Latent TGFß-binding proteins regulate UCP1 expression and function via TGFß2.

OBJECTIVE: Activation of brown adipose tissue (BAT) in humans has been proposed as a new treatment approach for combating obesity and its associated diseases, as BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood. METHODS: Profiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting that these proteins are capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes. RESULTS: We demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation, but diminishes UCP1 expression and function in the obtained mature adipocytes. We further show that these effects are dependent on TGFß2 but not TGFß1 signaling: TGFß2 deficiency decreases adipocyte UCP1 expression, whereas TGFß2 treatment increases it. The activity of the LTBP3-TGFß2 axis that we delineate herein also significantly correlates with UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well. CONCLUSIONS: These results provide evidence that LTBP3, via TGFß2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.

Elevated UCP1 levels are sufficient to improve glucose uptake in human white adipocytes.

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1+ and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.

Diet-dependent function of the extracellular matrix proteoglycan Lumican in obesity and glucose homeostasis.

OBJECTIVE: Extracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications. METHODS: Whole body ablation of Lumican (Lum-/-) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling. RESULTS: Lumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance. CONCLUSIONS: These data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.

Circulating levels of miR-122 and nonalcoholic fatty liver disease in pre-pubertal obese children.

OBJECTIVES: The liver-specific miR-122 was proposed as biomarker for NAFLD in adults. Here, we investigated the relationship between miR-122 levels, parameters of liver metabolism and NAFLD in pre-pubertal obese children. METHODS: Parameters of liver metabolism (ALT, AST and GGT) of three European cohorts were included (German cohort [n = 71; age: 11.53 ± 1.29 years; BMI z-score: 2.96 ± 0.64], Italian cohort [n = 45; age: 9.60 ± 2.11 years; BMI z-score: 3.57 ± 1.16], Slovenian cohort [n = 31; age: 7.53 ± 1.47 years; BMI z-score: 3.66 ± 0.88]). MiR-122 levels and CK18 concentrations were measured in fasting blood samples. In the German and Italian cohort, the diagnosis of NAFLD and grading of NAFLD was assessed by ultrasound. RESULTS: NAFLD was diagnosed in n = 50 patients of the German cohort (29.6%) and in n = 29 patients (72.5%) of the Italian cohort. In all three cohorts, miR-122 was positively correlated with ALT and AST as well as with CK18 concentrations. MiR-122 levels were higher in children with NAFLD compared with healthy controls. CONCLUSIONS: MiR-122 levels in pre-pubertal obese children could be a potential biomarker for paediatric NAFLD.

Teneurin-2 (TENM2) deficiency induces UCP1 expression in differentiating human fat cells.

Under certain conditions UCP1 expressing adipocytes arise in white adipose tissue depots of both mice and humans. It is still not fully understood whether these cells differentiate de novo from specific progenitor cells or if they transdifferentiate from mature white adipocytes. Performing expression pattern analysis comparing adipocyte progenitor cells from deep and subcutaneous neck adipose tissue, we recently identified teneurin-2 (TENM2) enriched in white adipocyte progenitor cells. Here we tested whether TENM2 deficiency in adipocyte progenitor cells would lead to a brown adipocyte phenotype. By targeting TENM2 in SGBS preadipocytes using siRNA, we demonstrate that TENM2 knockdown induces both UCP1 mRNA and protein expression upon adipogenic differentiation without affecting mitochondrial mass. Furthermore, TENM2 knockdown in human SGBS adipocytes resulted in increased basal and leak mitochondrial respiration. In line with our previous observation these data suggest that TENM2 deficiency in human adipocyte precursors leads to induction of brown adipocyte marker genes upon adipogenic differentiation.

Clozapine modifies the differentiation program of human adipocytes inducing browning.

