Acyl-Coenzyme A Thioesterase 9 Traffics Mitochondrial Short-Chain Fatty Acids Toward De Novo Lipogenesis and Glucose Production in the Liver.

BACKGROUND AND AIMS: Obesity-induced pathogenesis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is associated with increased de novo lipogenesis (DNL) and hepatic glucose production (HGP) that is due to excess fatty acids. Acyl-coenzyme A (CoA) thioesterase (Acot) family members control the cellular utilization of fatty acids by hydrolyzing (deactivating) acyl-CoA into nonesterified fatty acids and CoASH. APPROACH AND RESULTS: Using Caenorhabditis elegans, we identified Acot9 as the strongest regulator of lipid accumulation within the Acot family. Indicative of a maladaptive function, hepatic Acot9 expression was higher in patients with obesity who had NAFLD and NASH compared with healthy controls with obesity. In the setting of excessive nutrition, global ablation of Acot9 protected mice against increases in weight gain, HGP, steatosis, and steatohepatitis. Supportive of a hepatic function, the liver-specific deletion of Acot9 inhibited HGP and steatosis in mice without affecting diet-induced weight gain. By contrast, the rescue of Acot9 expression only in the livers of Acot9 knockout mice was sufficient to promote HGP and steatosis. Mechanistically, hepatic Acot9 localized to the inner mitochondrial membrane, where it deactivated short-chain but not long-chain fatty acyl-CoA. This unique localization and activity of Acot9 directed acetyl-CoA away from protein lysine acetylation and toward the citric acid (TCA) cycle. Acot9-mediated exacerbation of triglyceride and glucose biosynthesis was attributable at least in part to increased TCA cycle activity, which provided substrates for HGP and DNL. ß-oxidation and ketone body production, which depend on long-chain fatty acyl-CoA, were not regulated by Acot9. CONCLUSIONS: Taken together, our findings indicate that Acot9 channels hepatic acyl-CoAs toward increased HGP and DNL under the pathophysiology of obesity. Therefore, Acot9 represents a target for the management of NAFLD.

Mechanism of N-terminal modulation of activity at the melanocortin-4 receptor GPCR.

Most of our understanding of G protein-coupled receptor (GPCR) activation has been focused on the direct interaction between diffusible ligands and their seven-transmembrane domains. However, a number of these receptors depend on their extracellular N-terminal domain for ligand recognition and activation. To dissect the molecular interactions underlying both modes of activation at a single receptor, we used the unique properties of the melanocortin-4 receptor (MC4R), a GPCR that shows constitutive activity maintained by its N-terminal domain and is physiologically activated by the peptide α-melanocyte stimulating hormone (αMSH). We find that activation by the N-terminal domain and αMSH relies on different key residues in the transmembrane region. We also demonstrate that agouti-related protein, a physiological antagonist of MC4R, acts as an inverse agonist by inhibiting N terminus-mediated activation, leading to the speculation that a number of constitutively active orphan GPCRs could have physiological inverse agonists as sole regulators.

Submitochondrial Protein Translocation Upon Stress Inhibits Thermogenic Energy Expenditure.

Mitochondria-rich brown adipocytes dissipate cellular fuel as heat by thermogenic energy expenditure (TEE). Prolonged nutrient excess or cold exposure impair TEE and contribute to the pathogenesis of obesity, but the mechanisms remain incompletely understood. Here we report that stress-induced proton leak into the matrix interface of mitochondrial innermembrane (IM) mobilizes a group of proteins from IM into matrix, which in turn alter mitochondrial bioenergetics. We further determine a smaller subset that correlates with obesity in human subcutaneous adipose tissue. We go on to show that the top factor on this short list, acyl-CoA thioesterase 9 (ACOT9), migrates from the IM into the matrix upon stress where it enzymatically deactivates and prevents the utilization of acetyl-CoA in TEE. The loss of ACOT9 protects mice against the complications of obesity by maintaining unobstructed TEE. Overall, our results introduce aberrant protein translocation as a strategy to identify pathogenic factors. One-Sentence Summary: Thermogenic stress impairs mitochondrial energy utilization by forcing translocation of IM-bound proteins into the matrix.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Genetic ablation or chemical inhibition of phosphatidylcholine transfer protein attenuates diet-induced hepatic glucose production.

