Institute of Biomedical Sciences

Title

Endoplasmic Reticulum Stress in the Paraventricular Nucleus of the Hypothalamus During Diet-induced Non-alcoholic Fatty Liver Disease

Poster Number

22

Document Type

Poster

Keywords

Fatty liver disease, hypothalamus, obesity, oxytocin, steatosis

Publication Date

4-2017

Abstract

Non-alcoholic fatty liver disease (NAFLD), characterized by an accumulation of hepatic triglycerides, is directly related to obesity. Endoplasmic reticulum (ER) 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 rescues 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 several ER stress markers indicated 3- to 5-fold increases in UPR activation 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 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, including oxytocin and corticotrophin releasing factor (CRF) expressing neurons. Immunohistochemistry analyses of the PVN revealed HFD-mediated upregulation of protein disulfide isomerase (PDI), an ER chaperone that is increased during ER stress situations. Co-localization analysis in obese mice indicated that 91%±1% and 61%±3% of PVN oxytocin and CRF neurons, respectively, co-localized with PDI, whereas less co-expression was found in normal chow controls (oxytocin 82%±1%; CRF 53±2%). Collectively, these findings indicate that obesity-induced NAFLD is associated with PVN ER ultrastructural alterations and robust UPR activation, notably within PVN neuron subtypes that project to the liver. Moreover, these data suggest that PVN ER stress may be involved in the pathogenesis of NAFLD.

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To be presented at GW Annual Research Days 2017.

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Endoplasmic Reticulum Stress in the Paraventricular Nucleus of the Hypothalamus During Diet-induced Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD), characterized by an accumulation of hepatic triglycerides, is directly related to obesity. Endoplasmic reticulum (ER) 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 rescues 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 several ER stress markers indicated 3- to 5-fold increases in UPR activation 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 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, including oxytocin and corticotrophin releasing factor (CRF) expressing neurons. Immunohistochemistry analyses of the PVN revealed HFD-mediated upregulation of protein disulfide isomerase (PDI), an ER chaperone that is increased during ER stress situations. Co-localization analysis in obese mice indicated that 91%±1% and 61%±3% of PVN oxytocin and CRF neurons, respectively, co-localized with PDI, whereas less co-expression was found in normal chow controls (oxytocin 82%±1%; CRF 53±2%). Collectively, these findings indicate that obesity-induced NAFLD is associated with PVN ER ultrastructural alterations and robust UPR activation, notably within PVN neuron subtypes that project to the liver. Moreover, these data suggest that PVN ER stress may be involved in the pathogenesis of NAFLD.