IL-1 signaling in obesity-induced hepatic lipogenesis and steatosis

Abstract

Non-alcoholic fatty liver disease is prevalent in human obesity and type 2 diabetes, and is characterized by increases in both hepatic triglyceride accumulation (denoted as steatosis) and expression of pro-inflammatory cytokines such as IL-1β. We report here that the development of hepatic steatosis requires IL-1 signaling, which upregulates Fatty acid synthase to promote hepatic lipogenesis. Using clodronate liposomes to selectively deplete liver Kupffer cells in ob/ob mice, we observed remarkable amelioration of obesity-induced hepatic steatosis and reductions in liver weight, triglyceride content and lipogenic enzyme expressions. Similar results were obtained with diet-induced obese mice, although visceral adipose tissue macrophage depletion also occurred in response to clodronate liposomes in this model. There were no differences in the food intake, whole body metabolic parameters, serum β-hydroxybutyrate levels or lipid profiles due to clodronate-treatment, but hepatic cytokine gene expressions including IL-1β were decreased. Conversely, treatment of primary mouse hepatocytes with IL-1β significantly increased triglyceride accumulation and Fatty acid synthase expression. Furthermore, the administration of IL-1 receptor antagonist to obese mice markedly reduced obesity-induced steatosis and hepatic lipogenic gene expression. Collectively, our findings suggest that IL-1β signaling upregulates hepatic lipogenesis in obesity, and is essential for the induction of pathogenic hepatic steatosis in obese mice.

Figure 1. Clodronate liposomes effectively deplete KCs and VATMs of DIO mice.

Wild type mice were fed a HFD for 13 weeks and injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes i.p. over 6 days with a 3-day interval. We observed no changes in the total animal body weight of PBS liposome-treated mice (42.4±1.8 to 41.6±1.9) or clodronate-treated mice (44.01±2.2 to 41.5±2.6). VAT SVF was isolated and FACS analysis was performed. A.) Upper panels, PBS liposome-treated mice. Lower panels, clodronate-treated mice. Left panels, SiglecF-negative, CD11b- and F4/80-positive cells. Right panels, CD11c-positive cells. Panels are representative of 6 animals per group. B.) Percentage of the total number of cells counted for both CD11b- and F4/80-positive cells and CD11c-positive cells in the VAT of DIO mice. RNA was extracted and quantitative RT-PCR was performed for standard macrophage markers (F480, CD11c) in C.) VAT and D.) liver of DIO mice. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups. *P≤0.05, **P≤0.01.

Figure 2. Clodronate liposome-mediated KC and VATM depletion ameliorates hepatic steatosis in DIO mice.

Wild type mice were fed a HFD for 13 weeks and injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes by i.p. over 6 days with a 3-day interval. A.) Livers of DIO mice were isolated, fixed in 10% formalin, embedded in paraffin, and stained with H&E or frozen in OCT and stained with Oil-Red-O to assess steatosis. Images are representative of 12–16 animals at 10X and 20X magnification. B.) Livers of DIO animals were weighed and data is represented as a percentage of total body weight. C.) Total TGs were extracted and normalized to tissue weight. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups. *P≤0.05, **P≤0.01.

Figure 3. Clodronate-encapsulated liposomes deplete KCs but not VATMs in ob/ob mice.

8-week ob/ob mice were injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes by i.p. for 6 days with a 3-day interval. We observed no changes in the total animal body weight of PBS liposome-treated mice (44.2±0.7 to 45.2±0.6) or clodronate -treated mice (41.9±1.4 to 41.1±1.7). VAT SVF was isolated and FACS analysis was performed. A.) Upper panels, PBS liposome-treated mice. Lower panels, clodronate-treated animals. Left panels, SiglecF-negative, CD11b- and F4/80-positive cells. Right panels, CD11c-positive cells. Panels are representative of 4–6 animals per group. B.) Percentage of the total number of cells counted for both CD11b- and F4/80-positive cells and CD11c-positive cells in the VAT of ob/ob mice. RNA was extracted and quantitative RT-PCR was performed for standard macrophage markers (F480, CD11c) in C.) VAT and D.) liver of ob/ob mice. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups. *P≤0.05, ***P≤0.001.

Figure 4. Clodronate liposome-mediated KC depletion ameliorates hepatic steatosis in ob/ob mice.