UNLABELLED: Phosphatidylcholine transfer protein (PC-TP, synonym StARD2) is a highly specific intracellular lipid binding protein that is enriched in liver. Coding region polymorphisms in both humans and mice appear to confer protection against measures of insulin resistance. The current study was designed to test the hypotheses that Pctp-/- mice are protected against diet-induced increases in hepatic glucose production and that small molecule inhibition of PC-TP recapitulates this phenotype. Pctp-/- and wildtype mice were subjected to high-fat feeding and rates of hepatic glucose production and glucose clearance were quantified by hyperinsulinemic euglycemic clamp studies and pyruvate tolerance tests. These studies revealed that high-fat diet-induced increases in hepatic glucose production were markedly attenuated in Pctp-/- mice. Small molecule inhibitors of PC-TP were synthesized and their potencies, as well as mechanism of inhibition, were characterized in vitro. An optimized inhibitor was administered to high-fat-fed mice and used to explore effects on insulin signaling in cell culture systems. Small molecule inhibitors bound PC-TP, displaced phosphatidylcholines from the lipid binding site, and increased the thermal stability of the protein. Administration of the optimized inhibitor to wildtype mice attenuated hepatic glucose production associated with high-fat feeding, but had no activity in Pctp-/- mice. Indicative of a mechanism for reducing glucose intolerance that is distinct from commonly utilized insulin-sensitizing agents, the inhibitor promoted insulin-independent phosphorylation of key insulin signaling molecules. CONCLUSION: These findings suggest PC-TP inhibition as a novel therapeutic strategy in the management of hepatic insulin resistance.

Human hepatocyte PNPLA3-148M exacerbates rapid non-alcoholic fatty liver disease development in chimeric mice.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.

Association of functionally significant Melanocortin-4 but not Melanocortin-3 receptor mutations with severe adult obesity in a large North American case-control study.

Functionally significant heterozygous mutations in the Melanocortin-4 receptor (MC4R) have been implicated in 2.5% of early onset obesity cases in European cohorts. The role of mutations in this gene in severely obese adults, particularly in smaller North American patient cohorts, has been less convincing. More recently, it has been proposed that mutations in a phylogenetically and physiologically related receptor, the Melanocortin-3 receptor (MC3R), could also be a cause of severe human obesity. The objectives of this study were to determine if mutations impairing the function of MC4R or MC3R were associated with severe obesity in North American adults. We studied MC4R and MC3R mutations detected in a total of 1821 adults (889 severely obese and 932 lean controls) from two cohorts. We systematically and comparatively evaluated the functional consequences of all mutations found in both MC4R and MC3R. The total prevalence of rare MC4R variants in severely obese North American adults was 2.25% (CI(95%): 1.44-3.47) compared with 0.64% (CI(95%): 0.26-1.43) in lean controls (P < 0.005). After classification of functional consequence, the prevalence of MC4R mutations with functional alterations was significantly greater when compared with controls (P < 0.005). In contrast, the prevalence of rare MC3R variants was not significantly increased in severely obese adults [0.67% (CI(95%): 0.27-1.50) versus 0.32% (CI(95%): 0.06-0.99)] (P = 0.332). Our results confirm that mutations in MC4R are a significant cause of severe obesity, extending this finding to North American adults. However, our data suggest that MC3R mutations are not associated with severe obesity in this population.

Transcriptional Regulation in Non-Alcoholic Fatty Liver Disease.

Obesity is the primary risk factor for the pathogenesis of non-alcoholic fatty liver disease (NAFLD), the worldwide prevalence of which continues to increase dramatically. The liver plays a pivotal role in the maintenance of whole-body lipid and glucose homeostasis. This is mainly mediated by the transcriptional activation of hepatic pathways that promote glucose and lipid production or utilization in response to the nutritional state of the body. However, in the setting of chronic excessive nutrition, the dysregulation of hepatic transcriptional machinery promotes lipid accumulation, inflammation, metabolic stress, and fibrosis, which culminate in NAFLD. In this review, we provide our current understanding of the transcription factors that have been linked to the pathogenesis and progression of NAFLD. Using publicly available transcriptomic data, we outline the altered activity of transcription factors among humans with NAFLD. By expanding this analysis to common experimental mouse models of NAFLD, we outline the relevance of mouse models to the human pathophysiology at the transcriptional level.