Administration of second-generation antipsychotic drugs (SGAs) often leads to weight gain and consequent cardio-metabolic side effects. We observed that clozapine but not six other antipsychotic drugs reprogrammed the gene expression pattern of differentiating human adipocytes ex vivo, leading to an elevated expression of the browning marker gene UCP1, more and smaller lipid droplets and more mitochondrial DNA than in the untreated white adipocytes. Laser scanning cytometry showed that up to 40% of the differentiating single primary and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes had the characteristic morphological features of browning cells. Furthermore, clozapine significantly upregulated ELOVL3, CIDEA, CYC1, PGC1A and TBX1 genes but not ZIC1 suggesting induction of the beige-like and not the classical brown phenotype. When we tested whether browning induced by clozapine can be explained by its known pharmacological effect of antagonizing serotonin (5HT) receptors, it was found that browning cells expressed 5HT receptors 2A, 1D, 7 and the upregulation of browning markers was diminished in the presence of exogenous 5HT. Undifferentiated progenitors or completely differentiated beige or white adipocytes did not respond to clozapine administration. The clozapine-induced beige cells displayed increased basal and oligomycin-inhibited (proton leak) oxygen consumption, but these cells showed a lower response to cAMP stimulus as compared with control beige adipocytes indicating that they are less capable to respond to natural thermogenic anti-obesity cues. Our data altogether suggest that novel pharmacological stimulation of these masked beige adipocytes can be a future therapeutic target for the treatment of SGA-induced weight gain.

Comparative gene array analysis of progenitor cells from human paired deep neck and subcutaneous adipose tissue.

Brown and white adipocytes have been shown to derive from different progenitors. In this study we sought to clarify the molecular differences between human brown and white adipocyte progenitors cells. To this end, we performed comparative gene array analysis on progenitor cells isolated from paired biopsies of deep and subcutaneous neck adipose tissue from individuals (n = 6) undergoing neck surgery. Compared with subcutaneous neck progenitors, cells from the deep neck adipose tissue displayed marked differences in gene expression pattern, including 355 differentially regulated (>1.5 fold) genes. Analysis of highest regulated genes revealed that STMN2, MME, ODZ2, NRN1 and IL13RA2 genes were specifically expressed in white progenitor cells, whereas expression of LRRC17, CNTNAP3, CD34, RGS7BP and ADH1B marked brown progenitor cells. In conclusion, progenitors from deep neck and subcutaneous neck adipose tissue are characterized by a distinct molecular signature, giving rise to either brown or white adipocytes. The newly identified markers may provide potential pharmacological targets facilitating brown adipogenesis.

FTO deficiency induces UCP-1 expression and mitochondrial uncoupling in adipocytes.

Variants in the fat mass- and obesity-associated (FTO) gene are associated with obesity and body fat mass in genome-wide association studies. However, the mechanism by which FTO predisposes individuals to obesity is not clear so far. First mechanistic evidence was shown in Fto-negative mice. These mice are resistant to obesity due to enhanced energy expenditure, whereas the mass of brown adipose tissue remains unchanged. We hypothesize that FTO is involved in the induction of white adipose tissue browning, which leads to mitochondrial uncoupling and increases energy expenditure. Uncoupling protein 1 (Ucp-1) was significantly higher expressed in both gonadal and inguinal adipose depots of Fto(-/-) compared with Fto(+/+) littermates accompanied by the appearance of multivacuolar, Ucp-1-positive adipocytes in these tissues. By using lentiviral short hairpin RNA constructs, we established FTO-deficient human preadipocytes and adipocytes and analyzed key metabolic processes. FTO-deficient adipocytes showed an adipogenic differentiation rate comparable with control cells but exhibited a reduced de novo lipogenesis despite unchanged glucose uptake. In agreement with the mouse data, FTO-deficient adipocytes exhibited 4-fold higher expression of UCP-1 in mitochondria compared with control cells. The up-regulation of UCP-1 in FTO-deficient adipocytes resulted in enhanced mitochondrial uncoupling. We conclude that FTO deficiency leads to the induction of a brown adipocyte phenotype, thereby enhancing energy expenditure. Further understanding of the signaling pathway connecting FTO with UCP-1 expression might lead to new options for obesity and overweight treatment.

TRAIL (TNF-related apoptosis-inducing ligand) regulates adipocyte metabolism by caspase-mediated cleavage of PPARgamma.

Tumor necrosis factor α (TNFα) and other members of the TNF family affect adipose tissue metabolism and contribute to the obesity-related inflammation of adipose tissue. Here, we sought to identify the effects of TRAIL (TNF-related apoptosis-inducing ligand) on fat cell biology. TRAIL-receptor 2 (TRAIL-R2) and its mouse homolog DR5 were regulated upon acute and chronic energy imbalance in murine and human adipose tissue. TRAIL inhibited insulin-stimulated glucose uptake and de novo lipogenesis in human adipocytes. Interestingly, TRAIL did not interfere with the phosphorylation of insulin-stimulated kinases such as Akt or Erk and did not activate the NF-κB pathway. Instead, TRAIL activated cleavage of caspase-8 and caspase-3. The subsequent cleavage of PPARγ led to its inactivation and resulted in reduced expression of lipogenic genes, such as Glut-4, FASN, and ACC. Taken together, we discovered a so far unknown function of the death ligand TRAIL in regulating adipocyte metabolism. Our results imply that TRAIL/TRAIL-R system might provide a new target for the prevention and treatment of obesity and its co-morbidities.

