Erin E. Kershaw

ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-[alpha] and PGC-1

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate genes involved in energy metabolism and inflammation. For biological activity, PPARs require cognate lipid ligands, heterodimerization with retinoic X receptors, and coactivation by PPAR-γ coactivator-1α or PPAR-γ coactivator-1β (PGC-1α or PGC-1β, encoded by Ppargc1a and Ppargc1b, respectively). Here we show that lipolysis of cellular triglycerides by adipose triglyceride lipase (patatin-like phospholipase domain containing protein 2, encoded by Pnpla2; hereafter referred to as Atgl) generates essential mediator(s) involved in the generation of lipid ligands for PPAR activation. Atgl deficiency in mice decreases mRNA levels of PPAR-α and PPAR-δ target genes. In the heart, this leads to decreased PGC-1α and PGC-1β expression and severely disrupted mitochondrial substrate oxidation and respiration; this is followed by excessive lipid accumulation, cardiac insufficiency and lethal cardiomyopathy. Reconstituting normal PPAR target gene expression by pharmacological treatment of Atgl-deficient mice with PPAR-α agonists completely reverses the mitochondrial defects, restores normal heart function and prevents premature death. These findings reveal a potential treatment for the excessive cardiac lipid accumulation and often-lethal cardiomyopathy in people with neutral lipid storage disease, a disease marked by reduced or absent ATGL activity.

Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr)

The rat fatty (fa) mutation produces profound obesity of early onset caused by hyperphagia, defective nonshivering thermogenesis, and preferential deposition of energy into adipose tissue. Genetic mapping studies indicate that fa and diabetes (db) are homologous loci in the rat and mouse genomes, respectively. It has been shown that db alleles carry mutations in the Lepr (leptin receptor) gene. This paper describes a point mutation in the fatty allele of Lepr. A nucleotide substitution at position 880 (A→C) causes an amino acid substitution at position 269 (Gln → Pro). The mutation generates a novel Msp I site that cosegregates with fa in 1,028 meioses examined in obese F2 progeny from two crosses (BN×13M and WKY×13M) and is still segregating in three rat colonies. PCR-based mutagenesis was used to introduce the fa mutation into the mouse Lepr cDNA. Transient transfection studies indicate that the mutant Lepr cDNA has greatly reduced binding of leptin (Lep) at the cell surface. These data are strong evidence that the single nucleotide substitution in the fa allele of Lepr (Leprfa) is responsible for the obese phenotype.

Protein-tyrosine Phosphatase 1B Expression Is Induced by Inflammation in Vivo

Protein-tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin and leptin sensitivity. PTP1B overexpression in adipose tissue and skeletal muscle of humans and rodents may contribute to insulin resistance and obesity. The mechanisms mediating PTP1B overexpression in obese and diabetic states have been unclear. We find that adipose tissue inflammation and the pro-inflammatory cytokine tumor necrosis factor α (TNFα) regulate PTP1B expression in vivo. High fat feeding of mice increased PTP1B expression 1.5- to 7-fold in adipose tissue, liver, skeletal muscle, and arcuate nucleus of hypothalamus. PTP1B overexpression in high fat-fed mice coincided with increased adipose tissue expression of the macrophage marker CD68 and TNFα, which is implicated in causing obesity-induced insulin resistance. TNFα increased PTP1B mRNA and protein levels by 2- to 5-fold in a dose- and time-dependent manner in adipocyte and hepatocyte cell lines. TNFα administration in mice increased PTP1B mRNA 1.4- to 4-fold in adipose tissue, liver, skeletal muscle, and hypothalamic arcuate nucleus and PTP1B protein 2-fold in liver. Actinomycin D treatment blocked, and high dose salicylate treatment inhibited by 80%, TNFα-induced PTP1B expression in adipocyte cell lines, suggesting TNFα may induce PTP1B transcription via nuclear factor κB (NFκB) activation. Chromatin immunoprecipitation from adipocyte cell lines and liver of mice demonstrated TNFα-induced recruitment of NFκB subunit p65 to the PTP1B promoter in vitro and in vivo. In mice with diet-induced obesity, TNFα deficiency also partly blocked PTP1B overexpression in adipose tissue. Our data suggest that PTP1B overexpression in multiple tissues in obesity is regulated by inflammation and that PTP1B may be a target of anti-inflammatory therapies.

