Institute of Biomedical Sciences

Title

A Forebrain-Hypothalamic Circuit Mediates Hepatic Steatosis

Poster Number

21

Document Type

Poster

Status

Graduate Student - Doctoral

Abstract Category

Neuroscience

Keywords

Non-alcoholic fatty liver disease, Paraventricular nucleus of the hypothalamus, Subfornical organ, Intersectional viral technique

Publication Date

Spring 2018

Abstract

Non-alcoholic fatty liver disease (NAFLD), characterized by an accumulation of hepatic triglycerides (i.e. steatosis), is a growing health epidemic. We recently demonstrated a role for the brain in NAFLD. In particular, disruptions in the subfornical organ (SFO) - a small circumventricular forebrain nucleus - appear to play a key role in the development of hepatic steatosis. However, the neural network(s) through which the SFO contributes to NAFLD remains unknown. The paraventricular nucleus of the hypothalamus (PVN) is a central regulator of peripheral autonomic and endocrine function, and the SFO has dense excitatory projections to the PVN. Taken together, we hypothesized that activation of excitatory SFO PVN-projecting neurons would result in NAFLD development. We first confirmed that SFO PVN projecting neurons are excitatory by using retrograde viral labeling from the PVN (CAV2-GFP) combined with SFO immunohistochemistry for the excitatory neuronal marker calcium-calmodulindependent kinase II (CAMKII). Nearly 100% of the retrograde tracer co-localized with CAMKII in the SFO (not shown, n=3). Subsequently, we employed an intersectional viral strategy in which a retrograde transported canine adenovirus was targeted to the PVN to allow for expression of Cre-recombinase in SFO PVN-projecting neurons (CAV2-Cre-GFP), combined with SFO-targeted delivery of a Cre-inducible designer receptors engineered against designer drugs (DREADDs) excitatory construct (AAV2-DIO-hM3Gq-mCherry). With this approach, the pharmacological ligand clozapine-N-oxide (CNO; 3 mg/kg i.p.) was administered once daily over 6 days to activate SFO PVN-projecting neurons (n=4). Oil Red O staining of the liver demonstrated that, relative to control animals, 6-day activation of SFO PVN-projecting neurons resulted in a clear development of hepatic steatosis (2.69±0.02 vs. 2.92±0.02 au x 107 , saline vs. CNO, p<0.05). Real time qPCR analysis further indicated that activation of SFO neurons that project to the PVN resulted in a marked upregulation of liver markers of de novo lipogenesis and gluconeogenesis (e.g. DGAT1: 3.6±0.3 fold saline, p<0.05; G6Pase: 2.6±1.0 fold saline, p=0.12). Importantly, these changes occurred independent of differences in body weight (25±1 vs. 25+1 g, saline vs. CNO, p<0.05) and food intake. Collectively, these findings indicate that short-term activation of excitatory SFO PVN-projecting neurons results in a NAFLD phenotype characterized by elevated liver triglycerides and disruptions in liver metabolic markers. Furthermore, these findings suggest that manipulating this forebrain-hypothalamic network in the context of obesity may be a novel approach to target NAFLD.

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A Forebrain-Hypothalamic Circuit Mediates Hepatic Steatosis

Non-alcoholic fatty liver disease (NAFLD), characterized by an accumulation of hepatic triglycerides (i.e. steatosis), is a growing health epidemic. We recently demonstrated a role for the brain in NAFLD. In particular, disruptions in the subfornical organ (SFO) - a small circumventricular forebrain nucleus - appear to play a key role in the development of hepatic steatosis. However, the neural network(s) through which the SFO contributes to NAFLD remains unknown. The paraventricular nucleus of the hypothalamus (PVN) is a central regulator of peripheral autonomic and endocrine function, and the SFO has dense excitatory projections to the PVN. Taken together, we hypothesized that activation of excitatory SFO PVN-projecting neurons would result in NAFLD development. We first confirmed that SFO PVN projecting neurons are excitatory by using retrograde viral labeling from the PVN (CAV2-GFP) combined with SFO immunohistochemistry for the excitatory neuronal marker calcium-calmodulindependent kinase II (CAMKII). Nearly 100% of the retrograde tracer co-localized with CAMKII in the SFO (not shown, n=3). Subsequently, we employed an intersectional viral strategy in which a retrograde transported canine adenovirus was targeted to the PVN to allow for expression of Cre-recombinase in SFO PVN-projecting neurons (CAV2-Cre-GFP), combined with SFO-targeted delivery of a Cre-inducible designer receptors engineered against designer drugs (DREADDs) excitatory construct (AAV2-DIO-hM3Gq-mCherry). With this approach, the pharmacological ligand clozapine-N-oxide (CNO; 3 mg/kg i.p.) was administered once daily over 6 days to activate SFO PVN-projecting neurons (n=4). Oil Red O staining of the liver demonstrated that, relative to control animals, 6-day activation of SFO PVN-projecting neurons resulted in a clear development of hepatic steatosis (2.69±0.02 vs. 2.92±0.02 au x 107 , saline vs. CNO, p<0.05). Real time qPCR analysis further indicated that activation of SFO neurons that project to the PVN resulted in a marked upregulation of liver markers of de novo lipogenesis and gluconeogenesis (e.g. DGAT1: 3.6±0.3 fold saline, p<0.05; G6Pase: 2.6±1.0 fold saline, p=0.12). Importantly, these changes occurred independent of differences in body weight (25±1 vs. 25+1 g, saline vs. CNO, p<0.05) and food intake. Collectively, these findings indicate that short-term activation of excitatory SFO PVN-projecting neurons results in a NAFLD phenotype characterized by elevated liver triglycerides and disruptions in liver metabolic markers. Furthermore, these findings suggest that manipulating this forebrain-hypothalamic network in the context of obesity may be a novel approach to target NAFLD.