8-week old male ob/ob mice were injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes by i.p. over 6 days with a 3-day interval. A.) Livers of ob/ob animals were isolated, fixed in 10% formalin, embedded in paraffin, and stained with H&E or frozen in OCT and stained with Oil-Red-O to assess steatosis. Images are representative of 7–10 animals at 10X and 20X magnification. B.) Livers of ob/ob animals were weighed and data is represented as a percentage of total body weight. C.) Total TGs were extracted and normalized to tissue weight. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups. *P≤0.05, ***P≤0.001.

Figure 5. Clodronate liposome-mediated KC depletion in DIO and ob/ob mice significantly reduces hepatic expression of genes involved in inflammation and lipogenesis.

Wild type mice fed a HFD for 13 weeks or 8-week ob/ob mice were injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes by i.p. over 6 days with a 3-day interval. Data is representative of 12–16 DIO and 7–10 ob/ob animals. Liver was isolated, RNA was extracted and subjected to quantitative RT-PCR for inflammatory (TNF-α, IL-1β, IL-1α, IL-6, Nlrp3) and lipogenic (Fasn, Acc2, Dgat, Scd1, Elov6, PPARγ, Cidea) gene expression in A,C.) DIO and B,D) ob/ob mice. E.) Protein was extracted from the livers of DIO animals and Western blot analysis was performed to detect Fas expression. PBS and Clodronate samples are from the same film, cut due to sample separation. F.) Relative densitometry from E. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups. *P≤0.05, ***P≤0.001.

Figure 6. Physiological concentrations of IL-1β elicit a biological response in primary mouse hepatocytes to increase TG accumulation and lipogenic gene expression.

Primary hepatocytes from wild type mice were isolated via perfusion, stimulated with the indicated doses of recombinant mouse IL-1β and evaluated after 24-hours. A.) Cells were fixed in 10% formalin and stained with Oil-Red-O to assess TG accumulation. B.) Fas expression was analyzed in primary hepatocyte cell lysates after a 24-hour stimulation with the indicated doses of recombinant IL-1β and normalized to β-actin. C). Relative densitometry from B. D.) TGs were extracted and measured after a 24-hour stimulation with recombinant IL-1β (10 ng/mL) and normalized to the amount of total protein. The data are representative of 4 experiments. All stimulations were performed in duplicate. Values represent the mean ± SEM. A paired student’s t-test was used for comparisons between groups *P≤0.05, ***P≤0.001.

Figure 7. Pharmacological intervention via inhibition of IL-1 signaling ameliorates diet-induced steatosis in DIO mice.

Wild type mice were fed a HFD for 13 weeks. At 32 days prior to sacrifice, mice were started on daily injections of IL-1Ra (Anakinra; 32 mg/kg) or saline by i.p administration. Data are representative of 10 mice per group. A.) Livers were isolated, fixed in 10% formalin, embedded in paraffin, and stained with H&E or frozen in OCT and stained with Oil-Red-O to assess steatosis. B.) Livers were isolated, weighed and represented as a percentage of total animal body weight. C.) Total TGs were extracted and normalized to tissue weight. RNA was extracted and subjected to quantitative RT-PCR for expression of genes involved in D.) inflammation (TNF-α, IL-1β, IL-1α, IL-6, Nlrp3), E.) lipogenesis (Fasn, Acc2, Dgat, Scd1, Elov6, PPARγ, Cidea) and G.) fatty acid oxidation (PPARα, Cpt1a). F.) EDTA plasma was drawn via the retro orbital sinus and serum was analyzed for IL-1Ra by ELISA. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups *P≤0.05, **P≤0.01.

Figure 8. Pharmacological intervention via inhibition of IL-1 signaling improves the glucose tolerance in DIO mice.

Wild type mice were fed a HFD for 13 weeks. At 32 days prior to sacrifice, mice were started on daily injections of IL-1Ra (Anakinra; 32 mg/kg) or saline by i.p. administration. Data are representative of 10 mice per group. At the end of 13 weeks, Anakinra-treated mice displayed no changes in A.) total animal body weight and were more glucose tolerant as demonstrated by a reduction in B.) area under the curve (AUC) for the intraperitoneal GTT and C.) fasting insulin levels. Values represent the mean ± SEM. An unpaired student’s t-test was used for comparisons between groups. *P≤0.05, **P≤0.01.