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.

Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1^−/−) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1^−/− MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic metabolic-endocrine dysfunction.

Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA sequencing (snRNA-seq) analyses largely uncovered overlapping or similar cell-type landscapes. We hypothesized that adipocyte subtypes, differentiation trajectories and/or intercellular communication patterns could illuminate this depot similarity–difference gap. For this, we performed snRNA-seq of human subcutaneous or visceral adipose tissues (five or ten samples, respectively). Of 27,665 adipocyte nuclei in both depots, most were ‘classical’, namely enriched in lipid metabolism pathways. However, we also observed ‘nonclassical’ adipocyte subtypes, enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization or angiogenesis or ribosomal and mitochondrial processes. Pseudo-temporal analysis showed a developmental trajectory from adipose progenitor cells to classical adipocytes via nonclassical adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Last, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation and intercellular communication patterns to human fat depot differences.

Protein S-nitrosylation is a reversible protein modification implicated in both physiological and pathophysiological regulation of protein function. In obesity, skeletal muscle insulin resistance is associated with increased S-nitrosylation of insulin-signaling proteins. However, whether adipose tissue is similarly affected in obesity and, if so, what are the causes and functional consequences of increased S-nitrosylation in this tissue are unknown. Total protein S-nitrosylation was increased in intra-abdominal adipose tissue of obese humans and in high fat-fed or leptin-deficient ob/ob mice. Both the insulin receptor β-subunit and Akt were S-nitrosylated, correlating with body weight. Elevated protein and mRNA expression of inducible NO synthase and decreased protein levels of thioredoxin reductase were associated with increased adipose tissue S-nitrosylation. Cultured differentiated pre-adipocyte cell lines exposed to the NO donors S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine exhibited diminished insulin-stimulated phosphorylation of Akt but not of GSK3 nor of insulin-stimulated glucose uptake. Yet the anti-lipolytic action of insulin was markedly impaired in both cultured adipocytes and in mice injected with GSNO prior to administration of insulin. In cells, impaired ability of insulin to diminish phosphorylated PKA substrates in response to isoproterenol suggested impaired insulin-induced activation of PDE3B. Consistently, increased S-nitrosylation of PDE3B was detected in adipose tissue of high fat-fed obese mice. Site-directed mutagenesis revealed that Cys-768 and Cys-1040, two putative sites for S-nitrosylation adjacent to the substrate-binding site of PDE3B, accounted for ∼50% of its GSNO-induced S-nitrosylation. Collectively, PDE3B and the anti-lipolytic action of insulin may constitute novel targets for increased S-nitrosylation of adipose tissue in obesity.

Visceral adipose tissue foam cells are increased in human obesity, and were implicated in adipose dysfunction and increased cardio-metabolic risk. In the circulation, non-classical monocytes (NCM) are elevated in obesity and associate with atherosclerosis and type 2 diabetes. We hypothesized that circulating NCM correlate and/or are functionally linked to visceral adipose tissue foam cells in obesity, potentially providing an approach to estimate visceral adipose tissue status in the non-surgical obese patient. We preformed ex-vivo functional studies utilizing sorted monocyte subclasses from healthy donors. Moreover, we assessed circulating blood monocyte subclasses and visceral fat adipose tissue macrophage (ATM) lipid content by flow-cytometry in paired blood and omental-fat samples collected from patients (n = 65) undergoing elective abdominal surgery. Ex-vivo, NCM and NCM-derived macrophages exhibited lower lipid accumulation capacity compared to classical or intermediate monocytes/-derived macrophages. Moreover, of the three subclasses, NCM exhibited the lowest migration towards adipose tissue conditioned-media. In a cohort of n = 65, increased %NCM associated with higher BMI (r = 0.250,p<0.05) and ATM lipid content (r = 0.303,p<0.05). Among patients with BMI≥25Kg/m², linear regression models adjusted for age, sex or BMI revealed that NCM independently associate with ATM lipid content, particularly in men. Collectively, although circulating blood NCM are unlikely direct functional precursor cells for adipose tissue foam cells, their increased percentage in the circulation may clinically reflect higher lipid content in visceral ATMs.