Suneil K. Koliwad

DGAT enzymes and triacylglycerol biosynthesis

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases.

DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation

Diet-induced obesity (DIO) leads to inflammatory activation of macrophages in white adipose tissue (WAT) and subsequently to insulin resistance. PPARgamma agonists are antidiabetic agents known to suppress inflammatory macrophage activation and to induce expression of the triacylglycerol (TG) synthesis enzyme acyl CoA: diacylglycerol acyltransferase 1 (DGAT1) in WAT and in adipocytes. Here, we investigated in mice the relationship between macrophage lipid storage capacity and DIO-associated inflammatory macrophage activation. Mice overexpressing DGAT1 in both macrophages and adipocytes (referred to herein as aP2-Dgat1 mice) were more prone to DIO but were protected against inflammatory macrophage activation, macrophage accumulation in WAT, systemic inflammation, and insulin resistance. To assess the contribution of macrophage DGAT1 expression to this phenotype, we transplanted wild-type mice with aP2-Dgat1 BM. These mice developed DIO similar to that of control mice but retained the protection from WAT inflammation and insulin resistance seen in aP2-Dgat1 mice. In isolated macrophages, Dgat1 mRNA levels correlated directly with TG storage capacity and inversely with inflammatory activation by saturated fatty acids (FAs). Moreover, PPARgamma agonists increased macrophage Dgat1 mRNA levels, and the protective effects of these agonists against FA-induced inflammatory macrophage activation were absent in macrophages isolated from Dgat1-null mice. Thus, increasing DGAT1 expression in murine macrophages increases their capacity for TG storage, protects against FA-induced inflammatory activation, and is sufficient to reduce the inflammatory and metabolic consequences of DIO.

Microglia Dictate the Impact of Saturated Fat Consumption on Hypothalamic Inflammation and Neuronal Function

Diets rich in saturated fat produce inflammation, gliosis, and neuronal stress in the mediobasal hypothalamus (MBH). Here, we show that microglia mediate this process and its functional impact. Although microglia and astrocytes accumulate in the MBH of mice fed a diet rich in saturated fatty acids (SFAs), only the microglia undergo inflammatory activation, along with a buildup of hypothalamic SFAs. Enteric gavage specifically with SFAs reproduces microglial activation and neuronal stress in the MBH, and SFA treatment activates murine microglia, but not astrocytes, in culture. Moreover, depleting microglia abrogates SFA-induced inflammation in hypothalamic slices. Remarkably, depleting microglia from the MBH of mice abolishes inflammation and neuronal stress induced by excess SFA consumption, and in this context, microglial depletion enhances leptin signaling and reduces food intake. We thus show that microglia sense SFAs and orchestrate an inflammatory process in the MBH that alters neuronal function when SFA consumption is high.

Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-regulated triglyceride metabolism.

Glucocorticoids are important regulators of lipid homeostasis, and chronically elevated glucocorticoid levels induce hypertriglyceridemia, hepatic steatosis, and visceral obesity. The occupied glucocorticoid receptor (GR) is a transcription factor. However, those genes regulating lipid metabolism under GR control are not fully known. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting, when circulating glucocorticoid levels are physiologically increased. We therefore tested whether the ANGPTL4 gene (Angptl4) is transcriptionally controlled by GR. We show that treatment with the synthetic glucocorticoid dexamethasone increased Angptl4 mRNA levels in primary hepatocytes and adipocytes (2–3-fold) and in the livers and white adipose tissue of mice (∼4-fold). We tested the mechanism of this increase in H4IIE hepatoma cells and found that dexamethasone treatment increased the transcriptional rate of Angptl4. Using bioinformatics and chromatin immunoprecipitation, we identified a GR binding site within the rat Angptl4 sequence. A reporter plasmid containing this site was markedly activated by dexamethasone, indicative of a functional glucocorticoid response element. Dexamethasone treatment also increased histone H4 acetylation and DNase I accessibility in genomic regions near this site, further supporting that it is a glucocorticoid response element. Glucocorticoids promote the flux of triglycerides from white adipose tissue to liver. We found that mice lacking ANGPTL4 (Angptl4−/−) had reductions in dexamethasone-induced hypertriglyceridemia and hepatic steatosis, suggesting that ANGPTL4 is required for this flux. Overall, we establish that ANGPTL4 is a direct GR target that participates in glucocorticoid-regulated triglyceride metabolism.

Angiopoietin-like 4 (Angptl4) Protein Is a Physiological Mediator of Intracellular Lipolysis in Murine Adipocytes

Background: Angiopoietin-like 4 (Angptl4) is a secreted protein involved in triacylglycerol homeostasis. Results: Angptl4 was required for fasting, glucocorticoids, and catecholamines to stimulate cAMP-dependent signaling and triacylglycerol hydrolysis in murine fat, a response reproduced by treating adipocytes with purified ANGPTL4. Conclusion: Angptl4 is a physiological mediator of lipolysis. Significance: This finding may impact aberrant lipolytic states like insulin resistance. Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice, and TG hydrolysis induced by fasts of 6 or 24 h was greatly reduced in mice lacking Angptl4 (Angptl4−/−). Glucocorticoid treatment mimicked the lipolytic effects of fasting, although with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this, cAMP levels and TG hydrolysis were reduced in primary Angptl4−/− murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4. Remarkably, human ANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4 modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance.

Oxidant stress activates a non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells.

