Insomnia Promotes Hepatic Steatosis in Rats Possibly by Mediating Sympathetic Overactivation

Abstract

Background: Insomnia is a widespread problem that can lead to the occurrence of other diseases and correlates closely with sympathetic nerve hyperactivation. Obesity-induced hepatic steatosis is mediated by sympathetic overactivation. However, it remains unclear whether insomnia may cause hepatic steatosis. The goal of this study was to preliminarily investigate whether insomnia caused hepatic steatosis in rats via sympathetic hyperactivation. Methods: A total of 32 Sprague-Dawley male rats were divided randomly into four groups: model, sympathetic denervation (Sd), estazolam, and control (eight rats/group). Model group received sustained sleep deprivation using the modified multiple platform method. In the Sd group, rats underwent sleep deprivation after receiving Sd by 6-hydroxydopamine (6-OHDA). Estazolam group: the rats concurrently received sleep deprivation and treatment with estazolam. The other eight rats housed in cages and kept in a comfortable environment were used as control. Blood samples were obtained for analysis of plasma lipids and hepatic function. Sympathetic hyperactivation-related indexes and hepatic steatosis in liver tissues were tested. Results: Liver enzymes, plasma lipid levels, and hepatic steatosis were elevated in insomnia rats, and sympathetic hyperactivation was found. Insomnia-induced hepatic steatosis was effectively lowered with pharmacological ablation of the hepatic sympathetic nerves. Furthermore, the treatment of insomnia with estazolam inhibited sympathetic activation and reduced hepatic steatosis. Conclusion: Sustained sleep deprivation-induced insomnia promotes hepatic steatosis in rats possibly by mediating sympathetic overactivation.

Keywords: hepatic steatosis; insomnia; lipid droplets; sleep deprivation; sympathetic nerve.

Figure 1

Changes in hepatic function and blood lipids. (A) Serum was analyzed for liver enzyme [aspartate transaminase (ALT) and alanine aminotransferase (AST)] levels; (B) plasma triglyceride levels in insomnia rats were clearly higher than those in controls; and (C) Plasma free fatty acid (FFA) levels in insomnia rats were significantly lower than those in controls. The results were obtained from two independent experiments and are expressed as the mean±SD, n=8 per group, ^* p<0.05; ^** p<0.01; and ^*** p<0.001.

Figure 2

Histopathology, immunohistochemistry, and transmission electron microscopy (TEM) were performed to determine the degree of hepatic steatosis. (A) Hematoxylin-eosin (HE) staining of the liver showed steatosis of most hepatocytes in the model group (scale bar: 50μm). (B) Lipid droplets in hepatocytes were examined by TEM (scale bar: 1μm). (C) Immunohistochemical staining for Perilipin-2 was observed predominantly in hepatocytes, where positive staining was brown-yellow (scale bar: 50μm). The results were obtained from two independent experiments and are expressed as the mean±SD, n=8 per group, ^*** p<0.001.

Figure 3

Determination of nerve growth-related indexes in liver tissue. (A) Nerve growth factor (NGF) was determined by ELISA in liver tissues. (B) Norepinephrine (NE) in liver tissues was quantified by high-performance liquid chromatography-tandem mass spectrometry (HP-LC-MS/MS), presenting an increase in the model group. (C) Immunohistochemical staining of growth-associated protein-43 (GAP43) was used to assess nerve growth (scale bar: 50μm). The results were obtained from two independent experiments and are expressed as the mean±SD, n=8 per group, ^*** p<0.001. NE, norepinephrine.

Figure 4

Identification of sympathetic nerves in the liver by confocal laser scanning microscopy. GAP43/TH colocalization indicated hyperplastic sympathetic nerves in the livers of insomnia rats (scale bar: 75μm).

Figure 5

The effect of sympathetic denervation on hepatic steatosis. (A) NE of sympathetic neuromediators in liver was detected. (B) The plasma triglyceride levels in the liver tissues were analyzed. (C) The plasma free fatty acid levels were detected. (D) HE staining (scale bar: 50μm). (E) TEM was performed to observe lipid droplets (scale bar: 1μm). (F) Quantitative analysis of lipid droplets was measured by Perilipin-2 immunohistochemistry (scale bar: 50μm). The results were obtained from two independent experiments and are expressed as the mean±SD, n=8 per group, ^** p<0.01; and ^*** p<0.001. Sd, sympathetic denervation group; NE, norepinephrine.

Figure 6

Blood lipids and hepatic steatosis were detected to evaluate the effect of estazolam on fatty liver in insomnia rats. (A) The plasma triglyceride levels. (B) The plasma free fatty acid levels. (C) HE staining (scale bar: 50μm). (D) Detection of lipid droplets by TEM (scale bar: 1μm). (E) Expression of Perilipin-2 was determined by immunohistochemistry (scale bar: 50μm). The results were obtained from two independent experiments and are expressed as the mean±SD, n=8 per group, ^*** p<0.001.

Figure 7

Sympathetic innervation in liver was evaluated. (A) The NGF level was tested in liver tissues. (B) NE in liver tissues was detected by HP-LC-MS/MS. (C) GAP43 staining was observed in the liver as a qualitative metric of neural growth by immunohistochemistry (scale bar: 50μm). The results were obtained from two independent experiments and are expressed as the mean±SD, n=8 per group, ^** p<0.01 and ^*** p<0.001. NE, norepinephrine.