ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype

OBJECTIVE: Obesity variably disrupts human health, but molecular-based patients' health-risk stratification is limited. Adipose tissue (AT) stresses may link obesity with metabolic dysfunction, but how they signal in humans remains poorly-characterized. We hypothesized that a transcriptional AT stress-signaling cascade involving E2F1 and ASK1 (MAP3K5) molecularly defines high-risk obese subtype. METHODS: ASK1 expression in human AT biopsies was determined by real-time PCR analysis, and chromatin immunoprecipitation (ChIP) adopted to AT explants was used to evaluate the binding of E2F1 to the ASK1 promoter. Dual luciferase assay was used to measure ASK1 promoter activity in HEK293 cells. Effects of E2F1 knockout/knockdown in adipocytes was assessed utilizing mouse-embryonal-fibroblasts (MEF)-derived adipocyte-like cells from WT and E2F1-/- mice and by siRNA, respectively. ASK1 depletion in adipocytes was studied in MEF-derived adipocyte-like cells from WT and adipose tissue-specific ASK1 knockout mice (ASK1-ATKO). RESULTS: Human visceral-AT ASK1 mRNA (N = 436) was associated with parameters of obesity-related cardio-metabolic morbidity. Adjustment for E2F1 expression attenuated the association of ASK1 with fasting glucose, insulin resistance, circulating IL-6, and lipids (triglycerides, HDL-cholesterol), even after adjusting for BMI. Chromatin-immunoprecipitation in human-AT explants revealed BMI-associated increased occupancy of the ASK1 promoter by E2F1 (r2 = 0.847, p < 0.01). In adipocytes, siRNA-mediated E2F1-knockdown, and MEF-derived adipocytes of E2F1-knockout mice, demonstrated decreased ASK1 expression and signaling to JNK. Mutation/truncation of an E2F1 binding site in hASK1 promoter decreased E2F1-induced ASK1 promoter activity, whereas E2F1-mediated sensitization of ASK1 promoter to further activation by TNFα was inhibited by JNK-inhibitor. Finally, MEF-derived adipocytes from adipocyte-specific ASK1-knockout mice exhibited lower leptin and higher adiponectin expression and secretion, and resistance to the effects of TNFα. CONCLUSIONS: AT E2F1 -ASK1 molecularly defines a metabolically-detrimental obese sub-phenotype. Functionally, it may negatively affect AT endocrine function, linking AT stress to whole-body metabolic dysfunction

Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

<div><p>Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, <i>p<0.05</i>) and maximal (50%, <i>p<0.05</i>) OCR and gene expression of mitochondrial proteins and Bax without affecting cell viability or expression of glycolytic enzymes. Similar changes could be recapitulated by incubating cells with leptin, whereas, leptin-receptor specific antagonist inhibited the reduced OCR induced by conditioned media from obese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues.</p> </div

Leptin involvement in mediating obesity-reduced OCR.

<p>HCT116 cells were treated for 24 hours with CM collected from visceral AT of non-obese or obese subjects, with or without leptin antagonist (1 ng/ml). OCR levels were measured using the XF24 Analyzer. Non-obese (<i>n</i>=4), obese (<i>n</i>=7), non-obese antagonist (LepA non-obese, <i>n</i>=3), obese antagonist (LepA Obese, <i>n</i>=6). *, <i>P</i>< 0.05 vs. samples from obese (Student’s t-test). Results were normalized to protein concentration and expressed as percentage of control.</p

Effects of the obese CM on HCT116 glycolysis.

<p>HCT 116 cells were treated for 24 hours with CM collected from visceral AT of non-obese subjects (<i>n</i>=4) or obese subjects (<i>n</i>=9). (<i>A</i>) Bax gene expression levels were detected using quantitative real-time PCR. *, <i>P</i>< 0.05 vs. respective non-obese sample of each gene (Mann Whitney test). (<i>B</i>) Cell lysates were analyzed by Western blot. HM-7 and Caco₂ were used as controls (see Results) (<i>C</i>), Densitometric analysis of the Western blot data. *, <i>P</i>> 0.01, **, <i>P</i><0.05 (Two Way ANOVA, Bonferroni test).</p

Effect of leptin on HCT116 cells mitochondria.

<p>HCT116 cells were treated with DMEM (control) vs. leptin (100 ng/ml), for 24 hours. (<i>A</i>, <i>B</i>) Gene expression levels were detected using quantitative real-time PCR (<i>n</i>=4). *, <i>P</i>< 0.05, **, <i>P</i>< 0.01 vs. respective control of each gene (Student’s <i>t</i>-test). (<i>C</i>) Cell lysates were analyzed for cytochrome C (CytC, top panel) and β-actin (bottom panel) by Western blot, and densitometric analysis of the data was made. * <i>P</i>> 0.01, vs. control (Student’s <i>t</i>-test).</p

Interleukin-1β regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity

Effects of the obese CM on HCT116 cells mitochondria.

<p>HCT116 cells were treated for 24 hours with DMEM (control), CM collected from visceral AT of non-obese subjects. (<i>A</i>, <i>B</i>) Gene expression levels were detected using quantitative real-time PCR. Obese (<i>n</i>=8), non-obese (<i>n</i>=4). *, <i>P</i>< 0.05, **, <i>P</i>< 0.01 vs. respective non-obese sample of each gene (Mann Whitney test). (<i>C</i>) Gene expression levels were detected using quantitative real-time PCR. Control (<i>n</i>=3), non-obese (<i>n</i>=4), obese (<i>n</i>=9). *, <i>P</i>< 0.05, vs. control (Mann Whitney), ** <i>P</i>< 0.05, vs. non-obese (Mann Whitney). (<i>D</i>) Cell lysates were analyzed for Bax (top panel) or β-actin (bottom panel) antibodies, by Western blot and densitometric analysis was made. Vertical white lines denote image splicing to present only relevant bands, for clarity (shown is a single blot). Control (<i>n</i>=3), non-obese (<i>n</i>=3), obese (<i>n</i>=6). *, <i>P</i>> 0.01 vs. Control (One Way ANOVA, Tukey test). **, <i>P</i>< 0.001, vs. the non-obese samples (One Way ANOVA, Tukey test).</p

Role of adipose IL-1β in hepatocyte insulin resistance as revealed by co-culture approach.

<p>(<b>A</b>) Schematic representation of the fat explants – primary hepatocyte co-culture experimental design. (<b>B</b>) Insulin-stimulated Akt and GSK3 phosphorylation in primary hepatocytes from IL-1RIKO liver co-cultured or not with fat explants from WT-NC or WT-HFF and densitometry analysis of 2–5 mice per group. *p = 0.05 compared to incubation with fat explants from WT-HFF mice. (<b>C</b>) Insulin-stimulated Akt phosphorylation in primary hepatocytes from WT mice co-cultured with fat explants from WT-HFF, IL-1βKO-HFF, or WT-HFF in the presence of IL-1 receptor antagonist (WT-HFF+RA). The right graph depicts densitometry analysis of 7–9 mice per group. *p<0.05 compared to the signal obtained from primary hepatocytes incubated with fat explants from WT-HFF mice.</p