Adipogenic characterization of immortalized CD55+ progenitor cells from human white adipose tissue.

BACKGROUND: Mature adipocytes are notoriously difficult to study ex vivo and alternative cell culture systems have therefore been developed. One of the most common models are human adipose progenitor cells (hAPCs). Unfortunately, these display replicative senescence after prolonged culture conditions, which limits their use in mechanistic studies. METHODS: Herein, we knocked in human telomerase reverse transcriptase (TERT) into the AAVS1 locus of CD55+ hAPCs derived from abdominal subcutaneous adipose tissue and characterized the cells before and after differentiation into adipocytes. RESULTS: Immortalized TERT-hAPCs retained proliferative and adipogenic capacities comparable to those of early-passage wild type hAPCs for > 80 passages. In line with this, our integrative transcriptomic and proteomic analyses revealed that TERT-hAPCs displayed robust adipocyte expression profiles in comparison to wild type hAPCs. This was confirmed by functional analyses of lipid turnover where TERT-hAPCs exhibited pronounced responses to insulin and pro-lipolytic stimuli such as isoprenaline, dibutyrul cAMP and tumour necrosis factor alpha. In addition, TERT-hAPCs could be readily cultured in both standard 2D and 3D-cultures and proteomic analyses revealed that the spheroid culture conditions improved adipogenesis. CONCLUSION: Through descriptive and functional studies, we demonstrate that immortalization of human CD55+ hAPCs is feasible and results in cells with stable proliferative and adipogenic capacities over multiple passages. As these cells are cryopreservable, they provide the additional advantage over primary cells of allowing repeated studies in both 2D and 3D model systems with the same genetic background. (234/250).

Impaired phosphocreatine metabolism in white adipocytes promotes inflammation.

The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.

Human White Adipose Tissue Displays Selective Insulin Resistance in the Obese State.

Selective hepatic insulin resistance is a feature of obesity and type 2 diabetes. Whether similar mechanisms operate in white adipose tissue (WAT) of those with obesity and to what extent these are normalized by weight loss are unknown. We determined insulin sensitivity by hyperinsulinemic euglycemic clamp and insulin response in subcutaneous WAT by RNA sequencing in 23 women with obesity before and 2 years after bariatric surgery. To control for effects of surgery, women postsurgery were matched to never-obese women. Multidimensional analyses of 138 samples allowed us to classify the effects of insulin into three distinct expression responses: a common set was present in all three groups and included genes encoding several lipid/cholesterol biosynthesis enzymes; a set of obesity-attenuated genes linked to tissue remodeling and protein translation was selectively regulated in the two nonobese states; and several postobesity-enriched genes encoding proteins involved in, for example, one-carbon metabolism were only responsive to insulin in the women who had lost weight. Altogether, human WAT displays a selective insulin response in the obese state, where most genes are normalized by weight loss. This comprehensive atlas provides insights into the transcriptional effects of insulin in WAT and may identify targets to improve insulin action.

Age-Induced Reduction in Human Lipolysis: A Potential Role for Adipocyte Noradrenaline Degradation.

Gao et al. report that the observed reduction in adipose lipolysis with age in women could be explained by an upregulation of the catecholamine-degradation pathway in subcutaneous adipocytes. However, in contrast to findings in mice, these pathways are enriched in adipocytes and not in immune cells, suggesting species-specific differences in aging mechanisms.

Partitioning of MLX-Family Transcription Factors to Lipid Droplets Regulates Metabolic Gene Expression.

Lipid droplets (LDs) store lipids for energy and are central to cellular lipid homeostasis. The mechanisms coordinating lipid storage in LDs with cellular metabolism are unclear but relevant to obesity-related diseases. Here we utilized genome-wide screening to identify genes that modulate lipid storage in macrophages, a cell type involved in metabolic diseases. Among ∼550 identified screen hits is MLX, a basic helix-loop-helix leucine-zipper transcription factor that regulates metabolic processes. We show that MLX and glucose-sensing family members MLXIP/MondoA and MLXIPL/ChREBP bind LDs via C-terminal amphipathic helices. When LDs accumulate in cells, these transcription factors bind to LDs, reducing their availability for transcriptional activity and attenuating the response to glucose. Conversely, the absence of LDs results in hyperactivation of MLX target genes. Our findings uncover a paradigm for a lipid storage response in which binding of MLX transcription factors to LD surfaces adjusts the expression of metabolic genes to lipid storage levels.

