Endoplasmic Reticulum Stress in the Paraventricular Nucleus of the Hypothalamus During Diet‐Induced Non‐Alcoholic Fatty Liver Disease

Abstract

Non‐alcoholic fatty liver disease (NAFLD), characterized by an accumulation of hepatic triglycerides, is a growing health epidemic and directly related to obesity. Endoplasmic reticulum stress and activation of the unfolded protein response (UPR) has emerged as a key mechanism in the pathogenesis of NAFLD. We have recently shown that selectively reducing brain ER stress is sufficient to rescue obesity‐induced NAFLD, although the neural regions involved remain unclear. The paraventricular nucleus of the hypothalamus (PVN) is a critical autonomic and endocrine control area, and accumulating evidence suggests a role for the PVN in obesity‐related disorders. Taken together, we hypothesized that obesity‐induced NAFLD is associated with ER stress in the PVN, particularly in neuron subtypes that project to the liver. PVN ER stress was evaluated using a combination of mRNA measurements, electron microscopy imaging of ER ultrastructure, and immunohistochemical approaches in a model of obesity‐induced NAFLD, in which mice were fed high fat diet (HFD; 60% fat) or normal chow (5% fat) for a period of 10 weeks. Gene expression of the ER stress markers p58IPK, CHOP, GRP78 and the spliced isoform of X‐box‐binding‐protein in micropunches of the PVN indicated 3‐ to 5‐fold increases in all biomarkers in HFD fed mice, relative to normal chow (e.g. p58IPK: 1.1±0.3 vs. 3.4±0.2; CHOP: 1.0±0.2 vs. 4.7±0.8; normal chow vs. HFD, p<0.05; n=3–4/group). We also performed electron microscopy to examine the ultrastructure of the rough ER in PVN neurons (n=3/group; 3–4 PVN sections per animal). The majority of PVN ER from normal chow control animals exhibited flat, tube‐like cisternae with dense ribosomal attachment, highlighting minimal evidence of ER stress (4/30 neurons with stressed ER; 15%). In contrast, 58% (16/28 neurons) of PVN neurons from obese animals had ER that appeared bloated with evidence of ribosomal detachment. Building upon this, we investigated whether obesity‐induced NAFLD is associated with ER stress in PVN neuron subtypes that project to the liver. Of the heterogeneous cell populations in the PVN, only oxytocin and corticotrophin releasing factor (CRF) expressing neurons have been shown to spinally project to the liver. Immunohistochemistry analyses of the PVN revealed significant HFD‐mediated upregulation of protein disulfide isomerase (PDI), an ER chaperone that is increased during ER stress situations. Co‐localization analysis in obese mice (n=4) revealed that 91%±1% of PVN oxytocin neurons co‐localized with PDI (i.e. exhibited ER stress) and this was evident throughout the rostral to caudal extent of the PVN (rostral: 98±2%; medial: 95±1%; caudal: 83±3%). Similarly, significant co‐localization of CRF with PDI was found throughout the PVN of HFD fed mice (rostral: 43±5%; medial: 65±4%; caudal: 73±3%). Collectively, these findings indicate that obesity‐induced NAFLD is associated with PVN ER ultrastructural alterations and robust activation of the UPR, notably within PVN neuron subtypes that project to the liver (oxytocin and CRF). Moreover, these data suggest that ER stress in the PVN may be involved in the pathogenesis of NAFLD.Support or Funding InformationNIH HL116776