Fatty acid activation in thermogenic adipose tissue.

Channeling carbohydrates and fatty acids to thermogenic tissues, including brown and beige adipocytes, have garnered interest as an approach for the management of obesity-related metabolic disorders. Mitochondrial fatty acid oxidation (ß-oxidation) is crucial for the maintenance of thermogenesis. Upon cellular fatty acid uptake or following lipolysis from triglycerides (TG), fatty acids are esterified to coenzyme A (CoA) to form active acyl-CoA molecules. This enzymatic reaction is essential for their utilization in ß-oxidation and thermogenesis. The activation and deactivation of fatty acids are regulated by two sets of enzymes called acyl-CoA synthetases (ACS) and acyl-CoA thioesterases (ACOT), respectively. The expression levels of ACS and ACOT family members in thermogenic tissues will determine the substrate availability for ß-oxidation, and consequently the thermogenic capacity. Although the role of the majority of ACS and ACOT family members in thermogenesis remains unclear, recent proceedings link the enzymatic activities of ACS and ACOT family members to metabolic disorders and thermogenesis. Elucidating the contributions of specific ACS and ACOT family members to trafficking of fatty acids towards thermogenesis may reveal novel targets for modulating thermogenic capacity and treating metabolic disorders.

Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress.

The incorporation of excess saturated free fatty acids (SFAs) into membrane phospholipids within the ER promotes ER stress, insulin resistance, and hepatic gluconeogenesis. Thioesterase superfamily member 2 (Them2) is a mitochondria-associated long-chain fatty acyl-CoA thioesterase that is activated upon binding phosphatidylcholine transfer protein (PC-TP). Under fasting conditions, the Them2/PC-TP complex directs saturated fatty acyl-CoA toward ß-oxidation. Here, we showed that during either chronic overnutrition or acute induction of ER stress, Them2 and PC-TP play critical roles in trafficking SFAs into the glycerolipid biosynthetic pathway to form saturated phospholipids, which ultimately reduce ER membrane fluidity. The Them2/PC-TP complex activated ER stress pathways by enhancing translocon-mediated efflux of ER calcium. The increased cytosolic calcium, in turn, led to the phosphorylation of calcium/calmodulin-dependent protein kinase II, which promoted both hepatic insulin resistance and gluconeogenesis. These findings delineate a mechanistic link between obesity and insulin resistance and establish the Them2/PC-TP complex as an attractive target for the management of hepatic steatosis and insulin resistance.

Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity.

Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin-melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3-5% of all severe obesity cases in humans1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons 6 , organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation 7 . We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans.

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions.

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.

Thioesterase superfamily member 2 (Them2) and phosphatidylcholine transfer protein (PC-TP) interact to promote fatty acid oxidation and control glucose utilization.

Thioesterase superfamily member 2 (Them2) is a mitochondrion-associated long-chain fatty acyl coenzyme A (CoA) thioesterase that is highly expressed in the liver and oxidative tissues. Them2 activity in vitro is increased when it interacts with phosphatidylcholine transfer protein (PC-TP), a cytosolic lipid binding protein. Them2-/- and Pctp-/- mice exhibit enhanced hepatic insulin sensitivity and increased adaptive thermogenesis, and Them2-/- mice are also resistant to diet-induced hepatic steatosis. Although we showed previously that a Them2-PC-TP complex suppresses insulin signaling, the enzymatic activity of Them2 suggests additional direct involvement in regulating hepatic nutrient homeostasis. Here we used cultured primary hepatocytes to elucidate biochemical and cellular mechanisms by which Them2 and PC-TP regulate lipid and glucose metabolism. Under conditions simulating fasting, Them2-/- and Pctp-/- hepatocytes each exhibited decreased rates of fatty acid oxidation and gluconeogenesis. In results indicative of Them2-dependent regulation by PC-TP, chemical inhibition of PC-TP failed to reproduce these changes in Them2-/- hepatocytes. In contrast, rates of glucose oxidation and lipogenesis in the presence of high glucose concentrations were decreased only in Them2-/- hepatocytes. These findings reveal a primary role for Them2 in promoting mitochondrial oxidation of fatty acids and glucose in the liver.

Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling.

Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation after knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP-THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.