Identification and characterization of a novel DGAT1 missense mutation associated with congenital diarrhea.

Acyl-CoA:diacylglycerol acyltransferase (DGAT)1 and DGAT2 catalyze triglyceride (TG) biosynthesis in humans. Biallelic loss-of-function mutations in human DGAT1 result in severe congenital diarrhea and protein-losing enteropathy. Additionally, pharmacologic inhibition of DGAT1 led to dose-related diarrhea in human clinical trials. Here we identify a previously unknown DGAT1 mutation in identical twins of South Asian descent. These male patients developed watery diarrhea shortly after birth, with protein-losing enteropathy and failure to thrive. Exome sequencing revealed a homozygous recessive mutation in DGAT1, c.314T>C, p.L105P. We show here that the p.L105P DGAT1 enzyme produced from the mutant allele is less abundant, resulting in partial loss of TG synthesis activity and decreased formation of lipid droplets in patient-derived primary dermal fibroblasts. Thus, in contrast with complete loss-of-function alleles of DGAT1, the p.L105P missense allele partially reduces TG synthesis activity and causes a less severe clinical phenotype. Our findings add to the growing recognition of DGAT1 deficiency as a cause of congenital diarrhea with protein-losing enteropathy and indicate that DGAT1 mutations result in a spectrum of diseases.

Adipose and Circulating CCL18 Levels Associate With Metabolic Risk Factors in Women.

CONTEXT: Cardiometabolic complications in obesity may be linked to white adipose tissue (WAT) dysfunction. Transcriptomic studies of Sc WAT have reported that CCL18, encoding the CC chemokine ligand 18 (CCL18), is increased in obesity/insulin resistance but its functional role is unknown. OBJECTIVE: Our objectives were to determine if CCL18 is secreted from Sc WAT and if secreted and/or serum levels associate with metabolic phenotypes. We also planned to define the primary cellular source and if CCL18 exerts effects on adipocytes. DESIGN: This is a cohort study. SETTING: The study took place in an outpatient academic clinic. PARTICIPANTS: A total of 130 obese women scheduled for bariatric surgery and 35 nonobese controls were included. METHODS: Insulin sensitivity was assessed by hyperinsulinemic euglycemic clamp or homeostasis model assessment. CCL18 was analyzed in serum/WAT incubates by ELISA. Effects of recombinant CCL18 was determined in cultures of primary human adipocytes and the monocyte cell line THP-1 differentiated into M0/M1/M2 macrophages. MAIN OUTCOME MEASURE: Association with metabolic risk factors was measured. RESULTS: CCL18 was secreted from WAT and the levels correlated positively with insulin resistance, Adult Treatment Panel III risk score and plasma triglycerides, independent of body mass index and better than other established adipocytokines. In 80 obese women, S-CCL18 levels were significantly higher in insulin resistant compared with insulin sensitive subjects. In WAT CCL18 mRNA was expressed in macrophages and correlated positively with immune-related genes, particularly those enriched in M2 macrophages. While CCL18 increased cyto-/chemokine expression in M0/M2-THP-1 cells, human adipocytes showed no responses in vitro. CONCLUSIONS: Circulating and WAT-secreted CCL18 correlates with insulin resistance and metabolic risk score. Because CCL18 is macrophage-specific and associates with adipose immune gene expression, it may constitute a marker of WAT inflammation.

Transplanted Bone Marrow-Derived Cells Contribute to Human Adipogenesis.

Because human white adipocytes display a high turnover throughout adulthood, a continuous supply of precursor cells is required to maintain adipogenesis. Bone marrow (BM)-derived progenitor cells may contribute to mammalian adipogenesis; however, results in animal models are conflicting. Here we demonstrate in 65 subjects who underwent allogeneic BM or peripheral blood stem cell (PBSC) transplantation that, over the entire lifespan, BM/PBSC-derived progenitor cells contribute ∼10% to the subcutaneous adipocyte population. While this is independent of gender, age, and different transplantation-related parameters, body fat mass exerts a strong influence, with up to 2.5-fold increased donor cell contribution in obese individuals. Exome and whole-genome sequencing of single adipocytes suggests that BM/PBSC-derived progenitors contribute to adipose tissue via both differentiation and cell fusion. Thus, at least in the setting of transplantation, BM serves as a reservoir for adipocyte progenitors, particularly in obese subjects.

