Niklas Mejhert

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.

Partial Inhibition of Adipose Tissue Lipolysis Improves Glucose Metabolism and Insulin Sensitivity Without Alteration of Fat Mass

Partial inhibition of adipose tissue lipolysis does not increase fat mass but improves glucose metabolism and insulin sensitivity through modulation of fatty acid turnover and induction of fat cell de novo lipogenesis.

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.

Role of Follistatin in Promoting Adipogenesis in Women

CONTEXT Follistatin is a glycoprotein that binds and neutralizes biological activities of TGFbeta superfamily members including activin and myostatin. We previously identified by expression profiling that follistatin levels in white adipose tissue (WAT) were regulated by obesity. OBJECTIVE The objective of the study was to elucidate the role of follistatin in human WAT and obesity. DESIGN We measured secreted follistatin protein from WAT biopsies and fat cells in vitro. We also quantified follistatin mRNA expression in sc and visceral WAT and in WAT-fractionated cells and related it to obesity status, body region, and cellular origin. We investigated the effects of follistatin on adipocyte differentiation of progenitor cells in vitro. PARTICIPANTS Women (n = 66) with a wide variation in body mass index were recruited by advertisement and from a clinic for weight-reduction therapy. RESULTS WAT secreted follistatin in vitro. Follistatin mRNA levels in sc but not visceral WAT were decreased in obesity and restored to nonobese levels after weight reduction. Follistatin mRNA levels were high in the stroma-vascular fraction of WAT and low in adipocytes. Recombinant follistatin treatment promoted adipogenic differentiation of progenitor cells and neutralized the inhibitory action of myostatin on differentiation in vitro. Moreover, activin and myostatin signaling receptors were detected in WAT and adipocytes. CONCLUSION Follistatin is a new adipokine important for adipogenesis. Down-regulated WAT expression of follistatin in obesity may counteract adiposity but could, by inhibiting adipogenesis, contribute to hypertrophic obesity (large fat cells) 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.

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.

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.

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-α, 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.

Role of Receptor-Interacting Protein 140 in human fat cells

BackgroundMice lacking Receptor-interacting protein 140 (RIP140) have reduced body fat which at least partly is mediated through increased lipid and glucose metabolism in adipose tissue. In humans, RIP140 is lower expressed in visceral white adipose tissue (WAT) of obese versus lean subjects. We investigated the role of RIP140 in human subcutaneous WAT, which is the major fat depot of the body.MethodsMessenger RNA levels of RIP140 were measured in samples of subcutaneous WAT from women with a wide variation in BMI and in different human WAT preparations. RIP140 mRNA was knocked down with siRNA in in vitro differentiated adipocytes and the impact on glucose transport and mRNA levels of target genes determined.ResultsRIP140 mRNA levels in subcutaneous WAT were decreased among obese compared to lean women and increased by weight-loss, but did not associate with mitochondrial DNA copy number. RIP140 expression increased during adipocyte differentiation in vitro and was higher in isolated adipocytes compared to corresponding pieces of WAT. Knock down of RIP140 increased basal glucose transport and mRNA levels of glucose transporter 4 and uncoupling protein-1.ConclusionsHuman RIP140 inhibits glucose uptake and the expression of genes promoting energy expenditure in the same fashion as the murine orthologue. Increased levels of human RIP140 in subcutaneous WAT of lean subjects may contribute to economize on energy stores. By contrast, the function and expression pattern does not support that RIP140 regulate human obesity.

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.