Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state

Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin

In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1H−/−), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy and increased phosphorylated S6 kinase (S6K), a substrate of the mechanistic target of rapamycin, mTOR. Doppler echocardiography revealed evidence of significant diastolic dysfunction, indicated by a reduced E/A ratio and increased mean performance index, although the deceleration time and the expression of sarco/endoplasmic reticulum calcium ATPase and phospholambin showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1H−/− mice with rapamycin. Six to eight week old Acsl1H−/− mice and their littermate controls were given i.p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10 weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1H−/− hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1H−/− hearts exhibited an 8-fold higher uptake of 2-deoxy[1-14C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-14C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1H−/− mice

Acyl-CoA synthetase 1 deficiency alters cardiolipin species and impairs mitochondrial function

Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (Acsl1T−/−) have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. Acsl1T−/− hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in Acsl1T−/− hearts, control and Acsl1T−/− mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function

Inhibited Insulin Signaling in Mouse Hepatocytes Is Associated with Increased Phosphatidic Acid but Not Diacylglycerol

Although an elevated triacylglycerol content in non-adipose tissues is often associated with insulin resistance, the mechanistic relationship remains unclear. The data support roles for intermediates in the glycerol-3-phosphate pathway of triacylglycerol synthesis: diacylglycerol (DAG), which may cause insulin resistance in liver by activating PKCϵ, and phosphatidic acid (PA), which inhibits insulin action in hepatocytes by disrupting the assembly of mTOR and rictor. To determine whether increases in DAG and PA impair insulin signaling when produced by pathways other than that of de novo synthesis, we examined primary mouse hepatocytes after enzymatically manipulating the cellular content of DAG or PA. Overexpressing phospholipase D1 or phospholipase D2 inhibited insulin signaling and was accompanied by an elevated cellular content of total PA, without a change in total DAG. Overexpression of diacylglycerol kinase-θ inhibited insulin signaling and was accompanied by an elevated cellular content of total PA and a decreased cellular content of total DAG. Overexpressing glycerol-3-phosphate acyltransferase-1 or -4 inhibited insulin signaling and increased the cellular content of both PA and DAG. Insulin signaling impairment caused by overexpression of phospholipase D1/D2 or diacylglycerol kinase-θ was always accompanied by disassociation of mTOR/rictor and reduction of mTORC2 kinase activity. However, although the protein ratio of membrane to cytosolic PKCϵ increased, PKC activity itself was unaltered. These data suggest that PA, but not DAG, is associated with impaired insulin action in mouse hepatocytes

FGF21, a liver hormone that inhibits alcohol intake in mice, increases in human circulation after acute alcohol ingestion and sustained binge drinking at Oktoberfest

Objective: Excessive alcohol consumption is a leading cause of global morbidity and mortality. However, knowledge of the biological factors that influence ad libitum alcohol intake may be incomplete. Two large studies recently linked variants in the KLB locus with levels of alcohol intake in humans. KLB encodes β-klotho, co-receptor for the liver-derived hormone fibroblast growth factor 21 (FGF21). In mice, FGF21 reduces alcohol intake, and human Fgf21 variants are enriched among heavy drinkers. Thus, the liver may limit alcohol consumption by secreting FGF21. However, whether full-length, active plasma FGF21 (FGF21 (1–181)) levels in humans increase acutely or sub-chronically in response to alcohol ingestion is uncertain. Methods: We recruited 10 healthy, fasted male subjects to receive an oral water or alcohol bolus with concurrent blood sampling for FGF21 (1–181) measurement in plasma. In addition, we measured circulating FGF21 (1–181) levels, liver stiffness, triglyceride, and other metabolic parameters in three healthy Danish men before and after consuming an average of 22.6 beers/person/day (4.4 g/kg/day of ethanol) for three days during Oktoberfest 2017 in Munich, Germany. We further correlated fasting FGF21 (1–181) levels in 49 healthy, non-alcoholic subjects of mixed sex with self-reports of alcohol-related behaviors, emotional responses, and problems. Finally, we characterized the effect of recombinant human FGF21 injection on ad libitum alcohol intake in mice. Results: We show that alcohol ingestion (25.3 g or ∼2.5 standard drinks) acutely increases plasma levels of FGF21 (1–181) 3.4-fold in fasting humans. We also find that binge drinking for three days at Oktoberfest is associated with a 2.1-fold increase in baseline FGF21 (1–181) levels, in contrast to minor deteriorations in metabolic and hepatic biomarkers. However, basal FGF21 (1–181) levels were not correlated with differences in alcohol-related behaviors, emotional responses, or problems in our non-alcoholic subjects. Finally, we show that once-daily injection of recombinant human FGF21 reduces ad libitum alcohol intake by 21% in mice. Conclusions: FGF21 (1–181) is markedly increased in circulation by both acute and sub-chronic alcohol intake in humans, and reduces alcohol intake in mice. These observations are consistent with a role for FGF21 as an endocrine inhibitor of alcohol appetite in humans. Keywords: Fibroblast growth factor 21, FGF21, Alcohol, Alcohol appetit

Compartmentalized Acyl-CoA Metabolism in Skeletal Muscle Regulates Systemic Glucose Homeostasis

The impaired capacity of skeletal muscle to switch between the oxidation of fatty acid (FA) and glucose is linked to disordered metabolic homeostasis. To understand how muscle FA oxidation affects systemic glucose, we studied mice with a skeletal muscle–specific deficiency of long-chain acyl-CoA synthetase (ACSL)1. ACSL1 deficiency caused a 91% loss of ACSL-specific activity and a 60–85% decrease in muscle FA oxidation. Acsl1M−/− mice were more insulin sensitive, and, during an overnight fast, their respiratory exchange ratio was higher, indicating greater glucose use. During endurance exercise, Acsl1M−/− mice ran only 48% as far as controls. At the time that Acsl1M−/− mice were exhausted but control mice continued to run, liver and muscle glycogen and triacylglycerol stores were similar in both genotypes; however, plasma glucose concentrations in Acsl1M−/− mice were ∼40 mg/dL, whereas glucose concentrations in controls were ∼90 mg/dL. Excess use of glucose and the likely use of amino acids for fuel within muscle depleted glucose reserves and diminished substrate availability for hepatic gluconeogenesis. Surprisingly, the content of muscle acyl-CoA at exhaustion was markedly elevated, indicating that acyl-CoAs synthesized by other ACSL isoforms were not available for β-oxidation. This compartmentalization of acyl-CoAs resulted in both an excessive glucose requirement and severely compromised systemic glucose homeostasis

MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo

Erratum: https://link.springer.com/article/10.1007/s10863-014-9597-1MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1’s role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1−/− mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. This is an factual error as written; however, total oxygen consumption was decreased. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1−/− hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2’s regulation of mitochondrial function.Funding support from Medical Research Council, United Kingdom; National Institutes of Health, United States; British Heart Foundation, United Kingdo