Saturated fatty acids in human visceral adipose tissue are associated with increased 11- ß-hydroxysteroid-dehydrogenase type 1 expression.

BACKGROUND: Visceral fat accumulation is associated with metabolic disease. It is therefore relevant to study factors that regulate adipose tissue distribution. Recent data shows that overeating saturated fatty acids promotes greater visceral fat storage than overeating unsaturated fatty acids. Visceral adiposity is observed in states of hypercortisolism, and the enzyme 11-ß-hydroxysteroid-dehydrogenase type 1 (11ß-hsd1) is a major regulator of cortisol activity by converting inactive cortisone to cortisol in adipose tissue. We hypothesized that tissue fatty acid composition regulates body fat distribution through local effects on the expression of 11ß-hsd1 and its corresponding gene (HSD11B1) resulting in altered cortisol activity. FINDINGS: Visceral- and subcutaneous adipose tissue biopsies were collected during Roux-en-Y gastric bypass surgery from 45 obese women (BMI; 41±4 kg/m2). The fatty acid composition of each biopsy was measured and correlated to the mRNA levels of HSD11B1. 11ß-hsd1 protein levels were determined in a subgroup (n=12) by western blot analysis. Our main finding was that tissue saturated fatty acids (e.g. palmitate) were associated with increased 11ß-hsd1 gene- and protein-expression in visceral but not subcutaneous adipose tissue. CONCLUSIONS: The present study proposes a link between HSD11B1 and saturated fatty acids in visceral, but not subcutaneous adipose tissue. Nutritional regulation of visceral fat mass through HSD11B1 is of interest for the modulation of metabolic risk and warrants further investigation.

Early B cell factor 1 regulates adipocyte morphology and lipolysis in white adipose tissue.

White adipose tissue (WAT) morphology characterized by hypertrophy (i.e., fewer but larger adipocytes) associates with increased adipose inflammation, lipolysis, insulin resistance, and risk of diabetes. However, the causal relationships and the mechanisms controlling WAT morphology are unclear. Herein, we identified EBF1 as an adipocyte-expressed transcription factor with decreased expression/activity in WAT hypertrophy. In human adipocytes, the regulatory targets of EBF1 were enriched for genes controlling lipolysis and adipocyte morphology/differentiation, and in both humans and murine models, reduced EBF1 levels associated with increased lipolysis and adipose hypertrophy. Although EBF1 did not affect adipose inflammation, TNFα reduced EBF1 gene expression. High-fat diet intervention in Ebf1(+/-) mice resulted in more pronounced WAT hypertrophy and attenuated insulin sensitivity compared with wild-type littermate controls. We conclude that EBF1 is an important regulator of adipose morphology and fat cell lipolysis and may constitute a link between WAT inflammation, altered lipid metabolism, adipose hypertrophy, and insulin resistance.

Changes in subcutaneous fat cell volume and insulin sensitivity after weight loss.

OBJECTIVE: Large subcutaneous fat cells associate with insulin resistance and high risk of developing type 2 diabetes. We investigated if changes in fat cell volume and fat mass correlate with improvements in the metabolic risk profile after bariatric surgery in obese patients. RESEARCH DESIGN AND METHODS: Fat cell volume and number were measured in abdominal subcutaneous adipose tissue in 62 obese women before and 2 years after Roux-en-Y gastric bypass (RYGB). Regional body fat mass by dual-energy X-ray absorptiometry; insulin sensitivity by hyperinsulinemic-euglycemic clamp; and plasma glucose, insulin, and lipid profile were assessed. RESULTS: RYGB decreased body weight by 33%, which was accompanied by decreased adipocyte volume but not number. Fat mass in the measured regions decreased and all metabolic parameters were improved after RYGB (P < 0.0001). Whereas reduced subcutaneous fat cell size correlated strongly with improved insulin sensitivity (P = 0.0057), regional changes in fat mass did not, except for a weak correlation between changes in visceral fat mass and insulin sensitivity and triglycerides. The curve-linear relationship between fat cell size and fat mass was altered after weight loss (P = 0.03). CONCLUSIONS: After bariatric surgery in obese women, a reduction in subcutaneous fat cell volume associates more strongly with improvement of insulin sensitivity than fat mass reduction per se. An altered relationship between adipocyte size and fat mass may be important for improving insulin sensitivity after weight loss. Fat cell size reduction could constitute a target to improve insulin sensitivity.

