Endoplasmic reticulum stress in subfornical organ-hypothalamic paraventricular neurons mediates hepatic steatosis during obesity

Abstract

Non-alcoholic fatty liver disease (NAFLD), characterized by excess hepatic lipid accumulation (i.e. steatosis), increases the risk for cardiometabolic diseases. Evidence points to central nervous system alterations as key to NAFLD development. In this context, we reported that NAFLD is mediated by endoplasmic reticulum (ER) stress in the subfornical organ (SFO), a nucleus situated outside of the blood-brain-barrier. However, the neural networks through which SFO ER stress contributes to NAFLD remain unclear. The SFO sends dense neural projections to the hypothalamic paraventricular nucleus (PVN) - a key metabolic control region. Based on this, we hypothesized that ER stress in SFO→PVN projecting neurons contributes to hepatic steatosis during obesity. A model of NAFLD was used where male C57BL/6 mice were fed a high fat diet (HFD, 60% kcal fat) or normal chow for 8 weeks (n=4/group). The retrograde neuronal tracer cholera toxin subunit B (CTB) was then microinjected into the PVN and ER stress in SFO→PVN neurons was evaluated using immunohistochemistry 3 weeks later. During cellular stress, ER chaperone proteins, such as glucose-regulated peptide 78 (GRP78) and protein disulfide isomerase (PDI), are increased in an attempt to maintain homeostasis. Thus, they can be used as an indicator of ER stress. Immunohistochemistry showed clear upregulation of GRP78 in SFO→PVN neurons of HFD mice (GRP78+CTB+ cell (%)/total CTB+ cells: 39±5 vs 60±2, normal chow vs HFD, p<0.05) and this increase was found throughout the rostral to caudal extent of the SFO. Similarly, PDI was increased with HFD in SFO→PVN neurons (PDI+CTB+ cell (%)/total CTB+ cells: 34±5 vs 49±3, normal chow vs HFD, p<0.05). We next employed an intersectional viral strategy to selectively inhibit ER stress in SFO→PVN neurons. Male mice were fed a HFD for 7 wks (n=4/group) and then underwent SFO targeting of a Cre-inducible vector to overexpress GRP78 (AAV-FLEX-GRP78); an approach that we and others have shown is an effective way to reduce ER stress. Concurrently, a retrogradely transported Cre virus (CAV2-CRE-GFP) was targeted to the PVN (or CAV2-GFP as a control). Selective inhibition of ER stress in SFO→PVN neurons for 4 wks modestly dampened HFD induced body weight gain (Δbody weight: 7±1 vs 5±1g, CAV2 vs CAV2-CRE, p<0.05) although this was not related to differences in food intake, energy expenditure, activity, or regional adiposity. However, inhibition of ER stress in SFO→PVN neurons markedly reduced HFD induced hepatomegaly (2.1±0.2 vs 1.6±0.1g, CAV2 vs CAV2-CRE, p=0.06). Histological examination (Oil Red O staining) also revealed widespread hepatic steatosis in HFD fed mice, which was reduced by ~62% following ER stress inhibition in SFO→PVN neurons (37.5±5.4 vs 14.3±5.1% area; CAV2 vs CAV2-CRE, p<0.05). Collectively, these results indicate that ER stress occurs in SFO→PVN neurons during obesity and selectively lowering ER stress in this neural network is sufficient to reduce NAFLD. R01DK117007, R01HL141393, AHA932522 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.