A role for adipocyte-derived lipopolysaccharide-binding protein in inflammation- and obesity-associated adipose tissue dysfunction.

AIMS/HYPOTHESIS: Circulating lipopolysaccharide-binding protein (LBP) is an acute-phase reactant known to be increased in obesity. We hypothesised that LBP is produced by adipose tissue (AT) in association with obesity. METHODS: LBP mRNA and LBP protein levels were analysed in AT from three cross-sectional (n = 210, n = 144 and n = 28) and three longitudinal (n = 8, n = 25, n = 20) human cohorts; in AT from genetically manipulated mice; in isolated adipocytes; and in human and murine cell lines. The effects of a high-fat diet and exposure to lipopolysaccharide (LPS) and peroxisome proliferator-activated receptor (PPAR)γ agonist were explored. Functional in vitro and ex vivo experiments were also performed. RESULTS: LBP synthesis and release was demonstrated to increase with adipocyte differentiation in human and mouse AT, isolated adipocytes and human and mouse cell lines (Simpson-Golabi-Behmel syndrome [SGBS], human multipotent adipose-derived stem [hMAD] and 3T3-L1 cells). AT LBP expression was robustly associated with inflammatory markers and increased with metabolic deterioration and insulin resistance in two independent cross-sectional human cohorts. AT LBP also increased longitudinally with weight gain and excessive fat accretion in both humans and mice, and decreased with weight loss (in two other independent cohorts), in humans with acquired lipodystrophy, and after ex vivo exposure to PPARγ agonist. Inflammatory agents such as LPS and TNF-α led to increased AT LBP expression in vivo in mice and in vitro, while this effect was prevented in Cd14-knockout mice. Functionally, LBP knockdown using short hairpin (sh)RNA or anti-LBP antibody led to increases in markers of adipogenesis and decreased adipocyte inflammation in human adipocytes. CONCLUSIONS/INTERPRETATION: Collectively, these findings suggest that LBP might have an essential role in inflammation- and obesity-associated AT dysfunction.

Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance.

CONTEXT: Recently irisin (encoded by Fndc5 gene) has been reported to stimulate browning and uncoupling protein 1 expression in sc adipose tissue of mice. OBJECTIVE: The objective of the study was to investigate FNDC5 gene expression in human muscle and adipose tissue and circulating irisin according to obesity, insulin sensitivity, and type 2 diabetes. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURE: Adipose tissue FNDC5 gene expression and circulating irisin (ELISA) were analyzed in 2 different cohorts (n = 125 and n = 76); muscle FNDC5 expression was also evaluated in a subcohort of 34 subjects. In vitro studies in human preadipocytes and adipocytes and in induced browning of 3T3-L1 cells (by means of retinoblastoma 1 silencing) were also performed. RESULTS: In both sc and visceral adipose tissue, FNDC5 gene expression decreased significantly in association with obesity and was positively associated with brown adipose tissue markers, lipogenic, insulin pathway-related, mitochondrial, and alternative macrophage gene markers and negatively associated with LEP, TNFα, and FSP27 (a known repressor of brown genes). Circulating irisin and irisin levels in adipose tissue were significantly associated with FNDC5 gene expression in adipose tissue. In muscle, the FNDC5 gene was 200-fold more expressed than in adipose tissue, and its expression was associated with body mass index, PGC1α, and other mitochondrial genes. In obese participants, FNDC5 gene expression in muscle was significantly decreased in association with type 2 diabetes. Interestingly, muscle FNDC5 gene expression was significantly associated with FNDC5 and UCP1 gene expression in visceral adipose tissue. In men, circulating irisin levels were negatively associated with obesity and insulin resistance. Irisin was secreted from human adipocytes into the media, and the induction of browning in 3T3-L1 cells led to increased secreted irisin levels. CONCLUSIONS: Decreased circulating irisin concentration and FNDC5 gene expression in adipose tissue and muscle from obese and type 2 diabetic subjects suggests a loss of brown-like characteristics and a potential target for therapy.

OCT1 Expression in adipocytes could contribute to increased metformin action in obese subjects.

OBJECTIVE: Metformin has been well characterized in vitro as a substrate of liver-expressed organic cation transporters (OCTs). We investigated the gene expression and protein levels of OCT-1 and OCT-2 in adipose tissue and during adipogenesis and evaluated their possible role in metformin action on adipocytes. RESEARCH DESIGN AND METHODS: OCT1 and OCT2 gene expressions were analyzed in 118 adipose tissue samples (57 visceral and 61 subcutaneous depots) and during human preadipocyte differentiation. To test the possible role of OCT1 mediating the response of adipocytes to metformin, cotreatments with cimetidine (OCT blocker, 0.5 and 5 mmol/l) and metformin were made on human preadipocytes and subcutaneous adipose tissue (SAT). RESULTS: OCT1 gene was expressed in both subcutaneous and visceral adipose tissue. In both fat depots, OCT1 gene expression and protein levels were significantly increased in obese subjects. OCT1 gene expression in isolated preadipocytes significantly increased during differentiation in parallel to adipogenic genes. Metformin (5 mmol/l) decreased the expression of lipogenic genes and lipid droplets accumulation while increasing AMP-activated protein kinase (AMPK) activation, preventing differentiation of human preadipocytes. Cotreatment with cimetidine restored adipogenesis. Furthermore, metformin decreased IL-6 and MCP-1 gene expression in comparison with differentiated adipocytes. Metformin (0.1 and 1 mmol/l) decreased adipogenic and inflammatory genes in SAT. OCT2 gene expression was not detected in adipose tissue and was very small in isolated preadipocytes, disappearing during adipogenesis. CONCLUSIONS: OCT1 gene expression and protein levels are detectable in adipose tissue. Increased OCT1 gene expression in adipose tissue of obese subjects might contribute to increased metformin action in these subjects.