Ceruloplasmin is a novel adipokine which is overexpressed in adipose tissue of obese subjects and in obesity-associated cancer cells.

Obesity confers an increased risk of developing specific cancer forms. Although the mechanisms are unclear, increased fat cell secretion of specific proteins (adipokines) may promote/facilitate development of malignant tumors in obesity via cross-talk between adipose tissue(s) and the tissues prone to develop cancer among obese. We searched for novel adipokines that were overexpressed in adipose tissue of obese subjects as well as in tumor cells derived from cancers commonly associated with obesity. For this purpose expression data from human adipose tissue of obese and non-obese as well as from a large panel of human cancer cell lines and corresponding primary cells and tissues were explored. We found expression of ceruloplasmin to be the most enriched in obesity-associated cancer cells. This gene was also significantly up-regulated in adipose tissue of obese subjects. Ceruloplasmin is the body's main copper carrier and is involved in angiogenesis. We demonstrate that ceruloplasmin is a novel adipokine, which is produced and secreted at increased rates in obesity. In the obese state, adipose tissue contributed markedly (up to 22%) to the total circulating protein level. In summary, we have through bioinformatic screening identified ceruloplasmin as a novel adipokine with increased expression in adipose tissue of obese subjects as well as in cells from obesity-associated cancers. Whether there is a causal relationship between adipose overexpression of ceruloplasmin and cancer development in obesity cannot be answered by these cross-sectional comparisons.

Additive effects of microRNAs and transcription factors on CCL2 production in human white adipose tissue.

Adipose tissue inflammation is present in insulin-resistant conditions. We recently proposed a network of microRNAs (miRNAs) and transcription factors (TFs) regulating the production of the proinflammatory chemokine (C-C motif) ligand-2 (CCL2) in adipose tissue. We presently extended and further validated this network and investigated if the circuits controlling CCL2 can interact in human adipocytes and macrophages. The updated subnetwork predicted that miR-126/-193b/-92a control CCL2 production by several TFs, including v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (ETS1), MYC-associated factor X (MAX), and specificity protein 12 (SP1). This was confirmed in human adipocytes by the observation that gene silencing of ETS1, MAX, or SP1 attenuated CCL2 production. Combined gene silencing of ETS1 and MAX resulted in an additive reduction in CCL2 production. Moreover, overexpression of miR-126/-193b/-92a in different pairwise combinations reduced CCL2 secretion more efficiently than either miRNA alone. However, although effects on CCL2 secretion by co-overexpression of miR-92a/-193b and miR-92a/-126 were additive in adipocytes, the combination of miR-126/-193b was primarily additive in macrophages. Signals for miR-92a and -193b converged on the nuclear factor-κB pathway. In conclusion, TF and miRNA-mediated regulation of CCL2 production is additive and partly relayed by cell-specific networks in human adipose tissue that may be important for the development of insulin resistance/type 2 diabetes.

MicroRNAs regulate human adipocyte lipolysis: effects of miR-145 are linked to TNF-α.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.

MicroRNA profiling links miR-378 to enhanced adipocyte lipolysis in human cancer cachexia.

Cancer cachexia is associated with pronounced adipose tissue loss due to, at least in part, increased fat cell lipolysis. MicroRNAs (miRNAs) have recently been implicated in controlling several aspects of adipocyte function. To gain insight into the possible impact of miRNAs on adipose lipolysis in cancer cachexia, global miRNA expression was explored in abdominal subcutaneous adipose tissue from gastrointestinal cancer patients with (n = 10) or without (n = 11) cachexia. Effects of miRNA overexpression or inhibition on lipolysis were determined in human in vitro differentiated adipocytes. Out of 116 miRNAs present in adipose tissue, five displayed distinct cachexia-associated expression according to both microarray and RT-qPCR. Four (miR-483-5p/-23a/-744/-99b) were downregulated, whereas one (miR-378) was significantly upregulated in cachexia. Adipose expression of miR-378 associated strongly and positively with catecholamine-stimulated lipolysis in adipocytes. This correlation is most probably causal because overexpression of miR-378 in human adipocytes increased catecholamine-stimulated lipolysis. In addition, inhibition of miR-378 expression attenuated stimulated lipolysis and reduced the expression of LIPE, PLIN1, and PNPLA2, a set of genes encoding key lipolytic regulators. Taken together, increased miR-378 expression could play an etiological role in cancer cachexia-associated adipose tissue loss via effects on adipocyte lipolysis.