Regulation of FTO and FTM expression during human preadipocyte differentiation.

In genome-wide association studies (GWAS), polymorphisms in the first intron of FTO were shown to be associated with body fat mass. However, the functional properties of FTO and its nearby gene FTM are largely unknown. We examined the expression of these genes in subcutaneous adipose tissue and in isolated preadipocytes of lean and obese women. In in vitro differentiated primary human preadipocytes and in SGBS preadipocytes we found a decline in FTO and FTM expression during adipogenic differentiation. When investigating the hormonal regulation of FTO and FTM in adipocytes, insulin was identified as a key factor regulating FTM expression indicating a potential role of FTM in insulin regulated adipocyte metabolism.

LIGHT (TNFSF14) inhibits adipose differentiation without affecting adipocyte metabolism.

OBJECTIVE: The member of the tumor necrosis factor family LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells; TNFSF14 (tumor necrosis factor super family protein 14) is primarily expressed in lymphocytes, in which it induces the expression of pro-inflammatory cytokines and alterations of lipid homeostasis. Recently, the protein was shown to be upregulated in obesity and to induce cytokine secretion from adipocytes. RESEARCH METHODS AND PROCEDURES: Using an automated complementary DNA (cDNA) screen, LIGHT was identified to inhibit adipose differentiation. As cellular models for adipogenesis mouse 3T3-L1, human SGBS (Simpson-Golabi-Behmel syndrome) and primary human preadipocytes differentiated in vitro were used as well as primary human adipocytes to study adipocyte functions. Analysis of lipid deposition by Oil Red O staining, mRNA expression by quantitative reverse transcriptase-PCR, nuclear factor (NF)-κB activation as well as protein secretion by enzyme linked immunosorbent assay and Luminex technology was performed. RESULTS: LIGHT was found to inhibit lipid accumulation in the three models of preadipocytes in a dose-dependent manner without cytotoxic effects. This inhibition of differentiation was probably because of interference at early steps of adipogenesis, as early exposure during differentiation showed the strongest effect, as assessed by decreased peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα) mRNA expression. In contrast to TNFα, basal and insulin-stimulated glucose uptake and lipolysis of terminally differentiated mature adipocytes were not altered in the presence of LIGHT. At a concentration sufficient to inhibit differentiation, secretion of proinflammatory cytokines was not significantly induced and NF-κB activity was only modestly induced compared with TNFα. CONCLUSION: LIGHT is a novel inhibitor of human adipocyte differentiation without adversely influencing central metabolic pathways in adipocytes.

FTO - Friend or foe?

Fat mass and obesity associated gene ( FTO) is the most relevant polygene for obesity to date. It has been identified by genome wide association studies concerning body weight regulation. However, its functional relevance for the pathogenesis of obesity remains elusive. Studies in rodents provide data pointing to a central role of FTO through regulation of food intake. In addition, peripheral effects of FTO are also discussed in the literature. This review highlights the possible relevance of FTO for weight regulation and obesity development in central and peripheral tissues with a special focus on adipose tissue.

Influence of sex hormones on adiponectin expression in human adipocytes.

Adiponectin is an adipokine with profound antidiabetic and antiatherogenic effects. Circulating adiponectin concentrations are higher in women than in men. In order to study the molecular aspects of this sex-specific dimorphism, we examined the expression of adiponectin in human fat cells under the influence of sex hormones, using SGBS cells as an in vitro model. Androgen and estradiol receptor 1 and 2 (AR, ESR1, ESR2) mRNA expression increased dramatically during adipogenic differentiation. Stimulation with human male and female serum led to a downregulation of adiponectin expression, with male serum exerting significantly stronger inhibitory properties than female serum (p<0.05). Increasing concentrations of testosterone or estradiol influenced neither adiponectin mRNA expression and secretion nor intracellular protein expression of high-, middle-, and low-molecular-weight (HMW, MMW, LMW) adiponectin multimers. These data have been verified in in vitro-differentiated primary human adipocytes. We conclude that although human adipocytes express AR, ESR1, and ESR2 and respond to testosterone treatment with a decrease in leptin expression, expression and secretion of adiponectin is unaffected by sex steroids. We hypothesize, therefore, the existence of a serum factor that is differently regulated by sex steroids and subsequently causes the sex dimorphism in circulating adiponectin levels.

