Liver sympathetic denervation reverses obesity-induced hepatic steatosis

Abstract

Key points: Non-alcoholic fatty liver disease, characterized in part by elevated liver triglycerides (i.e. hepatic steatosis), is a growing health problem. In this study, we found that hepatic steatosis is associated with robust hepatic sympathetic overactivity. Removal of hepatic sympathetic nerves reduced obesity-induced hepatic steatosis. Liver sympathetic innervation modulated hepatic lipid acquisition pathways during obesity.

Abstract: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 Americans and is a significant risk factor for type II diabetes mellitus, insulin resistance and hepatic carcinoma. Characterized in part by excessive hepatic triglyceride accumulation (i.e. hepatic steatosis), the incidence of NAFLD is increasing - in line with the growing obesity epidemic. The role of the autonomic nervous system in NAFLD remains unclear. Here, we show that chronic hepatic sympathetic overactivity mediates hepatic steatosis. Direct multiunit recordings of hepatic sympathetic nerve activity were obtained in high fat diet and normal chow fed male C57BL/6J mice. To reduce hepatic sympathetic nerve activity we utilized two approaches including pharmacological ablation of the sympathetic nerves and phenol-based hepatic sympathetic nerve denervation. Diet-induced NAFLD was associated with a nearly doubled firing rate of the hepatic sympathetic nerves, which was largely due to an increase in efferent nerve traffic. Furthermore, established high fat diet-induced hepatic steatosis was effectively reduced with pharmacological or phenol-based removal of the hepatic sympathetic nerves, independent of changes in body weight, caloric intake or adiposity. Ablation of liver sympathetic nerves was also associated with improvements in liver triglyceride accumulation pathways including free fatty acid uptake and de novo lipogenesis. These findings highlight an unrecognized pathogenic link between liver sympathetic outflow and hepatic steatosis and suggest that manipulation of the liver sympathetic nerves may represent a novel therapeutic strategy for NAFLD.

Keywords: autonomic nervous system; diet-induced obesity; non-alcoholic fatty liver disease; sympathetic nerve activity.

Figure 1. High fat diet feeding evokes robust elevations in hepatic SNA.

Direct multiunit recordings of hepatic SNA were obtained in high fat diet and normal chow controls. (A) Representative total hepatic SNA and (B) group summary data calculated as a frequency and integrated voltage. (C) Representative efferent hepatic SNA (nerve sectioned distal to recording electrode to eliminate afferent input) and (D) group summary data. (n=5–6). Student’s two-tailed unpaired t-test. *p<0.05 vs. normal chow.

Figure 2. Pharmacological removal of sympathetic nerve activity reduces hepatic steatosis during diet-induced obesity.

6-hydroxydopamine (6) to ablate peripheral sympathetic nerves, or vehicle control (Veh), was administered (i.p.) once in high fat diet and normal chow fed mice and animals were sacrificed 3 days later. (A) Representative western blot and quantitative summary of liver tyrosine hydroxylase (TH) protein expression (n=4–8). (B) Liver mass (n=7–8). (C) Hepatic triglyceride content (n=4–7). (D) Representative liver hematoxylin and eosin staining. Scale bar=100 μm. (E) mRNA expression of liver gluconeogenesis and de novo lipogenesis markers (n-7–8). Two-way ANOVA with Tukey’s post-hoc test for all. *p<0.05 vs. normal chow, #p<0.05 vs. high fat diet vehicle.

Figure 3. Pharmacological removal of sympathetic nerve activity does not influence body weight, food/water intake, locomotor activity, adiposity or energy expenditure.

(A) Body mass, (B) caloric consumption, (C) water intake, (D) locomotor activity, and (E) regional adipose tissue mass, as well as indirect calorimetry measurements of (F) oxygen consumption (VO₂), (G) carbon dioxide production (VCO₂), (H) respiratory exchange ratio (RER), and (I) energy expenditure following administration (i.p.) of 6-OHDA (6) to ablate peripheral sympathetic nerves, or vehicle control (Veh), in high fat diet and normal chow fed mice. n=7–8 for all. Two-way ANOVA with Tukey’s post-hoc test for all. *p<0.05 vs. normal chow, #p<0.05 vs. high fat diet vehicle.

Figure 4. Hepatic denervation reduces diet-induced hepatic steatosis.

High fat diet and normal chow mice underwent selective hepatic denervation (D) or sham (S) surgery and were sacrificed 7 days later. (A) Representative western blot and quantitative summary of liver tyrosine hydroxylase (TH) protein expression (n=4–6). (B) Hepatic triglyceride content (n=4–8). (C) Liver Oil Red O stained area (n=3–4). (D) Representative Oil Red O and H&E liver staining. Scale bar=100 μm. (E) Body mass, (F) caloric consumption, (G) water intake, and (H) daily locomotor activity (n=4–8). Two-way ANOVA with Tukey’s post-hoc test for all. *p<0.05 vs. normal chow, #p<0.05 vs. high fat diet sham.

Figure 5. Short-term hepatic denervation does not affect adiposity, whole body energy utilization, and energy expenditure.

High fat diet and normal chow mice underwent selective hepatic denervation (D) or sham (S) surgery and were sacrificed 7 days later. (A) Masses of regional adipose tissue and organs that are innervated by the celiac and mesenteric ganglia (n=4–8). Indirect calorimetry measurements of (B) oxygen consumption (VO₂), (C) carbon dioxide production (VCO₂), (D) respiratory exchange ratio (RER), and (E) energy expenditure (n=4–8). Two-way ANOVA with Tukey’s post-hoc test for all. *p<0.05 vs. normal chow.

Figure 6. Hepatic denervation selectively decreases fatty acid uptake and de novo lipogenesis/gluconeogenesis pathways.

(A) Liver mRNA expression of fatty acid transporters and Pparα (n=4–7) and (B) de novo lipogenesis and gluconeogenesis markers (n=4–8) following sham (S) or hepatic denervation (D) surgery in normal chow control and high fat diet fed mice. (C) Representative immunohistochemistry for liver CD36 in sham and liver denervated animals. The right column represents a magnification of the white box in the corresponding image in the left column. Green; CD36, blue; DAPI, Scale bar=100 μm. (D) Triacylglycerol synthesis markers (n=4–6). Representative western blot and quantitative summary of liver mitochondrial (E) and peroxisomal (F) β-oxidation markers, as well as (G) MTTP as a marker of VLDL export (n=4–6). Two-way ANOVA followed by Tukey’s post-hoc test for all. *p<0.05 vs. normal chow, #p<0.05 vs. high fat diet sham.