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The endocannabinoid system (ECS) controls energy balance by regulating both energy intake and energy expenditure. Endocannabinoid levels are elevated in obesity suggesting a potential causal relationship. This study aimed to elucidate the rate of dysregulation of the ECS, and the metabolic organs involved, in diet-induced obesity. Eight groups of age-matched male C57Bl/6J mice were randomized to receive a chow diet (control) or receive a high fat diet (HFD, 45% of calories derived from fat) ranging from 1 day up to 18 weeks before euthanasia. Plasma levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA), and related N-acylethanolamines, were quantified by UPLC-MS/MS and gene expression of components of the ECS was determined in liver, muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) during the course of diet-induced obesity development. HFD feeding gradually increased 2-AG (+132% within 4 weeks, P < 0.05), accompanied by upregulated expression of its synthesizing enzymes Daglα and β in WAT and BAT. HFD also rapidly increased AEA (+81% within 1 week, P < 0.01), accompanied by increased expression of its synthesizing enzyme Nape-pld, specifically in BAT. Interestingly, Nape-pld expression in BAT correlated with plasma AEA levels (R2 = 0.171, β = 0.276, P < 0.001). We conclude that a HFD rapidly activates adipose tissue depots to increase the synthesis pathways of endocannabinoids that may aggravate the development of HFD-induced obesity..

Activation of free fatty acid receptor 1 (GPR40) by synthetic partial and full agonists occur via distinct allosteric sites. A crystal structure of GPR40-TAK-875 complex revealed the allosteric site for the partial agonist. Here we report the 2.76-Å crystal structure of human GPR40 in complex with a synthetic full agonist, compound 1, bound to the second allosteric site. Unlike TAK-875, which acts as a Gαq-coupled partial agonist, compound 1 is a dual Gαq and Gαs-coupled full agonist. compound 1 binds in the lipid-rich region of the receptor near intracellular loop 2 (ICL2), in which the stabilization of ICL2 by the ligand is likely the primary mechanism for the enhanced G protein activities. The endogenous free fatty acid (FFA), γ-linolenic acid, can be computationally modeled in this site. Both γ-linolenic acid and compound 1 exhibit positive cooperativity with TAK-875, suggesting that this site could also serve as a FFA binding site., GPR40 is a G-protein coupled receptor that binds to free fatty acids, mediating insulin and incretin secretion. Here, the authors present the crystal structure of human GPR40 with an agonist bound to an allosteric site located near the lipid-rich region that suggests a mechanism for biased agonism.

Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein‐coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120‐mediated signaling in BAT. We found that activation of GPR120 by the selective agonist TUG‐891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content and increased nutrient uptake by BAT, confirming increased BAT activity. Consistent with these observations, GPR120 deficiency reduced expression of genes involved in nutrient handling in BAT. Stimulation of brown adipocytes in vitro with TUG‐891 acutely induced O 2 consumption, through GPR120‐dependent and GPR120‐independent mechanisms. TUG‐891 not only stimulated GPR120 signaling resulting in intracellular calcium release, mitochondrial depolarization, and mitochondrial fission, but also activated UCP1. Collectively, these data suggest that activation of brown adipocytes with the GPR120 agonist TUG‐891 is a promising strategy to increase lipid combustion and reduce obesity.

As a part of our program to identify potent GPR40 agonists capable of being dosed orally once daily in humans, we incorporated fused heterocycles into our recently disclosed spiropiperidine and tetrahydroquinoline acid derivatives 1, 2, and 3 with the intention of lowering clearance and improving the maximum absorbable dose (Dabs). Hypothesis-driven structural modifications focused on moving away from the zwitterion-like structure. and mitigating the N-dealkylation and O-dealkylation issues led to triazolopyridine acid derivatives with unique pharmacology and superior pharmacokinetic properties. Compound 4 (LY3104607) demonstrated functional potency and glucose-dependent insulin secretion (GDIS) in primary islets from rats. Potent, efficacious, and durable dose-dependent reductions in glucose levels were seen during glucose tolerance test (GTT) studies. Low clearance, volume of distribution, and high oral bioavailability were observed in all species. The combination of enhanced pharmacology and pharmacokinetic properties supported further development of this compound as a potential glucose-lowering drug candidate.

