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    • Cy5 amine (non-sulfonated) Methods br Results br Discussion

    2022-05-20

    Methods
    Results
    Discussion The current study is the first to specifically investigate the role of hepatic GPR109A and GPR109B on HDL metabolism and response to niacin therapy. While niacin raises HDL-C levels in humans, it has been reported by others [20], [36] and confirmed by us here that niacin treatment reduced serum levels of HDL cholesterol in wild-type mice. This effect of reducing HDL-C was GPR109A dependent. GPR109A is a Gi-protein-coupled receptor that is known to reduce cAMP and lipase-mediated hydrolysis of triglyceride stores in adipose tissue. We found that niacin treatment also inhibited intracellular cAMP release response to forskolin stimulation in primary hepatocytes from wild-type mice. While hepatocyte expression of GPR109A in the basal state is low, it was markedly upregulated by very low dose endotoxin. GPR109A expression is upregulated in murine macrophages by inflammatory stimuli [37] but this phenomenon has not been reported in other cell types. While the basal expression of GPR109A in liver is low, we show that it is upregulated after LPS injection; furthermore, we show that this is due to upregulation in hepatocytes and not simply Kupffer cells. These studies establish that hepatocytes express the niacin receptor and upregulate its expression in response to inflammation. In order to study the specific effects of increased hepatocyte expression of GPR109A on lipid, and particularly HDL, metabolism, we made an AAV2/8-based vector encoding murine GPR109A driven by the hepatocyte-specific TBG promoter. Intraperitoneal injection of this vector into wild-type mice resulted in expression of human GPR109A at levels substantially greater than basal and in the range of what we observed after LPS injection. Several G-protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living Cy5 amine (non-sulfonated) and can exhibit ligand-independent activation, particularly when overexpressed [30], [31]. We found that overexpression of GPR109A in hepatocytes resulted in activation as evidenced by marked suppression of forskolin-stimulated cAMP. This activation may be caused by dimerization of the GPR109A, oligomerization with another GPCR in the setting of overexpression, or simply increased receptor activation [38]. Although GPR109A and GPR109B are highly similar at the protein level and share a similar expression pattern, they are the products of different genes and clearly differ in their ability to be activated by niacin and β-hydroxybutyrate [16]. Our data establish that GPR109B expressed in hepatocytes does not have the same effects on cAMP and ABCA1 activity as does the expression of GPR109A. Overexpression of GPR109A caused a reproducible and significant decrease in plasma HDL-C levels that we showed was not due to increased HDL catabolism but rather to reduced HDL cholesterol production. Hepatic ABCA1 plays a critical role in the biosynthesis of nascent HDL by promoting the efflux of cholesterol from hepatocytes to lipid-poor apoA-I [32]. Because activation of GPR109A is known to reduce cAMP in adipocytes, and ABCA1 protein and activity are known to be increased by cAMP in macrophages, we hypothesized that activation of GPR109A in hepatocytes would reduce cAMP and thus ABCA1 protein and activity. Our data support this model and provide a mechanistic basis for the decreased HDL-C levels with both niacin treatment and GPR109A overexpression in mice. In contrast, GPR109A deficiency was associated with significantly increased cholesterol efflux to apoA-I in primary hepatocytes, which is consistent with a role for GPR109A in modulating HDL-C metabolism in liver.
    Sources of funding This work was supported by NIH grant P50-HL-083799 from the National Heart Lung and Blood Institute (to D.J.R.).
    Disclosure
    Acknowledgments
    Nicotinic acid (niacin) has been a leading treatment for dyslipidemia and for the prevention of atherosclerosis for over 40years. Long term clinical studies have demonstrated niacin’s ability to reduce mortality from coronary heart disease. In spite of niacin’s clinical significance, patients treated with niacin show low compliance of use due to an intense flushing side effect., As a result, a number of drug discovery programs have focused on the development of a ‘flush-free’ niacin-like therapy. Despite considerable effort in the field, the absence of a niacin-related target and/or mechanism of action, has limited such investigations.