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

High fat feeding induces only a minor adipose tissue macrophage infiltration in IL-1βKO mice.

<p>(<b>A</b>) Quantitative real-time PCR analysis of adipose tissue (epididymal fat pad) of <i>IL-1b, il6</i> and <i>tnf</i> (normalized to <i>tbp</i>, <i>18S</i> and <i>36b4</i>). n≥3 per group. (<b>B–D</b>) Representative X20 light microscopy images of adipose tissue stained with H&E or with anti-Mac2 antibody. The mean±SEM number of crown like structures (CLS) per X10 microscopic field was counted as described in Materials and Methods. mRNA levels of <i>f4/80</i>, a macrophage marker, was assessed by quantitative real-time PCR. (<b>E–G</b>) Similar analysis as described above (B–D), but for IL-1βKO-NC and IL-1βKO-HFF mice. n = 3–6 animals per group were included in the analysis. *p<0.05 compared to IL-1βKO-NC; ***p<0.001 compared to WT-NC.</p

IL-1β impact on liver and adipose tissue mass and adipose tissue expandability.

<p>(<b>A</b>) Representative Computed Tomography (CT) scans (mid-coronal sections) of WT-NC and WT-HFF mice, and excised epididymal white adipose tissue (eWAT) and livers, and the mean±SEM of their weights. (<b>B</b>) Similar to A, but for IL-1βKO mice. ***p<0.001 compared to NC. (<b>C</b>) Spearman correlation between epididymal fat pads' weight and liver weight in HFF mice. (<b>D</b>) Adipocyte size distribution in WT and IL-1βKO mice, quantified as described in Methods. n = 3–6 mice per group. (<b>E</b>) Quantitative real-time PCR analysis of the indicated genes in epididymal adipose tissue in WT and IL-1βKO mice, respectively. n = 3–6 per group. *p<0.05 compared to IL-1βKO-NC ***p<0.0001 compared to WT-NC.</p

Role of adipose IL-1β in adipose-liver cross-talk as revealed by portally-drained mesenteric adipose tissue transplantation.

<p>(<b>A</b>) Serum IL-1β levels were measured in peripheral (systemic) or portal vein blood in WT mice fed normal chow (WT-NC) or high fat diet (WT-HFF). Connecting lines indicate the paired systemic-portal samples from a single mouse, n = 17–19. Red symbols represent≥20% higher IL-1β level in the portal compared to the systemic blood; (<b>B</b>) Schematic representation of the mesenteric adipose tissue transplantation experimental flow. (<b>C</b>)Portal blood levels of IL-1β were measured in sham-operated (n = 9) and in mice receiving mesenteric adipose tissue transplantation from a littermate WT mouse (Trans-WT, n = 13)*p<0.05. (<b>D, E</b>) Intra-peritoneal pyruvate tolerance test (PTT, 2 gr/Kg body weight) was performed in Sham (n = 9), Trans-WT (n = 13), and in mice receiving transplants from IL-1βKO mice (Trans-IL-1βKO, n = 7) four weeks post-transplantation. Area under the glucose levels curve (AUC) was calculated; *p<0.05 compared to Sham-operated controls.</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

Role of IL-1β in adipose tissue macrophage recruitment, ATM lipid content, and adipose inflammatory profile in dietary obesity.

<p>(<b>A</b>) FACS plots and gating of the stromal-vascular cells (SVCs) to detect adipose tissue macrophages (ATMs). Leucocytes (<b>B</b>), ATMs (<b>C</b>) in adipose tissue of WT-NC (n = 4), WT-HFF (n = 11), IL-1βKO-NC (n = 3) and IL-1βKO-HFF (n = 7). (<b>D</b>) Histogram of lipid content (determined with Bodipy) in representative mice of the 4 mouse groups (<b>E</b>)<b>.</b> Quantitative real-time PCR analysis of M1 or M2- genes in epididymal adipose tissue of (<b>F</b>) WT-NC, WT-HFF (n = 4, 11, respectively), and (<b>G</b>) IL-1βKO-NC and IL-1βKO-HFF (n = 3 and 7, respectively). The expression of each transcript was normalized to <i>tbp</i>, <i>18S</i> and <i>36b4</i> mRNA/rRNA, and a value of 1 was assigned to the normal chow group (NC) of each strain. *p<0.05, compared to NC; **p<0.01 compared to NC ***p<0.001.</p