Circulating pigment epithelium-derived factor levels are associated with insulin resistance and decrease after weight loss.

OBJECTIVE: We aimed to study circulating pigment epithelium-derived factor (PEDF) in vivo in association with insulin resistance and in vitro in human adipocytes. METHODS: Circulating PEDF (ELISA) and metabolic profile were assessed in 125 Caucasian men. PEDF levels were also assessed in an independent cohort of subjects (n = 33) to study the effects of changing insulin action. PEDF gene expression and secretion were measured during differentiation of human preadipocytes. RESULTS: In all subjects, PEDF was positively associated with body mass index (r = 0.326; P < 0.0001), waist-to-hip ratio (r = 0.380; P < 0.0001), HbA(1c), and fasting triglycerides and negatively with insulin sensitivity (r = -0.320; P < 0.0001). PEDF levels were significantly increased in subjects with altered glucose tolerance and type 2 diabetes. Of the inflammatory markers measured, PEDF levels were positively associated with serum soluble TNF-α receptor 1 and IL-10 in obese subjects. Interestingly, weight loss led to significantly decreased PEDF concentration from 34.8 ± 19.3 to 22.5 ± 14.2 µg/ml (P < 0.0001). Multiple linear regression analyses revealed that insulin sensitivity contributed independently to explain 14% of the variance in PEDF levels after controlling for the effects of body mass index, age, and log fasting triglycerides. Differences in PEDF observed after weight loss were related to changes in obesity, insulin resistance, and blood pressure measures. PEDF gene expression and secretion increased during differentiation of human preadipocytes. CONCLUSION: Circulating PEDF is associated with insulin sensitivity. The findings show, for the first time in humans, that PEDF concentrations decrease significantly after weight loss in association with blood pressure. PEDF seems to be involved in human adipocyte biology.

MiRNA expression profile of human subcutaneous adipose and during adipocyte differentiation.

BACKGROUND: Potential regulators of adipogenesis include microRNAs (miRNAs), small non-coding RNAs that have been recently shown related to adiposity and differentially expressed in fat depots. However, to date no study is available, to our knowledge, regarding miRNAs expression profile during human adipogenesis. Thereby, the aim of this study was to investigate whether miRNA pattern in human fat cells and subcutaneous adipose tissue is associated to obesity and co-morbidities and whether miRNA expression profile in adipocytes is linked to adipogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We performed a global miRNA expression microarray of 723 human and 76 viral mature miRNAs in human adipocytes during differentiation and in subcutaneous fat samples from non-obese (n = 6) and obese with (n = 9) and without (n = 13) Type-2 Diabetes Mellitus (DM-2) women. Changes in adipogenesis-related miRNAs were then validated by RT-PCR. Fifty of 799 miRNAs (6.2%) significantly differed between fat cells from lean and obese subjects. Seventy miRNAs (8.8%) were highly and significantly up or down-regulated in mature adipocytes as compared to pre-adipocytes. Otherwise, 17 of these 799 miRNAs (2.1%) were correlated with anthropometrical (BMI) and/or metabolic (fasting glucose and/or triglycerides) parameters. We identified 11 miRNAs (1.4%) significantly deregulated in subcutaneous fat from obese subjects with and without DM-2. Interestingly, most of these changes were associated with miRNAs also significantly deregulated during adipocyte differentiation. CONCLUSIONS/SIGNIFICANCE: The remarkable inverse miRNA profile revealed for human pre-adipocytes and mature adipocytes hints at a closely crosstalk between miRNAs and adipogenesis. Such candidates may represent biomarkers and therapeutic targets for obesity and obesity-related complications.

Val1483Ile in FASN gene is linked to central obesity and insulin sensitivity in adult white men.

The Val1483Ile polymorphism in the human fatty acid synthase (FASN) gene is located within the interdomain region of the FASN close to the two dynamic active centers of the FASN enzyme and putatively affects FASN action. We aimed to evaluate the association of this polymorphism with obesity phenotypes, insulin sensitivity, and adipose tissue FASN activity in adult white subjects. The polymorphism was evaluated in association with metabolic variables in two independent studies: in a case-control study of 457 men (229 with normal glucose tolerance (NGT) and 228 with altered glucose tolerance (AGT)); and in 600 population-based NGT subjects (274 men and 326 women). Adipose tissue FASN activity was analyzed using the method of Nepokroeff. The Ile variant was associated with a lower waist-to-hip ratio (WHR) and a lower increase in weight over a 7-year period in NGT men. In a subset of 147 men, carriers of the Ile variant showed significantly increased insulin sensitivity. BMI (P < 0.001), WHR (P = 0.03), and Val1483Ile (P = 0.03), contributed independently to 37% of insulin sensitivity variance. In men from the population-based study, the Ile variant was associated with a lower BMI, WHR, fasting glucose, and systolic blood pressure compared with carriers of the Val variant. In agreement with these results, the adipose tissue FASN activity was significantly lower in subjects with the Ile variant (P = 0.01). In summary, adult white men with the Ile 1483 variant of the FASN gene seem protected from developing central obesity through decreased adipose tissue FASN activity.