Characterization of the Wnt inhibitors secreted frizzled-related proteins (SFRPs) in human adipose tissue.

CONTEXT: Wnt signaling regulates adipogenesis and adipocyte function. Secreted frizzled-related proteins (SFRPs) are a family of secreted proteins (SFRP1-5) that bind and inhibit Wnts. Several members, including SFRP5, have recently been implicated in adipocyte dysfunction in obesity. OBJECTIVE: Our objective was to characterize the expression, secretion, and function of the SFRP family in human white adipose tissue (WAT) and fat cells. DESIGN: SFRP1-5 mRNA expression was measured in human sc and visceral WAT from lean and obese individuals and correlated to insulin sensitivity. SFRP secretion from WAT explants was assessed by ELISA. Gene expression of SFRPs in cultured adipocytes during and after differentiation was determined. Functional analyses were done by gene silencing or incubations with recombinant SFRPs. RESULTS: SFRP1-4, but not SFRP5, mRNA levels were altered in obesity. However, although SFRP1 was down-regulated and correlated positively with insulin sensitivity, SFRP2-4 were up-regulated, particularly in visceral WAT, and associated with insulin resistance. Only SFRP1, SFRP2, and SFRP4 were secreted from WAT, thereby constituting adipokines. Individual knockdowns of SFRP1, SFRP2, or SFRP4 during adipogenesis did not affect terminal differentiation. Incubations with SFRP1 reduced the secretion of the proinflammatory cytokines IL-6 and monocyte chemotactic protein-1 (MCP1) and increased the release of adiponectin. CONCLUSIONS: SFRP1, SFRP2, and SFRP4 are adipokines, the expression of which correlates with insulin sensitivity. For SFRP1, this may be related to effects on the secretion of IL-6, MCP1, and adiponectin. In contrast to recent murine findings implicating SFRP5 in metabolic dysfunction, this SFRP is neither regulated by obesity nor actively secreted from human WAT.

Adipose tissue microRNAs as regulators of CCL2 production in human obesity.

In obesity, white adipose tissue (WAT) inflammation is linked to insulin resistance. Increased adipocyte chemokine (C-C motif) ligand 2 (CCL2) secretion may initiate adipose inflammation by attracting the migration of inflammatory cells into the tissue. Using an unbiased approach, we identified adipose microRNAs (miRNAs) that are dysregulated in human obesity and assessed their possible role in controlling CCL2 production. In subcutaneous WAT obtained from 56 subjects, 11 miRNAs were present in all subjects and downregulated in obesity. Of these, 10 affected adipocyte CCL2 secretion in vitro and for 2 miRNAs (miR-126 and miR-193b), regulatory circuits were defined. While miR-126 bound directly to the 3'-untranslated region of CCL2 mRNA, miR-193b regulated CCL2 production indirectly through a network of transcription factors, many of which have been identified in other inflammatory conditions. In addition, overexpression of miR-193b and miR-126 in a human monocyte/macrophage cell line attenuated CCL2 production. The levels of the two miRNAs in subcutaneous WAT were significantly associated with CCL2 secretion (miR-193b) and expression of integrin, α-X, an inflammatory macrophage marker (miR-193b and miR-126). Taken together, our data suggest that miRNAs may be important regulators of adipose inflammation through their effects on CCL2 release from human adipocytes and macrophages.

Differential effects of 1α,25-dihydroxycholecalciferol on MCP-1 and adiponectin production in human white adipocytes.

