Mice deleted for GPAT3 have reduced GPAT activity in white adipose tissue and altered energy and cholesterol homeostasis in diet-induced obesity.

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4. To investigate the in vivo functions of GPAT3, we generated Gpat3-deficient (Gpat3(-/-)) mice. Total GPAT activity in white adipose tissue of Gpat3(-/-) mice was reduced by 80%, suggesting that GPAT3 is the predominant GPAT in this tissue. In liver, GPAT3 deletion had no impact on total GPAT activity but resulted in a 30% reduction in N-ethylmaleimide-sensitive GPAT activity. The Gpat3(-/-) mice were viable and fertile and exhibited no obvious metabolic abnormalities on standard laboratory chow. However, when fed a high-fat diet, female Gpat3(-/-) mice showed decreased body weight gain and adiposity and increased energy expenditure. Increased energy expenditure was also observed in male Gpat3(-/-) mice, although it was not accompanied by a significant change in body weight. GPAT3 deficiency lowered fed, but not fasted, glucose levels and tended to improve glucose tolerance in diet-induced obese male and female mice. On a high-fat diet, Gpat3(-/-) mice had enlarged livers and displayed a dysregulation in cholesterol metabolism. These data establish GPAT3 as the primary GPAT in white adipose tissue and reveal an important role of the enzyme in regulating energy, glucose, and lipid homeostasis.

Discovery of HSD-621 as a Potential Agent for the Treatment of Type 2 Diabetes.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) catalyzes the conversion of inactive glucocorticoid cortisone to its active form, cortisol. The glucocorticoid receptor (GR) signaling pathway has been linked to the pathophysiology of diabetes and metabolic syndrome. Herein, the structure-activity relationship of a series of piperazine sulfonamide-based 11ß-HSD1 inhibitors is described. (R)-3,3,3-Trifluoro-2-(5-(((R)-4-(4-fluoro-2-(trifluoromethyl)phenyl)-2-methylpiperazin-1-yl)sulfonyl)thiophen-2-yl)-2-hydroxypropanamide 18a (HSD-621) was identified as a potent and selective 11ß-HSD1 inhibitor and was ultimately selected as a clinical development candidate. HSD-621 has an attractive overall pharmaceutical profile and demonstrates good oral bioavailability in mouse, rat, and dog. When orally dosed in C57/BL6 diet-induced obesity (DIO) mice, HSD-621 was efficacious and showed a significant reduction in both fed and fasting glucose and insulin levels. Furthermore, HSD-621 was well tolerated in drug safety assessment studies.

A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia.

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXa(I16L)) rendered partially inactive by a defect in the transition from zymogen to active protease. Using mouse models of hemophilia, we show that FXa(I16L) has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXa(I16L) is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients. FXa(I16L) may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

Targeting Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases.

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in triglyceride synthesis. Findings from genetically modified mice as well as pharmacological studies suggest that inhibition of DGAT1 is a promising strategy for the treatment of obesity and type 2 diabetes. Here we characterize a tool DGAT1 inhibitor compound, T863. We found that T863 is a potent inhibitor for both human and mouse DGAT1 in vitro, which acts on the acyl-CoA binding site of DGAT1 and inhibits DGAT1-mediated triacylglycerol formation in cells. In an acute lipid challenge model, oral administration of T863 significantly delayed fat absorption and resulted in lipid accumulation in the distal small intestine of mice, mimicking the effects of genetic ablation of DGAT1. In diet-induced obese mice, oral administration of T863 for 2 weeks caused weight loss, reduction in serum and liver triglycerides, and improved insulin sensitivity. In addition to the expected triglyceride-lowering activity, T863 also lowered serum cholesterol. Hepatic IRS2 protein was dramatically up-regulated in mice treated with T863, possibly contributing to improved insulin sensitivity. In differentiated 3T3-L1 adipocytes, T863 enhanced insulin-stimulated glucose uptake, suggesting a possible role for adipocytes to improve insulin sensitivity upon DGAT1 inhibition. These results reveal novel mechanistic insights into the insulin-sensitizing effects of DGAT1 inhibition in mouse models. Taken together, our study provides a comprehensive evaluation of a small molecule inhibitor for DGAT1 and suggests that pharmacological inhibition of DGAT1 holds promise in treating diverse metabolic disorders.

GPAT3 and GPAT4 are regulated by insulin-stimulated phosphorylation and play distinct roles in adipogenesis.

