Jason J. Yuen

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

Intestinal DGAT1 deficiency reduces postprandial triglyceride and retinyl ester excursions by inhibiting chylomicron secretion and delaying gastric emptying

Acyl CoA:diacylglycerol acyltransferase (DGAT) 1 catalyzes the final step of triglyceride (TG) synthesis. We show that acute administration of a DGAT1 inhibitor (DGAT1i) by oral gavage or genetic deletion of intestinal Dgat1 (intestine-Dgat1−/−) markedly reduced postprandial plasma TG and retinyl ester excursions by inhibiting chylomicron secretion in mice. Loss of DGAT1 activity did not affect the efficiency of retinol esterification, but it did reduce TG and retinoid accumulation in the small intestine. In contrast, inhibition of microsomal triglyceride transfer protein (MTP) reduced chylomicron secretion after oral fat/retinol loads, but with accumulation of dietary TG and retinoids in the small intestine. Lack of intestinal accumulation of TG and retinoids in DGAT1i-treated or intestine-Dgat1−/− mice resulted, in part, from delayed gastric emptying associated with increased plasma levels of glucagon-like peptide (GLP)-1. However, neither bypassing the stomach through duodenal oil injection nor inhibiting the receptor for GLP-1 normalized postprandial TG or retinyl esters excursions in the absence of DGAT1 activity. In summary, intestinal DGAT1 inhibition or deficiency acutely delayed gastric emptying and inhibited chylomicron secretion; however, the latter occurred when gastric emptying was normal or when lipid was administered directly into the small intestine. Long-term hepatic retinoid metabolism was not impacted by DGAT1 inhibition.

Retinoids: Potent regulators of metabolism

Retinoids (vitamin A and its analogs) are highly potent regulators of cell differentiation, cell proliferation, and apoptosis. Because of these activities, retinoids have been most extensively studied in the contexts of embryonic development and of proliferative diseases, especially cancer and skin disease. Recently, there has been considerable new research interest focused on gaining understanding of the roles that retinoids and/or retinoid‐related proteins may have in the development of metabolic diseases, primarily obesity, diabetes, and dyslipidemia. This review will summarize recent advances that have been made in these areas, focusing on the role of retinoids in modulating adipogenesis, the roles of retinoids and retinoid‐related proteins as signaling molecules linking obesity with the development of type II diabetes, the roles of retinoids in pancreatic β‐cell biology/insulin secretion, and the actions of retinoids in hepatic steatosis.

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.

Adipocyte‐specific overexpression of retinol‐binding protein 4 causes hepatic steatosis in mice

There is considerable evidence that both retinoids and retinol‐binding protein 4 (RBP4) contribute to the development of liver disease. To understand the basis for this, we generated and studied transgenic mice that express human RBP4 (hRBP4) specifically in adipocytes. When fed a chow diet, these mice show an elevation in adipose total RBP4 (mouse RBP4 + hRBP4) protein levels. However, no significant differences in plasma RBP4 or retinol levels or in hepatic or adipose retinoid (retinol, retinyl ester, and all‐trans‐retinoic acid) levels were observed. Strikingly, male adipocyte‐specific hRBP4 mice fed a standard chow diet display significantly elevated hepatic triglyceride levels at 3‐4 months of age compared to matched littermate controls. When mice were fed a high‐fat diet, this hepatic phenotype, as well as other metabolic phenotypes (obesity and glucose intolerance), worsened. Because adipocyte‐specific hRBP4 mice have increased tumor necrosis factor‐α and leptin expression and crown‐like structures in adipose tissue, our data are consistent with the notion that adipose tissue is experiencing RBP4‐induced inflammation that stimulates increased lipolysis within adipocytes. Our data further establish that elevated hepatic triglyceride levels result from increased hepatic uptake of adipose‐derived circulating free fatty acids. We obtained no evidence that elevated hepatic triglyceride levels arise from increased hepatic de novo lipogenesis, decreased hepatic free fatty acid oxidation, or decreased very‐low‐density lipoprotein secretion. Conclusion: Our investigations establish that RBP4 expressed in adipocytes induces hepatic steatosis arising from primary effects occurring in adipose tissue. (Hepatology 2016;64:1534‐1546)

Retinoic acid receptor signaling is required to maintain glucose-stimulated insulin secretion and β-cell mass

