase in Rarres2 mRNA expression. ATG7 mRNA expression in human subcutaneous adipose tissue correlated with body mass index, fat mass (r > 0.27; P < 0.01), and adipocyte cell size (r > 0.24; P < 0.02). Atg7 knockdown in 3T3-L1 adipocytes decreased chemerin secretion by 22% (P < 0.04). Rs2606729 in ATG7 was predicted to alter ATG7 transcription and induced higher luciferase activity in vitro (P < 0.0001). CONCLUSIONS: Human adipose ATG7 mRNA expression relates to measures of adiposity. Atg7 knockdown reduces chemerin secretion from adipocytes in vitro, supportive of a functional interplay between ATG7 and chemerin in autophagy-mediated adipocyte dysfunction.

Vaspin suppresses cytokine-induced inflammation in 3T3-L1 adipocytes via inhibition of NFκB pathway.

Vaspin expression is increased in white adipose tissue (WAT) of diet-induced obese mice and rats and is supposed to compensate HFD-induced inflammatory processes and insulin resistance in adipose tissue by counteracting pro-inflammatory gene expression in obesity. Multiple studies have also demonstrated strong anti-inflammatory effects in vascular and skin cells. Here, we used vaspin treated 3T3-L1 murine adipocytes as well as 3T3-L1 cells with stable vaspin expression to investigate the effect of exogenous and endogenous vaspin on inflammatory processes and insulin signaling in adipocytes. Our stably transfected cells secreted significant amounts of vaspin which was in the physiological range of ∼0.5 ng/ml in cell supernatants. Adipocyte differentiation was not affected by vaspin as expression of adipogenic marker genes as well as lipid accumulation after full differentiation was similar to control cells. We found that IL-1ß induced expression and secretion of pro-inflammatory cytokines, such as IL-6, MCP1 and TNFα was significantly blunted in vaspin expressing 3T3-L1 cells. Treatment of 3T3-L1 cells with exogenous vaspin resulted in reduced cytokine-induced activation of the intracellular and pro-inflammatory NFκB signaling cascades (IKKα/ß, IκB and NFκB). Moreover, endogenous vaspin positively affected insulin signaling by increasing insulin-stimulated phosphorylation of the key mediator protein kinase B (AKT). Together, we demonstrate anti-inflammatory effects of vaspin in 3T3-L1 adipocytes as well as increased insulin signaling by endogenous expression or exogenous treatment. The results provide evidence for potent anti-inflammatory action of vaspin not only in vascular cells but also in adipose tissue.

Thyroid hormones and browning of adipose tissue.

Thyroid hormone (TH) disorders are associated with profound changes in whole body energy metabolism. A major TH target is thermogenic brown adipose tissue (BAT), which can be stimulated directly through thyroid hormone receptors (TRs) expressed in brown adipocytes and indirectly, through TRs expressed in hypothalamic neurons. White adipose tissue (WAT) adopts BAT characteristics by a diverse range of stimuli in a process referred to as browning. It is now understood that TH also induce WAT browning through peripheral and central mechanisms. In this review, we discuss evidence from animal and human studies that TH disorders are associated with changes in both BAT thermogenesis and WAT browning, thereby influencing body temperature and body weight regulation.