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of glycerolipids. Mammals have at least four GPAT isoforms. Here we report the further characterization of the two recently identified microsomal GPAT3 and GPAT4. Both enzymes are highly expressed in adipose tissues. However, while GPAT3 is highly (approximately 60-fold) induced during adipocyte differentiation, GPAT4 induction is only modest (approximately 5-fold), leading to a lower abundance of GPAT4 mRNA in adipocytes. While overexpression of GPAT3 and GPAT4 in either insect or mammalian cells results in a comparable increase of GPAT activity, shRNA-mediated knockdown of GPAT3, but not GPAT4, in 3T3-L1 adipocytes led to a significant decrease in GPAT activity, a profound inhibition of lipid accumulation, and a lack of expression of several adipogenic markers during adipocyte differentiation. These data suggest that GPAT3 may encode the major GPAT isoform in adipocytes and play an important role in adipogenesis. Furthermore, we have shown that both GPAT3 and GPAT4 are phosphorylated by insulin at Ser and Thr residues, leading to increased GPAT activity that is sensitive to wortmannin. Our results reveal a link between the lipogenic effects of insulin and microsomal GPAT3 and GPAT4, implying their importance in glycerolipid biosynthesis.

Efficacious 11beta-hydroxysteroid dehydrogenase type I inhibitors in the diet-induced obesity mouse model.

Cortisol and the glucocorticoid receptor signaling pathway have been implicated in the development of diabetes and obesity. The reduction of cortisone to cortisol is catalyzed by 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1). 2,4-Disubsituted benzenesulfonamides were identified as potent inhibitors of both the human and mouse enzymes. The lead compounds displayed good pharmacokinetics and ex vivo inhibition of the target in mice. Cocrystal structures of compounds 1 and 20 bound to human 11beta-HSD1 were obtained. Compound 20 was found to achieve high concentrations in target tissues, resulting in 95% inhibition in the ex vivo assay when dosed with a food mix (0.5 mg of drug per g of food) after 4 days. Compound 20 was efficacious in a mouse diet-induced obesity model and significantly reduced fed glucose and fasted insulin levels. Our findings suggest that 11beta-HSD1 inhibition may be a valid target for the treatment of diabetes.

Scaffold-based discovery of indeglitazar, a PPAR pan-active anti-diabetic agent.

In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARalpha, PPARgamma, and PPARdelta. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARgamma, to circumvent the clinically observed side effects of full PPARgamma agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARgamma. Compared with full PPARgamma-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).

H6PDH interacts directly with 11beta-HSD1: implications for determining the directionality of glucocorticoid catalysis.

Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11beta-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system. Interestingly, both full-length H6PDH and the truncated NTD are secreted into the culture medium in the absence of 11beta-HSD1. Purified full-length H6PDH is a bi-functional enzyme with glucose-6-phosphate dehydrogenase (G6PDH) activity as well as 6-phosphogluconolactonase (6PGL) activity. Using co-immunoprecipitation experiments with purified H6PDH and 11beta-HSD1, and with cell lysates expressing H6PDH and 11beta-HSD1, we observe direct physical interaction between the two enzymes. We also show the modulation of 11beta-HSD1 directionality by H6PDH using overexpression and siRNA knockdown systems. The NTD retains the ability to interact with 11beta-HSD1 physically as well as modulate 11beta-HSD1 directionality indicating that the NTD of H6PDH is sufficient for the regulation of the 11beta-HSD1 activity.

Bidirectional modulation of adipogenesis by the secreted protein Ccdc80/DRO1/URB.

Adipocyte-secreted proteins play important roles in metabolic regulation through autocrine, paracrine, and endocrine mechanisms. Using transcriptional profiling, we identified coiled-coil domain containing 80 (Ccdc80; also known as DRO1 and URB) as a novel secreted protein highly expressed in white adipose tissue. In 3T3-L1 cells Ccdc80 is expressed and secreted in a biphasic manner with high levels in postconfluent preadipocytes and terminally differentiated adipocytes. To determine whether Ccdc80 regulates adipocyte differentiation, Ccdc80 expression was manipulated using both knockdown and overexpression approaches. Small hairpin RNA-mediated silencing of Ccdc80 in 3T3-L1 cells inhibits adipocyte differentiation. This phenotype was partially reversed by treating the knockdown cells with Ccdc80-containing conditioned medium from differentiated 3T3-L1 cells. Molecular studies indicate that Ccdc80 is required for the full inhibition of T-cell factor-mediated transcriptional activity, down-regulation of Wnt/beta-catenin target genes during clonal expansion, and the subsequent induction of C/EBPalpha and peroxisome proliferator-activated receptor gamma. Surprisingly, overexpression of Ccdc80 in 3T3-L1 cells also inhibits adipocyte differentiation without affecting the repression of the Wnt/beta-catenin signaling pathway. Taken together, these data suggest that Ccdc80 plays dual roles in adipogenesis by mechanisms that involve at least in part down-regulation of Wnt/beta-catenin signaling and induction of C/EBPalpha and peroxisome proliferator-activated receptor gamma.

PPARdelta Agonism for the Treatment of Obesity and Associated Disorders: Challenges and Opportunities.