1. In vascular endothelial cells, oxidant stress increases cell Na+ content and inhibits the agonist‐stimulated influx of external Ca2+. Further, oxidant stress increases uptake of Ca2+ into otherwise quiescent endothelial cells. To determine the mechanism responsible for altered Na+ and Ca2+ homeostasis, the present study examined the effect of oxidant stress on ionic current and channel activity in calf pulmonary artery endothelial cells. 2. Voltage‐clamped control cells had a zero‐current potential of ‐60 mV. Incubation of cells with the oxidant tert‐butylhydroperoxide (tBuOOH; 0.4 mM, 1 h) caused depolarization to ‐4 mV and activation of ionic current equally selective for Na+ and K+. 3. Cell‐attached membrane patches made on tBuOOH‐treated cells contained ion channels that had a bidirectional conductance of 30 pS and that were not present in patches from control cells. Inside‐out patches excised from oxidant‐treated cells showed the channel to be equally selective for Na+ and K+ and to allow inward Ca2+ current. 4. Oxidant‐activated channels were observed to display two gating modalities that were further evident during analysis of single‐channel open probability. Neither modality was significantly affected by altering internal [Ca2+] (1 microM‐10 nM). 5. Activation of non‐selective channels provides a possible mechanism by which oxidants may increase endothelial cell Na+ content. Channel permeability to Ca2+ may account in part for the elevation of cytosolic free [Ca2+] that occurs in oxidant‐treated cells. 6. Channel activation is associated with membrane depolarization, a mechanism that may contribute to oxidant inhibition of the agonist‐stimulated Ca2+ influx pathway.

Hypothalamic Inflammation in the Control of Metabolic Function

Diet-induced obesity leads to devastating and common chronic diseases, fueling ongoing interest in determining new mechanisms underlying both obesity and its consequences. It is now well known that chronic overnutrition produces a unique form of inflammation in peripheral insulin target tissues, and efforts to limit this inflammation have met with some success in preserving insulin sensitivity in obese individuals. Recently, the activation of inflammatory pathways by dietary excess has also been observed among cells located in the mediobasal hypothalamus, a brain area that exerts central control over peripheral glucose, fat, and energy metabolism. Here we review progress in the field of diet-induced hypothalamic inflammation, drawing key distinctions between metabolic inflammation in the hypothalamus and that occurring in peripheral tissues. We focus on specific stimuli of the inflammatory response, the roles of individual hypothalamic cell types, and the links between hypothalamic inflammation and metabolic function under normal and pathophysiological circumstances. Finally, we explore the concept of controlling hypothalamic inflammation to mitigate metabolic disease.

Oxidized glutathione mediates cation channel activation in calf vascular endothelial cells during oxidant stress.

1. The oxidant, tert‐butylhydroperoxide (tBuOOH) depolarizes calf pulmonary artery endothelial cells by activating a non‐selective cation channel. To identify the molecular mediator of channel activation during oxidant stress, the patch‐clamp technique was used to compare tBuOOH‐induced changes in membrane potential and channel activity with those induced by oxidized glutathione (GSSG), a cytosolic product of oxidant metabolism. 2. When recording pipettes contained GSSG (2 mM), whole‐cell zero‐current potential measured immediately following pipette break‐in was not different from control values (‐57 mV). However, within 20 min of break‐in, zero‐current potential was depolarized to ‐7 mV. The time course of depolarization was dependent on the concentration of GSSG and was accelerated by inhibition of GSSG metabolism. 3. In excised membrane patches, channels were activated by internal GSSG, but not by internal tBuOOH, reduced glutathione (GSH), or external GSSG. Channels were equal in size (28 pS) and in ionic selectivity to those activated by incubation of intact cells with tBuOOH. As little as 20 microM GSSG was sufficient to maximally activate channels. However, the time course of channel activation was concentration dependent between 20 microM and 2 mM GSSG. 4. Channel activation by GSSG was reversed by GSH and by increasing the [GSH]:[GSSG] ratio. Likewise, channel activation by pre‐incubation of intact cells with tBuOOH was reversed by GSH applied after patch excision. 5. These results strongly suggest that GSSG is an endogenous intracellular mediator of channel activation and depolarization during oxidant stress.

Oxidant stress and endothelial membrane transport

The endothelium modulates vascular tone, vasoreactivity, and permeability in response to agonist-stimulation. Much of the pathophysiology of oxidant-induced vascular injury can be attributed to endothelial cell dysfunction. In the past several years, the effects of oxidant stress on agonist-stimulated Ca(2+)-channels have been described. More recently, the effects of oxidant stress on several other endothelial membrane-transport systems have been elucidated. It now appears that inhibition of the agonist-stimulated Ca2+ channel is due at least in part to membrane depolarization via oxidant-activation of a Na(+)-permeable, nonselective cation channel. In this review, the effects of oxidant stress on ion transport through the agonist-stimulated Ca2+ influx channel, Na+ and K+ channels, Na+/K(+)-ATPase, Ca(2+)-ATPase, and the Na+/K+/2Cl- cotransporter are discussed. The interrelated effects of oxidant stress on these endothelial membrane transport pathways are considered, and the net effect on Ca2+ signaling is described.

A screen in mice uncovers repression of lipoprotein lipase by microRNA‐29a as a mechanism for lipid distribution away from the liver

Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy‐dependent cells like myocytes, LPL is normally repressed in adult hepatocytes. We identified miR‐29a as the miRNA responsible for repressing LPL in hepatocytes, and found that decreasing hepatic miR‐29a levels causes lipids to accumulate in mouse livers. Conclusion: Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR‐29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR‐29as known antifibrotic effect, suggest miR‐29a is a therapeutic target in fatty liver disease. (Hepatology 2015;61:141–152)