Mechanism by Which Hepatic Lipids Drive the Development of Hypertension and a Decline in Lung Function

Abstract

More than 1 in 4 Americans have hepatic steatosis, or fatty liver. Hepatic steatosis is closely associated with obesity and obesity associated pathophysiologies, including type 2 diabetes, hypertension, and decreased lung function. Identifying a mechanism by which hepatic steatosis causes these disorders would provide pharmacological targets to improve treatment options for those affected by these conditions. In lipid laden hepatocytes, gamma aminobutyric acid (GABA) is produced by metabolic flux through the GABA shunt. Production of GABA is driven by the alterations in reducing equivalents, decrease in FAD+ to FADH2 and NAD+ to NADH2, which drive the production of GABA. This production of GABA is dependent on the enzyme GABA-Transaminase, which also breaks down GABA (Figure 1). GABA release and re-uptake by hepatocytes is regulated by the Slc6 family of electrogenic transporters. These transporters co-transport 2-3 Na+ and 1 Cl- with GABA, thus 1-2 net positive charges are transported with GABA (Figure 1). Hepatic lipid accumulation also induces hepatocyte depolarization. Thus, as GABA is co-transported with a net positive charge, depolarization would favor the export of GABA and discourage the re-uptake. GABA signaling decreases hepatic afferent vagal nerve activity. Inhibiting hepatic GABA release improves insulin sensitivity and glucose clearance in high-fat diet-induced obese mice dependent on an intact hepatic afferent vagal nerve. We hypothesized that liver GABA was driving the obesity-induced development of pulmonary and cardiovascular dysfunction and within this dissertation have investigated this hypothesis (Figure 1). In Chapter 1 is a review of the afferent and efferent branch of the vagal nerve which synapse with the liver. Our proposed mechanism is dependent on efferent signaling from the hepatic afferent vagal nerve, this chapter also details how our work fits into previous research. Because of the ability to genetically manipulate mice, they are a valued model in assessing physiological function and tissue cross talk. However, until recently the pulmonary disease and function field hadn’t fully exploited the advantages of this small rodent model. Tools to assess lung function repeatedly and accurately in mice are limited. In fact, the gold-standard method for assessing lung function in mice (forced oscillation technique) is terminal and only assesses lung mechanics, not the ventilatory output. In turn, before conducting studies on the mechanism by which obesity alters pulmonary function, we developed and validated a novel system to measure pulmonary function accurately and repeatedly in the conscious mouse (Chapter 2). Validation of our head-out plethysmography system included measuring tidal breathing, the bronchoconstrictive response to methacholine, and the effect of sex and age on pulmonary function. We further assessed alterations in lung function in an asthmatic mouse model. Chapter 3 describes studies that assess the effect of obesity on lung function in mice. Obesity increased volume, minute ventilation (volume per minute), mid-expiratory flow (flow rate at 50% expiratory volume), and end-inspiratory pause (pause at end of inspiration) and decreased expiratory time (P<0.0001). When comparing measures using head-out plethysmography with measures taken in the same mice using the forced oscillation technique, we found that multiple variables were significantly correlated. Minute ventilation was most significantly associated with maximal airway resistance, maximal airway elastance, tissue damping, and tissue elastance, while, volume, corrected for energy expenditure, was most significantly associated with maximal resistance of the conducting airways. Finally, we investigated the role of hepatic GABA production and the hepatic vagal nerve on obesity-associated declines in lung function. Neither hepatic vagotomy nor inhibiting hepatocyte GABA production affected breathing in diet induced obese mice. Blood Pressure Hepatic steatosis is closely associated with development of hypertension. Hepatic steatosis is associated with elevations in blood pressure, even in individuals with normal blood pressure. We set out to investigate the mechanism driving this association in Chapter 4. We investigated the role of hepatocyte GABA release and production in regulating blood pressure. We further evaluated the role of the hepatic afferent vagal nerve. Depolarization of hepatocytes, known to encourage GABA release, increased systolic, diastolic, and mean arterial blood pressure without affecting heart rate. This response was dependent on an intact hepatic afferent vagal nerve. Inhibition of hepatic GABA production with the GABA transaminase inhibitor ethanolamine-O-sulfate or knockdown of GABA transaminase using an antisense oligonucleotide decreased systolic, diastolic, and mean arterial blood pressure without affecting heart rate in high fat diet induced obese mice. Conclusion Liver fat is positively associated with the severity and incidence of pulmonary and cardiovascular dysfunction. In Chapters 2 and 3 we found that obesity-induced alterations in lung function are independent of liver GABA production and release. These findings encourage future studies of alternative hepatokines that may affect breathing and the evaluation of the potential mechanical effects of an enlarged liver on pulmonary function. An ideal model for these studies of mechanical effects would be the glycogen phosphorylase knockout mouse, which would have an enlarged liver in the absence of obesity. In chapter 4, we describe how obesity-induced increases in blood pressure can be resolved by limiting obesity-induced liver GABA production. These studies further encourage additional studies aimed at understanding the regulation of hepatic GABA production and release to fully appreciate the role in blood pressure. Future work should additionally focus on the development of peripherally restricted GABA transaminase inhibitors to safely limit the negative effects of hepatic lipid accumulation on vascular health.