Background Cold exposure and β3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influence lipoprotein metabolism by enhancing lipoprotein lipase–mediated catabolism of triglyceride-rich lipoproteins and increasing plasma high-density lipoprotein (HDL) levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive. Objective The objective was to assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men. Methods Body mass index–matched young, lean men were exposed to a personalized cooling protocol for 2 hours. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1 H-nuclear magnetic resonance. Adenosine triphosphate–binding cassette A1 (ABCA1)-mediated cholesterol efflux capacity of HDL was measured using [ 3 H]cholesterol-loaded ABCA1-transfected Chinese hamster ovary cells. Results Short-term cooling increased serum levels of free fatty acids, triglycerides, and cholesterol. Cooling increased the concentration of large very low–density lipoprotein (VLDL) particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux in vitro . Conclusions Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.

LY2881835 is a selective, potent, and efficacious GPR40 agonist. The objective of the studies described here was to examine the pharmacological properties of LY2881835 in preclinical models of T2D. Significant increases in insulin secretion were detected when LY2881835 was tested in primary islets from WT mice but not in islets from GPR40 KO mice. Furthermore, LY2881835 potentiated glucose stimulated insulin secretion in normal lean mice. Acute administration of LY2881835 lowered glucose during OGTTs in WT mice but not in GPR40 KO mice. These findings demonstrate that LY2881835 induces GPR40‐mediated activity ex vivo and in vivo. LY2881835 was administered orally at 10 mg/kg to diet‐induced obese (DIO) mice (an early model of T2D due to insulin resistance) for 14 days. Statistically significant reductions in glucose were seen during OGTTs performed on days 1 and 15. When a study was done for 3 weeks in Zucker fa/fa rats, a rat model of insulin resistance, normalization of blood glucose levels equivalent to those seen in lean rats was observed. A similar study was performed in streptozotocin (STZ)‐treated DIO mice to explore glucose control in a late model of T2D. In this model, pancreatic insulin content was reduced ~80% due to STZ‐treatment plus the mice were insulin resistant due to their high fat diet. Glucose AUCs were significantly reduced during OGTTs done on days 1, 7, and 14 compared to control mice. In conclusion, these results demonstrate that LY2881835 functions as a GPR40‐specific insulin secretagogue mediating immediate and durable glucose control in rodent models of early‐ and late‐stage T2D.

The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet β-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing. A clinical study with 3 administered to subjects with T2DM provided proof of concept of 3 as a potential glucose-lowering therapy. This manuscript summarizes the scientific rationale, medicinal chemistry, preclinical, and early development data of this new class of GPR40 agonists.

For over a decade, scientists have been investigating the sub-family of nuclear hormone receptors known as the peroxisome proliferator-activated receptors (PPARs) as potential targets for improved therapies for type 2 diabetes (T2D). Initial efforts focused on compounds with the thiazolidinedione (TZD) structure, synthesizing compounds based on structural differences followed by testing in rodent models without knowing the molecular target. Once the receptor for the TZDs was identified as PPARy, more extensive efforts began to identify and develop improved versions of the TZDs. Many biotechnology and pharmaceutical companies using this information established assays that were used in high through-put screening efforts to identify compounds with greater affinity and selectivity to the receptor. Surprising, these extensive efforts have not led to improved therapies but, in fact, have identified molecules with improved efficacy but with more severe and diverse liabilities. Today, scientists continue to pursue the PPAR subfamily as a target for advanced treatments for T2D and the metabolic syndrome. New approaches include selective PPAR modulators, expansion into the PPARα and PPARδ subtypes, and partial agonists with dual or pan PPAR properties. These drug discovery efforts will continue, as T2D and the associated metabolic diseases become severe medical problems in nations worldwide. Eventually, new and improved therapies will emerge based on the extensive preclinical and clinical knowledge that has accumulated from research and discovery on PPARs by dedicated and thoughtful scientists in search of improved therapies for people with T2D.