The prevalence of obesity in the USA and worldwide has reached epidemic proportions during the last two decades. Drugs currently available for the treatment of obesity provide no more than 5% placebo-adjusted weight loss and are associated with undesirable side effects. Peroxisome proliferator-activated receptor (PPAR) modulators offer potential benefits for the treatment of obesity and its associated complications but their development has been complicated by biological, technical, and regulatory challenges. Despite significant challenges, PPAR modulators are attractive targets for the treatment of obesity and could offer a viable alternative to the millions of patients who fail to lose weight following rigorous dieting and exercise protocols. In addition, PPAR modulators have the potential-added benefit of ameliorating the associated comorbidities.

Molecular identification of a novel mammalian brain isoform of acyl-CoA:lysophospholipid acyltransferase with prominent ethanolamine lysophospholipid acylating activity, LPEAT2.

Acyl-CoA-dependent lysophospholipid acyltransferases play an important role in attaining the appropriate molecular species of phospholipids. A number of genes encoding these activities were recently identified. It has become clear that multiple genes can encode one enzymatic activity and that a given gene may encode multiple activities. Here we report the identification of a gene encoding a mammalian acyl-CoA-dependent lysophospholipid acyltransferase with prominent activity toward ethanolamine-containing lysophospholipids, which we termed acyl-CoA:lysophosphatidylethanolamine acyltransferase 2, LPEAT2 (previously annotated as AYTL3 or AGPAT7). LPEAT2 is predominantly expressed in brain, coinciding with an enrichment of phosphatidylethanolamine in this tissue. Ectopic expression of LPEAT2 in mammalian HEK293T cells led to a dramatic increase (up to 9-fold) in LPEAT activity when compared with cells transfected with empty vector or an unrelated acyltransferase. LPEAT2 also exhibited significant acyl-CoA-dependent acyltransferase activity toward 1-O-alkenyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, 1-O-alkyl-lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylcholine but lacked appreciable acylating activity toward glycerol 3-phosphate, lysophosphatidic acid, lysophosphatidylinositol, and diacylglycerol, demonstrating multiple but selective functions of LPEAT2 as an enzyme involved in phospholipid remodeling. LPEAT2 recognizes a broad range of medium and long chain fatty acyl-CoA, and its activity was not affected by Ca(2+). When overexpressed in mammalian cells, LPEAT2 is localized to the endoplasmic reticulum. siRNA-mediated knockdown of LPEAT2 in HEK293T cells significantly decreased LPEAT and 1-alkenyl-LPEAT activities but did not affect other lysophospholipid acylating activities. These findings identify LPEAT2 as an important enzyme in the biosynthesis of ethanolamine-containing phospholipids, especially in brain.

An enzyme-linked immunosorbent assay to measure insulin receptor dephosphorylation by PTP1B.

Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.

Normal food intake and body weight in mice lacking the G protein-coupled receptor GPR39.

It has been recently proposed that obestatin, a peptide encoded by the ghrelin gene, reduces food intake by activating the orphan G protein-coupled receptor GPR39. To gain further insights into the role of GPR39 in body weight homeostasis, we characterized the phenotype of mice with targeted disruption of the GPR39 gene. Body weight, adiposity, and food intake were found to be similar between GPR39(+/+) and GPR39(-/-) mice. Furthermore, fasting glucose and insulin levels were similar between both genotypes. Injection of obestatin peptide (1 micromol/kg, ip) obtained from multiple sources did not consistently inhibit food intake in wild-type mice after an overnight fast, and no difference in food intake was observed between wild-type and GPR39 knockout mice after injection of the peptide. Finally, ectopic expression of GPR39 in HEK293T cells revealed a constitutive activation of the receptor that was unaffected by stimulation with obestatin. Our phenotypic characterization suggests that GPR39 is not a major modulator of food intake in mice, although a more subtle role cannot be excluded. The role of GPR39 in normal physiology requires further study and should be conducted independently of the function of obestatin.

Molecular identification of microsomal acyl-CoA:glycerol-3-phosphate acyltransferase, a key enzyme in de novo triacylglycerol synthesis.

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of triacylglycerol. It has been well recognized that mammals possess multiple enzymatically distinct proteins with GPAT activity. Although the mitochondrial-associated GPAT has been cloned and extensively characterized, the molecular identity of the endoplasmic reticulum (ER)-associated GPAT, which accounts for the majority of total GPAT activity in most tissues, has remained elusive. Here we report the identification of genes encoding human and mouse ER-associated GPAT (termed GPAT3). GPAT3 is a member of the acyltransferase family predominantly expressed in tissues characterized by active lipid metabolism, such as adipose tissue, small intestine, kidney, and heart. Ectopic expression of GPAT3 leads to a significant increase in N-ethylmaleimide-sensitive GPAT activity, whereas acyltransferase activity toward a variety of other lysophospholipids, as well as neutral lipid substrates, is not altered. Overexpression of GPAT3 in mammalian cells results in increased triacylglycerol, but not phospholipid, formation. GPAT3 is localized to the ER when overexpressed in COS-7 cells. GPAT3 mRNA is dramatically up-regulated during adipocyte differentiation, is reciprocally regulated in adipose tissue and liver of ob/ob mice, and is up-regulated in mice treated with a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist. A substantial loss of GPAT activity in 3T3-L1 adipocytes was achieved by reducing GPAT3 mRNA levels through GPAT3-specific siRNA knockdown. These findings identify GPAT3 as a previously uncharacterized triacylglycerol biosynthetic enzyme. Similar to other lipogenic enzymes, GPAT3 may be useful as a target for the treatment of obesity.