Transcriptional control of adipose lipid handling by IRF4.

Adipocytes store triglyceride during periods of nutritional affluence and release free fatty acids during fasting through coordinated cycles of lipogenesis and lipolysis. While much is known about the acute regulation of these processes during fasting and feeding, less is understood about the transcriptional basis by which adipocytes control lipid handling. Here, we show that interferon regulatory factor 4 (IRF4) is a critical determinant of the transcriptional response to nutrient availability in adipocytes. Fasting induces IRF4 in an insulin- and FoxO1-dependent manner. IRF4 is required for lipolysis, at least in part due to direct effects on the expression of adipocyte triglyceride lipase and hormone-sensitive lipase. Conversely, reduction of IRF4 enhances lipid synthesis. Mice lacking adipocyte IRF4 exhibit increased adiposity and deficient lipolysis. These studies establish a link between IRF4 and the disposition of calories in adipose tissue, with consequences for systemic metabolic homeostasis.

Adiponutrin Functions as a Nutritionally Regulated Lysophosphatidic Acid Acyltransferase

Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.

Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome

PNPLA3 (adiponutrin, calcium-independent phospholipase A2 epsilon [iPLA2ε]) is an adipose-enriched, nutritionally regulated protein that belongs to the patatin-like phospholipase domain containing (PNPLA) family of lipid metabolizing proteins. Genetic variations in the human PNPLA3 gene (i.e., the rs738409 I148M allele) has been strongly and repeatedly associated with fatty liver disease. Although human PNPLA3 has triacylglycerol (TAG) hydrolase and transacylase activities in vitro, its in vivo function and physiological relevance remain controversial. The objective of this study was to determine the metabolic consequences of global targeted deletion of the Pnpla3 gene in mice. We found that Pnpla3 mRNA expression is altered in adipose tissue and liver in response to acute and chronic nutritional challenges. However, global targeted deletion of the Pnpla3 gene in mice did not affect TAG hydrolysis, nor did it influence energy/glucose/lipid homoeostasis or hepatic steatosis/injury. Experimental interventions designed to increase Pnpla3 expression (refeeding, high-sucrose diet, diet-induced obesity, and liver X receptor agonism) likewise failed to reveal differences in the above-mentioned metabolic phenotypes. Expression of the Pnpla3 paralog, Pnpla5, was increased in adipose tissue but not in liver of Pnpla3-deficient mice, but compensatory regulation of genes involved in TAG metabolism was not identified. Together these data argue against a role for Pnpla3 loss-of-function in fatty liver disease or metabolic syndrome in mice.

Adipose triglyceride lipase deficiency causes tissue-specific changes in insulin signaling.

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL−/−) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL−/− mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL−/− mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.

Coupling of lipolysis and de novo lipogenesis in brown, beige, and white adipose tissues during chronic β3-adrenergic receptor activation