Peroxisome proliferator-activated receptors (PPARs) are ideal targets for the identification and development of oral agents to treat metabolic syndrome because this family of ligand-activated transcription factors modulates multiple aspects of lipid and carbohydrate metabolism. The potential exists that a single molecule could have beneficial effects on insulin resistance, obesity, dyslipidemia, hypertension and/or inflammation.

Herein we describe a series of potent and selective PPARgamma agonists with moderate PPARalpha affinity and little to no affinity for other nuclear receptors. In vivo studies in a NIDDM animal model (ZDF rat) showed that these compounds are efficacious at low doses in glucose normalization and plasma triglyceride reduction. Compound 1b (LY519818) was selected from our SAR studies to be advanced to clinical evaluation for the treatment of type II diabetes.

Insulin-induced gene 1 (INSIG-1) is a key regulator in the processing of the sterol regulatory element-binding proteins (SREBPs). We demonstrated that Insig-1 is regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) providing a link between insulin sensitization/glucose homeostasis and lipid homeostasis. Insig-1 was identified as a PPARgamma target gene using microarray analysis of mRNA from the white adipose tissue of diabetic (db/db) animals treated with PPARgamma agonists. Insig-1 was induced in subcutaneous (9-fold) and epididymal (4-fold) fat pads from db/db mice treated for 8 days with the PPARgamma agonist rosiglitazone (30 mg/kg/day). This in vivo response was confirmed in differentiated C3H10T1/2 adipocytes treated with rosiglitazone. To elucidate the molecular mechanisms regulating INSIG-1 expression, we cloned and characterized the human INSIG-1 promoter. Co-expression of PPARgamma and RXRalpha transactivated the INSIG-1 promoter in the presence of PPARgamma agonists. This induction was attenuated when a dominant negative PPARgamma construct was transfected into cells. Furthermore, a PPARgamma antagonist repressed the transactivation of the INSIG-1 promoter-reporter construct. Truncations of the promoter resulted in the identification of a PPAR response element that mediated the regulation of the promoter. We demonstrated with recombinant proteins that the PPARgamma/RXRalpha heterodimer binds directly to this PPAR response element. In addition to regulation by PPARgamma/RXRalpha, we demonstrated that the INSIG-1 promoter is regulated by transcriptionally active SREBP. The sterol response element was identified 380 base pairs upstream of the transcriptional start site. These findings suggest that the regulation of Insig-1 by PPARgamma agonists could in turn regulate SREBP processing and thus couple insulin sensitizers with the regulation of lipid homeostasis.

As the incidence of Type 2 diabetes has reached near epidemic proportions, the quest for novel therapies to combat this disorder has intensified dramatically. In recent years, the peroxisome proliferator-activated receptor (PPAR) family has received tremendous attention as perhaps an ideal target class to address the multiple metabolic anomalies associated with the diabetic state. This review focuses on a variety of novel PPAR approaches currently being investigated for Type 2 diabetes or the metabolic syndrome, including the highly potent selective PPAR agonists, PPAR combination agonists and alternative PPAR ligands.

Mitogenic signal-transduction pathways have not been well defined in pancreatic β-cells. In the glucose-sensitive rat β-cell line, INS-1, glucose (6-18 mM) increased INS-1 cell proliferation (> 20-fold at 15 mM glucose). Rat growth hormone (rGH) also induced INS-1 cell proliferation, but this was glucose-dependent in the physiologically relevant concentration range (6-18 mM glucose). The combination of rGH (10 nM) and glucose (15 mM) was synergistic, maximally increasing INS-1 cell proliferation by > 50-fold. Moreover, glucose-dependent rGH-induced INS-1 cell proliferation was increased further by addition of insulin-like growth factor 1 (IGF-1; 10 nM) to > 90-fold at 12 mM glucose. Glucose metabolism and phosphatidylinositol-3ʹ-kinase (PI3ʹK) activation were necessary for both glucose- and rGH-stimulated INS-1 cell proliferation. Glucose (> 3 mM) independently increased tyrosine-phosphorylation-mediated recruitment of growth-factor-bound protein 2 (Grb2)/murine sons of sevenless-1 protein (mSOS) and PI3ʹK to insulin receptor substrate (IRS)-1 and IRS-2, as well as SH2-containing protein (Shc) association with Grb2/mSOS and downstream activation of mitogen-activated protein kinase and 70 kDa S6 kinase. Glucose-induced IRS- and Shc-mediated signal transduction was enhanced further by the addition of IGF-1, but not rGH. In contrast, rGH was able to activate Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signal transduction at glucose concentrations above 3 mM, but neither glucose independently, nor glucose with added IGF-1, were able to activate the JAK2/STAT5 signalling pathway. Thus rGH-mediated proliferation of β-cells is directly via the JAK2/STAT5 pathway without engaging the Shc or IRS signal-transduction pathways, although activation of PI3ʹK may play an important permissive role in the glucose-dependent aspect of rGH-induced β-cell mitogensis. The additive effect of rGH and IGF-1 on glucose-dependent β-cell proliferation is therefore reflective of rGH and IGF-1 activating distinctly different mitogenic signalling pathways in β-cells with minimal crosstalk between them.

