Identification of a Metabolic, Transcriptomic, and Molecular Signature of Patatin-Like Phospholipase Domain Containing 3-Mediated Acceleration of Steatohepatitis.

BACKGROUND AND AIMS: The mechanisms by which the I148M mutant variant of the patatin-like phospholipase domain-containing 3 (PNPLA3I148M ) drives development of nonalcoholic steatohepatitis (NASH) are not known. The aim of this study was to obtain insights on mechanisms underlying PNPLA3I148M -induced acceleration of NASH. APPROACH AND RESULTS: Hepatocyte-specific overexpression of empty vector (luciferase), human wild-type PNPLA3, or PNPLA3I148M was achieved using adeno-associated virus 8 in a diet-induced mouse model of nonalcoholic fatty liver disease followed by chow diet or high-fat Western diet with ad libitum administration of sugar in drinking water (WDSW) for 8 weeks. Under WDSW, PNPLA3I148M overexpression accelerated steatohepatitis with increased steatosis, inflammation ballooning, and fibrosis (P < 0.001 versus other groups for all). Silencing PNPLA3I148M after its initial overexpression abrogated these findings. PNPLA3I148M caused 22:6n3 docosahexanoic acid depletion and increased ceramides under WDSW in addition to increasing triglycerides and diglycerides, especially enriched with unsaturated fatty acids. It also increased oxidative stress and endoplasmic reticulum stress. Increased total ceramides was associated with signature of transducer and activator of transcription 3 (STAT3) activation with downstream activation of multiple immune-inflammatory pathways at a transcriptomic level by network analyses. Silencing PNPLA3I148M reversed STAT3 activation. Conditioned media from HepG2 cells overexpressing PNPLA3I148M increased procollagen mRNA expression in LX2 cells; this was abrogated by hepatocyte STAT3 inhibition. CONCLUSIONS: Under WDSW, PNPLA3I148M overexpression promotes steatosis and NASH by metabolic reprogramming characterized by increased triglycerides and diglycerides, n3 polyunsaturated fatty acid depletion, and increased ceramides with resultant STAT3 phosphorylation and downstream inflammatory pathway activation driving increased stellate cell fibrogenic activity.

Author Correction: The Transcriptomic Signature Of Disease Development And Progression Of Nonalcoholic Fatty Liver Disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting a ceramide double bond improves insulin resistance and hepatic steatosis.

Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.

GPR119 Agonism Increases Glucagon Secretion During Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia in diabetes is associated with impaired glucagon secretion. In this study, we tested whether stimulation of GPR119, a G-protein-coupled receptor expressed in pancreatic islet as well as enteroendocrine cells and previously shown to stimulate insulin and incretin secretion, might enhance glucagon secretion during hypoglycemia. In the study, GPR119 agonists were applied to isolated islets or perfused pancreata to assess insulin and glucagon secretion during hypoglycemic or hyperglycemic conditions. Insulin infusion hypoglycemic clamps were performed with or without GPR119 agonist pretreatment to assess glucagon counterregulation in healthy and streptozotocin (STZ)-induced diabetic rats, including those exposed to recurrent bouts of insulin-induced hypoglycemia that leads to suppression of hypoglycemia-induced glucagon release. Hypoglycemic clamp studies were also conducted in GPR119 knockout (KO) mice to evaluate whether the pharmacological stimulatory actions of GPR119 agonists on glucagon secretion during hypoglycemia were an on-target effect. The results revealed that GPR119 agonist-treated pancreata or cultured islets had increased glucagon secretion during low glucose perfusion. In vivo, GPR119 agonists also significantly increased glucagon secretion during hypoglycemia in healthy and STZ-diabetic rats, a response that was absent in GPR119 KO mice. In addition, impaired glucagon counterregulatory responses were restored by a GPR119 agonist in STZ-diabetic rats that were exposed to antecedent bouts of hypoglycemia. Thus, GPR119 agonists have the ability to pharmacologically augment glucagon secretion, specifically in response to hypoglycemia in diabetic rodents. Whether this effect might serve to diminish the occurrence and severity of iatrogenic hypoglycemia during intensive insulin therapy in patients with diabetes remains to be established.

The Transcriptomic Signature Of Disease Development And Progression Of Nonalcoholic Fatty Liver Disease.