Chronic activation of β3-adrenergic receptors (β3-ARs) expands the catabolic activity of both brown and white adipose tissue by engaging uncoupling protein 1 (UCP1)-dependent and UCP1-independent processes. The present work examined de novo lipogenesis (DNL) and TG/glycerol dynamics in classic brown, subcutaneous “beige,” and classic white adipose tissues during sustained β3-AR activation by CL 316,243 (CL) and also addressed the contribution of TG hydrolysis to these dynamics. CL treatment for 7 days dramatically increased DNL and TG turnover similarly in all adipose depots, despite great differences in UCP1 abundance. Increased lipid turnover was accompanied by the simultaneous upregulation of genes involved in FAS, glycerol metabolism, and FA oxidation. Inducible, adipocyte-specific deletion of adipose TG lipase (ATGL), the rate-limiting enzyme for lipolysis, demonstrates that TG hydrolysis is required for CL-induced increases in DNL, TG turnover, and mitochondrial electron transport in all depots. Interestingly, the effect of ATGL deletion on induction of specific genes involved in FA oxidation and synthesis varied among fat depots. Overall, these studies indicate that FAS and FA oxidation are tightly coupled in adipose tissues during chronic adrenergic activation, and this effect critically depends on the activity of adipocyte ATGL.

Myocardial ATGL Overexpression Decreases the Reliance on Fatty Acid Oxidation and Protects against Pressure Overload-Induced Cardiac Dysfunction

ABSTRACT Alterations in myocardial triacylglycerol content have been associated with poor left ventricular function, suggesting that enzymes involved in myocardial triacylglycerol metabolism play an important role in regulating contractile function. Myocardial triacylglycerol catabolism is mediated by adipose triglyceride lipase (ATGL), which is rate limiting for triacylglycerol hydrolysis. To address the influence of triacylglycerol hydrolysis on myocardial energy metabolism and function, we utilized mice with cardiomyocyte-specific ATGL overexpression (MHC-ATGL). Biochemical examination of MHC-ATGL hearts revealed chronically reduced myocardial triacylglycerol content but unchanged levels of long-chain acyl coenzyme A esters, ceramides, and diacylglycerols. Surprisingly, fatty acid oxidation rates were decreased in ex vivo perfused working hearts from MHC-ATGL mice, which was compensated by increased rates of glucose oxidation. Interestingly, reduced myocardial triacylglycerol content was associated with moderately enhanced in vivo systolic function in MHC-ATGL mice and increased isoproterenol-induced cell shortening of isolated primary cardiomyocytes. Most importantly, MHC-ATGL mice were protected from pressure overload-induced systolic dysfunction and detrimental structural remodeling following transverse aortic constriction. Overall, this study shows that ATGL overexpression is sufficient to alter myocardial energy metabolism and improve cardiac function.

Myocardial Adipose Triglyceride Lipase Overexpression Protects Diabetic Mice From the Development of Lipotoxic Cardiomyopathy

Although diabetic cardiomyopathy is associated with enhanced intramyocardial triacylglycerol (TAG) levels, the role of TAG catabolizing enzymes in this process is unclear. Because the TAG hydrolase, adipose triglyceride lipase (ATGL), regulates baseline cardiac metabolism and function, we examined whether alterations in cardiomyocyte ATGL impact cardiac function during uncontrolled type 1 diabetes. In genetic (Akita) and pharmacological (streptozotocin) murine models of type 1 diabetes, cardiac ATGL protein expression and TAG content were significantly increased. To determine whether increased ATGL expression during diabetes is detrimental or beneficial to cardiac function, we studied streptozotocin-diabetic mice with heterozygous ATGL deficiency and cardiomyocyte-specific ATGL overexpression. After diabetes, streptozotocin-diabetic mice with heterozygous ATGL deficiency displayed increased TAG accumulation, lipotoxicity, and diastolic dysfunction comparable to wild-type mice. In contrast, myosin heavy chain promoter (MHC)-ATGL mice were resistant to diabetes-induced increases in intramyocardial TAG levels, lipotoxicity, and cardiac dysfunction. Moreover, hearts from diabetic MHC-ATGL mice exhibited decreased reliance on palmitate oxidation and blunted peroxisome proliferator--activated receptor-α activation. Collectively, this study shows that after diabetes, increased cardiac ATGL expression is an adaptive, albeit insufficient, response to compensate for the accumulation of myocardial TAG, and that overexpression of ATGL is sufficient to ameliorate diabetes-induced cardiomyopathy.