Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease

Abstract

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independent of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficient mice (InsR+/-), in the pathogenesis of liver fibrosis in C57BL/6mice.We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation inMCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independent of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization

Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver

“…In addition, the causal effect of hepatic fat on fibrosis was partly independent of disease activity at the time of biopsy, supporting the active role of hepatic fat accumulation in inducing progressive liver disease over the entire lifetime. These data are consistent with experimental evidence linking hepatic fat accumulation and insulin resistance with fibrosis in NAFLD, independent of inflammatory pathways .…”

Effect of Orally Administered Relaxin in Experimental Nash <strong> </strong>

“…Each mouse was then treated with an amount of RLX equivalent to 200 U per day by oral administration. With the measured EC50, 200U are equivalent to 25µg [17]. In addition, stability of RLX in water at room temperature (20°C ± 5°C) was assessed a) by measuring the EC50 of a 25 ug/ml concentration at time 0, 44 and 164 hours and b) by analyzing the RP-HPLC RLX profile at time 0, 24, 48, 72, 144 hours.…”

“…Gene expression was evaluated in triplicate by real-time quantitative PCR (qPCR), using the SYBR green chemistry (Fast SYBR Green Master Mix, Life Technologies), for Tumor Necrosis Factor-α (TNF-α), Collagen type I A1 (Col1A1), α-smooth muscle actin (α-SMA), MMP-8, MMP-9, MMP-13 and TIMP1 (Table 1). Data were normalized using the β-actin housekeeping gene and expressed as arbitrary units (AU) [17].…”

“…Liver tissue samples were fixed in 10% PBS buffered formalin and embedded in paraffin within 48 hours of formalin fixation. Histological staining was performed using H&E and hepatic fibrosis was evaluated by Sirius Red staining as previously described [21]. Diagnosis of NASH was based on the presence of steatosis with lobular necro-inflammation and ballooning or fibrosis.…”

Pathogenesis of Nonalcoholic Steatohepatitis and Hormone-Based Therapeutic Approaches

“…Thus, one of the potential strategies to inhibit NASH-related fibrosis is to suppress accumulation of extracellular matrix proteins including collagen or to directly inhibit HSC activation. Lysyl oxidase-like 2 (LOXL2), an enzyme promoting collagen cross-linking, is upregulated in the livers of animals with fibrosis (70) and of diabetic patients with NAFLD (71) and is essential in hepatic fibrogenesis (72). Phase 2b clinical studies in NASH patients using an antibody against LOXL2 (Simtuzumab/GS-6624; NCT01672866 and NCT01672879) were recently terminated.…”