A longitudinal molecular model of the development and progression of nonalcoholic fatty liver disease (NAFLD) over time is lacking. We have recently validated a high fat/sugar water-induced animal (an isogenic strain of C57BL/6 J:129S1/SvImJ mice) model of NAFLD that closely mimics most aspects of human disease. The hepatic transcriptome of such mice with fatty liver (8 weeks), steatohepatitis with early fibrosis (16-24 weeks) and advanced fibrosis (52 weeks) after initiation of the diet was evaluated and compared to mice on chow diet. Fatty liver development was associated with transcriptional activation of lipogenesis, FXR-RXR, PPAR-α mediated lipid oxidation and oxidative stress pathways. With progression to steatohepatitis, metabolic pathway activation persisted with additional activation of IL-1/inhibition of RXR, granulocyte diapedesis/adhesion, Fc macrophage activation, prothrombin activation and hepatic stellate cell activation. Progression to advanced fibrosis was associated with dampening of metabolic, oxidative stress and cell stress related pathway activation but with further Fc macrophage activation, cell death and turnover and activation of cancer-related networks. The molecular progression of NAFLD involves a metabolic perturbation which triggers subsequent cell stress and inflammation driving cell death and turnover. Over time, inflammation and fibrogenic pathways become dominant while in advanced disease an inflammatory-oncogenic profile dominates.

Glucagon like receptor 1/ glucagon dual agonist acutely enhanced hepatic lipid clearance and suppressed de novo lipogenesis in mice.

Lipid lowering properties of glucagon have been reported. Blocking glucagon signaling leads to rise in plasma LDL levels. Here, we demonstrate the lipid lowering effects of acute dosing with Glp1r/Gcgr dual agonist (DualAG). All the experiments were performed in 25 week-old male diet-induced (60% kCal fat) obese mice. After 2 hrs of fasting, mice were injected subcutaneously with vehicle, liraglutide (25nmol/kg) and DualAG (25nmol/kg). De novo cholesterol and palmitate synthesis was measured by deuterium incorporation method using D2O. 13C18-oleate infusion was used for measuring fatty acid esterification. Simultaneous activation of Glp1r and Gcgr resulted in decrease in plasma triglyceride and cholesterol levels. DualAG enhanced hepatic LDLr protein levels, along with causing decrease in content of plasma ApoB48 and ApoB100. VLDL secretion, de novo palmitate synthesis and fatty acid esterification decreased with acute DualAG treatment. On the other hand, ketone levels were elevated with DualAG treatment, indicating increased fatty acid oxidation. Lipid relevant changes were absent in liraglutide treated group. In an acute treatment, DualAG demonstrated significant impact on lipid homeostasis, specifically on hepatic uptake, VLDL secretion and de novo synthesis. These effects collectively reveal that lipid lowering abilities of DualAG are primarily through glucagon signaling and are liver centric.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Cyp8b1 ablation prevents Western diet-induced weight gain and hepatic steatosis because of impaired fat absorption.

Bile acids (BAs) are cholesterol derivatives that regulate lipid metabolism, through their dual abilities to promote lipid absorption and activate BA receptors. However, different BA species have varying abilities to perform these functions. Eliminating 12α-hydroxy BAs in mice via Cyp8b1 knockout causes low body weight and improved glucose tolerance. The goal of this study was to determine mechanisms of low body weight in Cyp8b1-/- mice. We challenged Cyp8b1-/- mice with a Western-type diet and assessed body weight and composition. We measured energy expenditure, fecal calories, and lipid absorption and performed lipidomic studies on feces and intestine. We investigated the requirement for dietary fat in the phenotype using a fat-free diet. Cyp8b1-/- mice were resistant to Western diet-induced body weight gain, hepatic steatosis, and insulin resistance. These changes were associated with increased fecal calories, due to malabsorption of hydrolyzed dietary triglycerides. This was reversed by treating the mice with taurocholic acid, the major 12α-hydroxylated BA species. The improvements in body weight and steatosis were normalized by feeding mice a fat-free diet. The effects of BA composition on intestinal lipid handling are important for whole body energy homeostasis. Thus modulating BA composition is a potential tool for obesity or diabetes therapy.

Phenotyping of adipose, liver, and skeletal muscle insulin resistance and response to pioglitazone in spontaneously obese rhesus monkeys.