[Conjugated linoleic acids (CLA) and their relevance in the reduction of body fat. A critical review of the currently available data]. / Konjugierte Linolsäuren (CLA) und ihre Bedeutung für die Reduktion des Körperfetts. Eine kritische Betrachtung der momentanen Datenlage.

Various experiments on animals have shown that conjugated linoleic acids (CLA) supposedly have numerous positive effects on health including reducing body fat. Although previous studies on humans did not lead to consistent results, the popularity of CLA as a weight loss product for overweight people and as a muscle-building substance for athletes is increasing. Numerous internet providers or drugstores market CLA supplements or CLA-containing products. This article presents background information on the occurrence and biological effect of CLA and provides an up-to-date summary of studies on human subjects. Finally, whether supplementation with CLA for the reduction of body fat is useful will be evaluated.

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.

Endocrinology of adipose tissue - an update.

Adipose tissue is the body's largest repository of energy and it plays an important role in total energy homeostasis. Moreover, it is now well recognized as an endocrine organ. A wide range of different factors including complex proteins as well as fatty acids, prostaglandins, and steroids are either synthesized de novo or converted in adipose tissue and released into the blood stream. These so-called adipokines contribute to the development of obesity-related disorders, particularly type-2 diabetes (T2D) and cardiovascular disease. In this review, we present an overview on the endocrine functions of adipose tissue with a special focus on discoveries reported within the past 5 years.

Conjugated linoleic acids promote human fat cell apoptosis.

Conjugated linoleic acids (CLAs) are conjugated dienoic isomers of linoleic acid. Some isomers have been shown to reduce fat mass in animal and cell culture models. However, controversial results were obtained in studies of supplementation of CLAs in human subjects. In order to get more insights into the direct effects of CLAs on human fat cells, we have studied the influence of cis-9, trans-11 CLA and trans-10, cis-12 CLA on the biology of human SGBS preadipocytes and adipocytes. Both CLA isomers equally inhibited the proliferation of preadipocytes in a dose-dependent manner. Continuous treatment with 1-10 microM trans-10, cis-12 CLA, and to a weaker extent cis-9, trans-11 CLA, inhibited accumulation of lipids during adipogenic differentiation. Treatment with higher doses of CLA induced apoptosis in preadipocytes, in differentiating cells, and adipocytes. The trans-10, cis-12 isomer had a higher apoptotic potency in adipocytes than cis-9, trans-11 CLA. Taken together, the treatment of human preadipocytes and adipocytes with physiological relevant concentrations of CLAs resulted in an impairment of proliferation and differentiation and induction of apoptosis. The trans-10, cis-12 isomer was more potent than the cis-9, trans-11 isomer. Further clinical studies are needed to evaluate the effects of CLAs on human fat mass and metabolism in vivo.

The effect of the HIV protease inhibitor ritonavir on proliferation, differentiation, lipogenesis, gene expression and apoptosis of human preadipocytes and adipocytes.

HIV patients in highly active antiretroviral therapy (HAART) develop lipodystrophy and insulin resistance. Protease inhibitors have been shown to alter adipocyte metabolism in murine cell lines. In this study, biological effects of the HIV protease inhibitor, ritonavir, were investigated on human SGBS preadipocytes and adipocytes. Ritonavir dose-dependently impaired preadipocyte proliferation and adipogenic differentiation. Gene expression analysis measured by real-time PCR, showed no effect of ritonavir (up to 20 microM) on expression of mRNA of PPARgamma2 and SREBP1c, but suppressed adiponectin mRNA while increasing IL-6 mRNA expression. In human adipocytes, ritonavir at therapeutic concentrations inhibited insulin-stimulated lipogenesis, reduced GLUT4 mRNA, fatty acid synthase and adiponectin expression, while increasing IL-6 mRNA expression. Finally, long-term treatment (72 and 120 h) of SGBS adipocytes but not preadipocytes with ritonavir induced apoptosis in up to 15% of the cells. All together, these data show effects of ritonavir on human preadipocytes and adipocytes aiming at reducing adipose tissue mass and increasing insulin resistance. These in vitro findings may partly explain the clinical findings in patients under HAART. Furthermore, SGBS cells may serve as a useful tool in further investigation of the mechanism of protease inhibitor action in human adipocytes.