BACKGROUND/AIM: Obesity is characterized by a low-grade inflammation in white adipose tissue (WAT), which promotes insulin resistance. Low serum levels of 1α,25-dihydroxycholecalciferol (DHCC) associate with insulin resistance and higher body mass index although it is unclear whether vitamin D supplementation improves insulin sensitivity. We investigated the effects of DHCC on adipokine gene expression and secretion in adipocytes focusing on two key factors with pro-inflammatory [monocyte chemoattractant protein-1 (MCP-1/CCL2)] and anti-inflammatory [adiponectin (ADIPOQ)] effects. METHODS: Pre-adipocytes were isolated from human subcutaneous WAT and cultured until full differentiation. Differentiated adipocytes were either pre-treated with DHCC (10(-7) M) and subsequently incubated with tumor necrosis factor-α (TNFα, 100 ng/mL) or concomitantly incubated with TNFα/DHCC. MCP1 and adiponectin mRNA expression was measured by RT-PCR and protein release by ELISA. RESULTS: DHCC was not toxic and did not affect adipocyte morphology or the mRNA levels of adipocyte-specific genes. TNFα induced a significant increase in CCL2 mRNA and protein secretion, while DHCC alone reduced CCL2 mRNA expression (~25%, p < 0.05). DHCC attenuated TNFα-induced CCL2 mRNA expression in both pre-incubation (~15%, p < 0.05) and concomitant (~60%, p < 0.01) treatments. TNFα reduced ADIPOQ mRNA (~80%) and secretion (~35%). DHCC alone decreased adiponectin secretion to a similar degree (~35%, p < 0.05). Concomitant treatment with DHCC/TNFα for 48 h had an additive effect, resulting in a pronounced reduction in adiponectin secretion (~70%). CONCLUSIONS: DHCC attenuates MCP-1 and adiponectin production in human adipocytes, thereby reducing the expression of both pro- and anti-inflammatory factors. These effects may explain the difficulties so far in determining the role of DHCC in insulin sensitivity and obesity in humans.

Twist1 in human white adipose tissue and obesity.

CONTEXT: Twist1 is a transcription factor implicated in the regulation of TNFα signaling and was recently shown to be highly expressed in both human and murine adipose tissue, but its role in obesity is unknown. OBJECTIVE: Our objective was to assess the expression of twist1 in human white adipose tissue (WAT), its relationship to obesity and insulin sensitivity, and how it modifies TNFα-mediated inflammation in adipocytes. PROCEDURE: Twist mRNA levels were measured in WAT from 130 nonobese and obese subjects, and its relation to clinical parameters was assessed. Twist1 expression was measured before and after weight loss as well as in different adipose regions. Human in vitro differentiated adipocytes were treated with TNFα under control conditions or after twist1 gene silencing by RNA interference. Gene expression and secretion of proinflammatory proteins were measured. RESULTS: Twist1 expression was low in obese subjects and increased after weight loss. Twist1 mRNA levels correlated with adiponectin levels and inversely with insulin resistance as well as adipocyte volume (P < 0.001 for all). Low twist1 expression associated with a hypertrophic adipose tissue and high secretion of TNFα and monocyte chemoattractant protein-1 from WAT. Finally, twist1 silencing in human adipocytes enhanced TNFα-induced monocyte chemoattractant protein-1 expression and secretion, which was paralleled by an increase in the mRNA expression of the nuclear factor-κB gene RelA. CONCLUSIONS: Low twist1 expression in human WAT correlates with obesity and an insulin-resistant phenotype, which may be mediated by an increased sensitivity to the proinflammatory effect of TNFα.

A possible inflammatory role of twist1 in human white adipocytes.

OBJECTIVE: Twist1 is a transcription factor that is highly expressed in murine brown and white adipose tissue (WAT) and negatively regulates fatty acid oxidation in mice. The role of twist1 in WAT is not known and was therefore examined. RESEARCH DESIGN AND METHODS: The expression of twist1 was determined by quantitative real-time PCR in different tissues and in different cell types within adipose tissue. The effect of twist1 small interfering RNA on fatty acid oxidation, lipolysis, adipokine secretion, and mRNA expression was determined in human adipocytes. The interaction between twist1 and specific promoters in human adipocytes was investigated by chromatin immunoprecipitation (ChIP) and reporter assays. RESULTS: Twist1 was highly expressed in human WAT compared with a set of other tissues and found predominantly in adipocytes. Twist1 levels increased during in vitro differentiation of human preadipocytes. Gene silencing of twist1 in human white adipocytes had no effect on lipolysis or glucose transport. Unexpectedly, and in contrast with results in mice, twist1 RNA interference reduced fatty acid oxidation. Furthermore, the expression and secretion of the inflammatory factors tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein-1 were downregulated by twist1 silencing. ChIP and reporter assays confirmed twist1 interaction with the promoters of these genes. CONCLUSIONS: Twist1 may play a role in inflammation of human WAT because it can regulate the expression and secretion of inflammatory adipokines via direct transcriptional effects in white adipocytes. Furthermore, twist1 may, in contrast to findings in mice, be a positive regulator of fatty acid oxidation in human white adipocytes.