Intervention with a caspase-1 inhibitor reduces obesity-associated hyperinsulinemia, non-alcoholic steatohepatitis and hepatic fibrosis in LDLR-/-.Leiden mice

Abstract

Background/objectives: Non-alcoholic steatohepatitis (NASH) is a serious liver condition, closely associated with obesity and insulin resistance. Recent studies have suggested an important role for inflammasome/caspase-1 in the development of NASH, but the potential therapeutic value of caspase-1 inhibition remains unclear. Therefore, we aimed to investigate the effects of caspase-1 inhibition in the ongoing disease process, to mimic the clinical setting.

Subjects/methods: To investigate effects of caspase-1 inhibition under therapeutic conditions, male LDLR-/-.Leiden mice were fed a high-fat diet (HFD) for 9 weeks to induce a pre-diabetic state before start of treatment. Mice were then continued on HFD for another 12 weeks, without (HFD) or with (HFD-YVAD) treatment with the caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg(-1) per day).

Results: Nine weeks of HFD feeding resulted in an obese phenotype, with obesity-associated hypertriglyceridemia, hypercholesterolemia, hyperglycemia and hyperinsulinemia. Treatment with Ac-YVAD-cmk did not affect further body weight gain or dyslipidemia, but did attenuate further progression of insulin resistance. Histopathological analysis of livers clearly demonstrated prevention of NASH development in HFD-YVAD mice: livers were less steatotic and neutrophil infiltration was strongly reduced. In addition, caspase-1 inhibition had a profound effect on hepatic fibrosis, as assessed by histological quantification of collagen staining and gene expression analysis of fibrosis-associated genes Col1a1, Acta2 and Tnfa.

Conclusions: Intervention with a caspase-1 inhibitor attenuated the development of NASH, liver fibrosis and insulin resistance. Our data support the importance of inflammasome/caspase-1 in the development of NASH and demonstrate that therapeutic intervention in the already ongoing disease process is feasible.

Figure 1

Effects of caspase-1 inhibition on high-fat diet (HFD)-induced body weight, adiposity and adipose tissue inflammation. LDLR−/−.Leiden mice were fed a HFD for 21 weeks (n=15) or HFD+caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg⁻¹ daily) starting treatment after 9 weeks of HFD (HFD+YVAD; n=15). Low-fat diet (LFD)-fed mice (n=15) were included as a reference. (a) HFD feeding significantly induced body weight relative to LFD, which was not affected by caspase-1 inhibition. (b) Body fat percentage was induced by HFD and was not affected in HFD+YVAD mice. (c) HFD induced pronounced adipose tissue inflammation specifically in the epididymal depot, which tended to be reduced in HFD+YVAD mice. (d) Representative photomicrographs of HPS-stained epididymal adipose tissue sections. All data are from the t=21 weeks time point and are mean±s.e.m. *P<0.05, ***P<0.001 compared with HFD.

Figure 2

Effects of caspase-1 inhibition on fasting glucose and insulin and glucose tolerance. LDLR−/−.Leiden mice were fed a high-fat diet for 21 weeks (HFD; n=15) or HFD+caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg⁻¹ daily) starting treatment after 9 weeks of HFD (HFD+YVAD; n=15). Low-fat diet (LFD)-fed mice (n=15) were included as a reference. (a) HFD-induced increases in fasted blood glucose relative to treatment start (t=9 weeks) were reduced in HFD+YVAD. (b) HFD-induced increases in fasted plasma insulin relative to treatment start (t=9 weeks) were reduced in HFD+YVAD. (c) The glucose response to an intraperitoneal glucose tolerance test (ipGTT; performed at t=20 weeks) was modified by caspase-1 inhibition. (d) HFD mice showed a clear insulin response during the ipGTT, whereas HFD+YVAD mice showed no response in plasma insulin. Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 compared with HFD at the same time point.

Figure 3

Effects of caspase-1 inhibition on hepatic steatosis. LDLR−/−.Leiden mice were fed a high-fat diet for 21 weeks (HFD; n=15) or HFD+caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg⁻¹ daily) starting treatment after 9 weeks of HFD (HFD+YVAD; n=15). Low-fat diet (LFD)-fed mice (n=15) were included as a reference. (a) Representative photomicrographs of hematoxylin and eosin-stained liver sections. (b) Macrovesicular steatosis did not differ between groups. (c) HFD feeding induced pronounced microvesicular steatosis, which was reduced in HFD+YVAD. (d) HFD-induced hepatic triglycerides tended to be reduced in HFD+YVAD. All data are from the t=21 weeks time point and are mean±s.e.m. **P<0.01 compared with HFD.

Figure 4

Effects of caspase-1 inhibition on hepatic inflammation. LDLR−/−.Leiden mice were fed a high-fat diet for 21 weeks (HFD; n=15) or HFD+caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg⁻¹ daily) starting treatment after 9 weeks of HFD (HFD+YVAD; n=15). Low-fat diet (LFD)-fed mice (n=15) were included as a reference. (a) HFD-induced plasma alanine aminotransferase (ALAT) was reduced in HFD+YVAD. (b) Lobular inflammation (number of inflammatory foci per x100 field) was induced by HFD, this induction was prevented by Ac-YVAD-cmk treatment. (c) F4/80 mRNA expression did not differ between groups. (d) Representative photomicrographs of immunohistochemical analysis of MPO-positive neutrophils. (e) Quantification of the number of MPO-positive inflammatory foci (per x100 field) revealed a clear induction in HFD, which was not apparent in YVAD. (f) Hepatic Tnf-α mRNA was reduced in HFD+YVAD. All data are from the t=21 weeks time point and are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 compared with HFD.

Figure 5

Effects of caspase-1 inhibition on hepatic fibrosis. LDLR−/−.Leiden mice were fed a high-fat diet for 21 weeks (HFD; n=15) or HFD+caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg⁻¹ daily) starting treatment after 9 weeks of HFD (HFD+YVAD; n=15). Low-fat diet (LFD)-fed mice (n=15) were included as a reference. (a) Representative photomicrographs of Picro-Sirius red-stained liver sections show hepatopericellular fibrosis in HFD animals, which is reduced in HFD+YVAD. (b) Quantification of positively stained area in Picro-Sirius red-stained liver sections show a clear induction of fibrosis in HFD, which is reduced in HFD+YVAD. (c) Col1a1 mRNA expression is reduced in HFD+YVAD. (d) mRNA expression of hepatic stellate cell activation marker Acta2 (α-SMA) tended to be induced in HFD and was reduced in HFD+YVAD. All data are from the t=21 weeks time point and are mean±s.e.m. *P<0.05, **P<0.01 compared with HFD.