Retinoic acid signaling is required for maintaining a range of cellular processes, including cell differentiation, proliferation, and apoptosis. We investigated the actions of all‐trans‐retinoic acid (atRA) signaling in pancreatic β‐cells of adult mice. atRA signaling was ablated in β‐cells by overexpressing a dominant‐negative retinoic acid receptor (RAR)‐α mutant (RARdn) using an inducible Cre‐Lox system under the control of the pancreas duodenal homeobox gene promoter. Our studies establish that hypomorphism for RAR in β‐cells leads to an age‐dependent decrease in plasma insulin in the fed state and in response to a glucose challenge. Glucose‐stimulated insulin secretion was also impaired in islets isolated from mice expressing RARdn. Among genes that are atRA responsive, Glut2 and Gck mRNA levels were decreased in isolated islets from RARdn‐expressing mice. Histologic analyses of RARdn‐expressing pancreata revealed a decrease in β‐cell mass and insulin per β‐cell 1 mo after induction of the RARdn. Our results indicate that atRA signaling mediated by RARs is required in the adult pancreas for maintaining both β‐cell function and mass, and provide insights into molecular mechanisms underlying these actions.—Brun, P.‐J., Grijalva, A., Rausch, R., Watson, E., Yuen, J. J., Das, B. C., Shudo, K., Kagechika, H., Leibel, R. L., Blaner, W. S. Retinoic acid receptor signaling is required to maintain glucose‐stimulated insulin secretion and β‐cell mass. FASEB J. 29, 671‐683 (2015). www.fasebj.org

Vitamin A Absorption, Storage and Mobilization

It is well established that chylomicron remnant (dietary) vitamin A is taken up from the circulation by hepatocytes, but more than 80 % of the vitamin A in the liver is stored in hepatic stellate cells (HSC). It presently is not known how vitamin A is transferred from hepatocytes to HSCs for storage. Since retinol-binding protein 4 (RBP4), a protein that is required for mobilizing stored vitamin A, is synthesized solely by hepatocytes and not HSCs, it similarly is not known how vitamin A is transferred from HSCs to hepatocytes. Although it has long been thought that RBP4 is absolutely essential for delivering vitamin A to tissues, recent research has proven that this notion is incorrect since total RBP4-deficiency is not lethal. In addition to RBP4, vitamin A is also found in the circulation bound to lipoproteins and as retinoic acid bound to albumin. It is not known how these different circulating pools of vitamin A contribute to the vitamin A needs of different tissues. In our view, better insight into these three issues is required to better understand vitamin A absorption, storage and mobilization. Here, we provide an up to date synthesis of current knowledge regarding the intestinal uptake of dietary vitamin A, the storage of vitamin A within the liver, and the mobilization of hepatic vitamin A stores, and summarize areas where our understanding of these processes is incomplete.

Retinoic Acid Mediates Visceral-specific Adipogenic Defects of Human Adipose-derived Stem Cells

Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots.

DGAT1-deficiency affects the cellular distribution of hepatic retinoid and attenuates the progression of CCl4-induced liver fibrosis

BACKGROUND Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final step of triglyceride synthesis, transferring an acyl group from acyl-CoA to diacylglycerol. DGAT1 also catalyzes the acyl-CoA-dependent formation of retinyl esters in vitro and in mouse intestine and skin. Although DGAT1 is expressed in both hepatocytes and hepatic stellate cells (HSCs), we reported genetic and nutritional studies that established that DGAT1 does not contribute to retinyl ester formation in the liver. METHODS We now have explored in more depth the role(s) of DGAT1 in hepatic retinoid metabolism and storage. RESULTS Our data show that DGAT1 affects the cellular distribution between hepatocytes and HSCs of stored and newly absorbed dietary retinol. For livers of Dgat1-deficient mice, a greater percentage of stored retinyl ester is present in HSCs at the expense of hepatocytes. This is also true for newly absorbed oral [(3)H]retinol. These differences are associated with significantly increased expression, by 2.8-fold, of cellular retinol-binding protein, type I (RBP1) in freshly isolated HSCs from Dgat1-deficient mice, raising the possibility that RBP1, which contributes to retinol uptake into cells and retinyl ester synthesis, accounts for the differences. We further show that the retinyl ester-containing lipid droplets in HSCs are affected in Dgat1-null mice, being fewer in number but, on average, larger than in wild type (WT) HSCs. Finally, we demonstrate that DGAT1 affects experimentally induced HSC activation in vivo but that this effect is independent of altered retinoic acid availability or effects on gene expression. CONCLUSIONS Our studies establish that DGAT1 has a role in hepatic retinoid storage and metabolism, but this does not involve direct actions of DGAT1 in retinyl ester synthesis.

Carotenoid Metabolism and Enzymology

Carotenoids are plant-derived lipophilic compounds with a common chemical structure of eight isoprenoid units. The major carotenoids that are present in human tissues are primarily the polyunsaturated hydrocarbon carotenes β-carotene and lycopene and the oxygen-containing xanthophylls β-cryptoxanthin, lutein, and zeaxanthin. These carotenoids function in the body as retinoid (vitamin A) precursors and antioxidants.