Nuclear receptors as drug targets in metabolic diseases: new approaches to therapy.

Nuclear receptors represent novel targets for the development of therapeutic agents for the treatment of numerous diseases, including type 2 diabetes, obesity dyslipidemia, atherosclerosis and the metabolic syndrome. There have been many recent advances in the development of new therapeutic agents for a subset of these receptors, including the peroxisome proliferator-activated receptors, the liver X receptors and the farnesoid X receptor. To date, the synthesis of selective modulators that regulate the activity of these receptors has been empirical. However, a detailed understanding of the molecular basis for selective modulation, as well as new insights into the biology of these receptors, might open the door to the rational design of a new generation of therapeutic agents with improved safety and efficacy.

Molecular activation of PPARgamma by angiotensin II type 1-receptor antagonists.

OBJECTIVE AND DESIGN: Elevated blood pressure and insulin resistance are strongly associated in patients. We explored the potential for the anti-hypertensive angiotensin II type 1-receptor (ATR(1)) antagonists to improve insulin sensitivity through modulation of the nuclear receptor PPARgamma, in vitro and in vivo compared to the potent insulin sensitizer, rosiglitazone. METHODS: PPARgamma modulation by ATR(1) antagonists was measured first by direct recruitment of PGC-1, followed by trans-activation reporter assays in cells, and promotion of adipogenesis in fibroblast and pre-adipocyte cell lines. Improvement of insulin sensitivity was measured as changes in levels of glucose, insulin, and adiponectin in ob/ob mice. RESULTS: Telmisartan, candesartan, irbesartan, and losartan (but not valsartan or olmesartan) each served as bona fide PPARgamma ligands in vitro, with EC(50) values between 3 and 5 micro mol/l. However, only telmisartan, and to a lesser extent candesartan, resulted in significant PPARgamma agonism in cells. In vivo, although rosiglitazone significantly lowered both glucose (33%, p<0.01) and insulin (61%, p<0.01) levels and increased expression of adiponectin (74%, p<0.001), sartan treatment had no effect. CONCLUSIONS: Many members of the sartan family of ATR(1) antagonists are PPARgamma ligands in cell-free assays but their modulation of PPARgamma in cells is relatively weak. Furthermore, none appear to improve insulin sensitivity in a rodent model under conditions where other insulin sensitizers, including rosiglitazone, do. These results question whether reported effects of sartans on insulin sensitivity may be through other means, and should guide further efforts to develop dual agents to treat hypertension and insulin resistance.

Bone morphogenetic proteins and growth differentiation factors as drug targets in cardiovascular and metabolic disease.

Bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) control the development and homeostasis of multiple tissue types in many organisms, from humans to invertebrates. These morphogens are expressed in a tissue-specific manner and they signal by binding to serine-threonine kinase receptors, resulting in coordinated changes in gene expression that regulate the differentiation and development of multiple tissue types. In addition, these proteins are regulated post-transcriptionally through binding to several soluble proteins. In this review we focus on a subset of BMPs and GDFs that have been implicated in the pathophysiology of type 2 diabetes and cardiovascular disease.

Synthesis and biological evaluation of sulfonamidooxazoles and beta-keto sulfones: selective inhibitors of 11beta-hydroxysteroid dehydrogenase type I.

The design, synthesis, and biological evaluation of arylsulfonamidooxazoles as 11beta-HSD1 inhibitors and the serendipitous discovery of beta-keto sulfones as potent 11beta-HSD1 inhibitors are described here. These two classes of compounds are not active against 11beta-HSD2 and therefore may have significant therapeutic potential for metabolic syndrome, type 2 diabetes and related metabolic dysfunctions.

Myostatin, a negative regulator of muscle mass: implications for muscle degenerative diseases.

Myostatin is a secreted protein that negatively regulates skeletal muscle mass determining both muscle fiber number and size. The myostatin pathway is conserved and regulates muscle mass in a number of animal species ranging from fish to humans. Inhibition of myostatin using a variety of therapeutic approaches can increase muscle mass in a number of animal models of human disease, including muscular dystrophy.