Insulin resistance and diabetes can develop spontaneously with obesity and aging in rhesus monkeys, highly similar to the natural history of obesity, insulin resistance, and progression to type 2 diabetes in humans. The current studies in obese rhesus were undertaken to assess hepatic and adipose contributions to systemic insulin resistance-currently, a gap in our knowledge-and to benchmark the responses to pioglitazone (PIO). A two-step hyperinsulinemic-euglycemic clamp, with tracer-based glucose flux estimates, was used to measure insulin resistance, and in an intervention study was repeated following 6 wk of PIO treatment (3 mg/kg). Compared with lean healthy rhesus, obese rhesus has a 60% reduction of glucose utilization during a high insulin infusion and markedly impaired suppression of lipolysis, which was evident at both low and high insulin infusion. However, obese dysmetabolic rhesus manifests only mild hepatic insulin resistance. Six-week PIO treatment significantly improved skeletal muscle and adipose insulin resistance (by ~50%). These studies strengthen the concept that insulin resistance in obese rhesus closely resembles human insulin resistance and indicate the value of obese rhesus for appraising new insulin-sensitizing therapeutics.

Correction: The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

[This corrects the article DOI: 10.1371/journal.pone.0164133.].

The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

OBJECTIVES: Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. METHODS: We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. RESULTS: The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. CONCLUSIONS: These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

Duodenal-jejunal bypass surgery induces hepatic lipidomic alterations associated with ameliorated hepatic steatosis in mice.

OBJECTIVE: Bariatric surgery induces weight loss and improvement of insulin resistance; one aspect of both is an amelioration of hepatic steatosis. This study was undertaken to assess the changes in the hepatic lipidome after duodenal-jejunal bypass (DJB) surgery. METHODS: A DJB surgical model was developed and characterized in diet-induced obese mice. In comparison with sham-operated mice, an unbiased lipidomic profiling of hepatic lipids was performed together with measurements of gene expression within key pathways of hepatic lipid metabolism. RESULTS: In the liver of DJB mice, a dramatic reduction (by 77%) in hepatic triacylglycerols was observed. Global lipidomic profiling identified marked decreases of triacylglycerols comprised of medium length fatty acids and with low double bond content. Specific diacylglycerol species were also among the most dramatic decreases in hepatic lipids, whereas lysophosphatidic acids and phosphatidic acids were increased. Expression of fatty acid transporter and lipogenic genes was down-regulated. CONCLUSIONS: From in-depth analysis of hepatic lipid composition, specific lipid intermediates were identified that are preferentially changed following DJB surgery. These changes were most likely due to DJB-induced weight loss, and only further studies will be able to distinguish weight loss-dependent from weight loss-independent changes.

Increased Bile Acid Synthesis and Impaired Bile Acid Transport in Human Obesity.

CONTEXT: Alterations in bile acid (BA) synthesis and transport have the potential to affect multiple metabolic pathways in the pathophysiology of obesity. OBJECTIVE: The objective of the study was to investigate the effects of obesity on serum fluctuations of BAs and markers of BA synthesis. DESIGN: We measured BA fluctuations in 11 nonobese and 32 obese subjects and BA transporter expression in liver specimens from 42 individuals and specimens of duodenum, jejunum, ileum, colon, and pancreas from nine individuals. MAIN OUTCOME MEASURES: We analyzed serum BAs and markers of BA synthesis after overnight fasting, during a hyperinsulinemic-euglycemic clamp, or a mixed-meal tolerance test and the association of BA transporter expression with body mass index. RESULTS: BA synthesis markers were 2-fold higher (P < .01) and preferentially 12α-hydroxylated (P < .05) in obese subjects, and both measures were correlated with clamp-derived insulin sensitivity (r = -0.62, P < .0001, and r = -0.39, P = .01, respectively). Insulin infusion acutely reduced serum BAs in nonobese subjects, but this effect was blunted in obese subjects (δBAs -44.2% vs -4.2%, P < .05). The rise in serum BAs postprandially was also relatively blunted in obese subjects (δBAs +402% vs +133%, P < .01). Liver expression of the Na+-taurocholate cotransporting polypeptide and the bile salt export pump were negatively correlated with body mass index (r = -0.37, P = .02, and r = -0.48, P = .001, respectively). CONCLUSIONS: Obesity is associated with increased BA synthesis, preferential 12α-hydroxylation, and impaired serum BA fluctuations. The findings reveal new pathophysiological aspects of BA action in obesity that may lend themselves to therapeutic targeting in metabolic disease.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Gene expression in WAT from healthy humans and monkeys correlates with FGF21-induced browning of WAT in mice.

OBJECTIVE: Identify a gene expression signature in white adipose tissue (WAT) that reports on WAT browning and is associated with a healthy phenotype. METHODS: RNA from several different adipose depots across three species were analyzed by whole transcriptome profiling, including 1) mouse subcutaneous white fat, brown fat, and white fat after in vivo treatment with FGF21; 2) human subcutaneous and omental fat from insulin-sensitive and insulin-resistant patients; and 3) rhesus monkey subcutaneous fat from healthy and dysmetabolic individuals. RESULTS: A "browning" signature in mice was identified by cross-referencing the FGF21-induced signature in WAT with the brown adipose tissue (BAT) vs. WAT comparison. In addition, gene expression levels in WAT from insulin-sensitive/healthy vs. insulin-resistant/dysmetabolic humans and rhesus monkeys, respectively, correlated with the gene expression levels in mouse BAT vs. WAT. A subset of 49 genes were identified that were consistently regulated or differentially expressed in the mouse and human data sets that could be used to monitor browning of WAT across species. CONCLUSIONS: Gene expression profiles of WATs from healthy insulin-sensitive individuals correlate with those of BAT and FGF21-induced browning of WAT.

Improved Stability of Proline-Derived Direct Thrombin Inhibitors through Hydroxyl to Heterocycle Replacement.

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

Increased Bile Acid Synthesis and Deconjugation After Biliopancreatic Diversion.

Biliopancreatic diversion (BPD) improves insulin sensitivity and decreases serum cholesterol out of proportion with weight loss. Mechanisms of these effects are unknown. One set of proposed contributors to metabolic improvements after bariatric surgeries is bile acids (BAs). We investigated the early and late effects of BPD on plasma BA levels, composition, and markers of BA synthesis in 15 patients with type 2 diabetes (T2D). We compared these to the early and late effects of Roux-en-Y gastric bypass (RYGB) in 22 patients with T2D and 16 with normal glucose tolerance. Seven weeks after BPD, insulin sensitivity had doubled and serum cholesterol had halved. At this time, BA synthesis markers and total plasma BAs, particularly unconjugated BAs, had markedly risen; this effect could not be entirely explained by low FGF19. In contrast, after RYGB, insulin sensitivity improved gradually with weight loss and cholesterol levels declined marginally; BA synthesis markers were decreased at an early time point (2 weeks) after surgery and returned to the normal range 1 year later. These findings indicate that BA synthesis contributes to the decreased serum cholesterol after BPD. Moreover, they suggest a potential role for altered enterohepatic circulation of BAs in improving insulin sensitivity and cholesterol metabolism after BPD.

Potentiation of insulin-mediated glucose lowering without elevated hypoglycemia risk by a small molecule insulin receptor modulator.

Insulin resistance is the key feature of type 2 diabetes and is manifested as attenuated insulin receptor (IR) signaling in response to same levels of insulin binding. Several small molecule IR activators have been identified and reported to exhibit insulin sensitization properties. One of these molecules, TLK19781 (Cmpd1), was investigated to examine its IR sensitizing action in vivo. Our data demonstrate that Cmpd1, at doses that produced minimal efficacy in the absence of insulin, potentiated insulin action during an OGTT in non-diabetic mice and enhanced insulin-mediated glucose lowering in diabetic mice. Interestingly, different from insulin alone, Cmpd1 combined with insulin showed enhanced efficacy and duration of action without increased hypoglycemia. To explore the mechanism underlying the apparent glucose dependent efficacy, tissue insulin signaling was compared in healthy and diabetic mice. Cmpd1 enhanced insulin's effects on IR phosphorylation in both healthy and diabetic mice. In contrast, the compound potentiated insulin's effects on Akt phosphorylation in diabetic but not in non-diabetic mice. These differential effects on signaling corresponding to glucose levels could be part of the mechanism for reduced hypoglycemia risk. The in vivo efficacy of Cmpd1 is specific and dependent on IR expression. Results from these studies support the idea of targeting IR for insulin sensitization, which carries low hypoglycemia risk by standalone treatment and could improve the effectiveness of insulin therapies.

Racing to define pharmaceutical R&D external innovation models.

The pharmaceutical industry continues to face fundamental challenges because of issues with research and development (R&D) productivity and rising customer expectations. To lower R&D costs, move beyond me-too therapies, and create more transformative portfolios, pharmaceutical companies are actively capitalizing on external innovation through precompetitive collaboration with academia, cultivation of biotech start-ups, and proactive licensing and acquisitions. Here, we review the varying innovation strategies used by pharmaceutical companies, compare and contrast these models, and identify the trends in external innovation. We also discuss factors that influence these external innovation models and propose a preliminary set of metrics that could be used as leading indicators of success.

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans.

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.