To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.

The anterior cingulate cortex (ACC) has been implicated in alcohol and drug addiction. We recently identified the small G protein K-ras as an alcohol-regulated gene in the ACC by gene expression analysis. We show here that the adiponectin receptor 2 (AdipoR2) was differentially regulated by alcohol in the ACC in a K-ras-dependent manner. Additionally, withdrawal-associated increased drinking was attenuated in AdipoR2 null mice. Intracellular recordings revealed that adiponectin increased the excitability of ACC neurons and that this effect was more pronounced during alcohol withdrawal, suggesting that AdipoR2 signaling may contribute to increased ACC activity. Altogether, the data implicate K-ras-regulated pathways involving AdipoR2 in the cellular and behavioral actions of alcohol that may contribute to overactivity of the ACC during withdrawal and excessive alcohol drinking.

The increasing prevalence of obesity is a fundamental contributor to the growing prevalence of the metabolic syndrome. Rexinoids, a class of compounds that selectively bind and activate RXR, are being studied as a potential option for the treatment of metabolic syndrome. These compounds have glucose-lowering, insulin-sensitizing, and antiobesity effects in animal models of insulin resistance and type 2 diabetes. However, undesirable side effects such as hypertriglyceridemia and suppression of the thyroid hormone axis also occur. This review examines and compares the effects of four RXR-selective ligands: LGD1069, LG100268, AGN194204, and LG101506, a selective RXR modulator. Similar to selective modulators of other nuclear receptors such as the estrogen receptor (SERMs), LG101506 binding to RXR selectively maintains the desirable characteristic effects of rexinoids while minimizing the undesirable effects. These recent findings suggest that, with continued research efforts, RXR-specific ligands with improved pharmacological profiles may eventually be available as additional treatment options for the current epidemic of obesity, insulin resistance, type 2 diabetes, and all of the associated metabolic sequelae.

Adiponectin/adiponectin receptors (AdipoR) are involved in energy homeostasis and inflammatory pathways. To investigate the role of AdipoR2 in metabolic control, we studied the lipid and glucose metabolic phenotypes in AdipoR2-deficient mice. AdipoR2 deletion diminished high-fat diet-induced dyslipidemia and insulin resistance yet deteriorated glucose homeostasis as high-fat feeding continued, which resulted from the failure of pancreatic β-cells to adequately compensate for the moderate insulin resistance. A defect in the AdipoR2 gene may represent a mechanism underlying the etiology of certain subgroups of type 2 diabetic patients who eventually develop overt diabetes, whereas other obese patients do not.

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. The precise mechanisms whereby FGF-21 regulates metabolism remain to be determined. Here we describe the early signaling events triggered by FGF-21 treatment of 3T3-L1 adipocytes and reveal a functional interplay between FGF-21 and peroxisome proliferator-activated receptor gamma (PPARγ) pathways that leads to a marked stimulation of glucose transport. While the early actions of FGF-21 on 3T3-L1 adipocytes involve rapid accumulation of intracellular calcium and phosphorylation of Akt, GSK-3, p70S6K, SHP-2, MEK1/2, and Stat3, continuous treatment for 72 h induces an increase in PPARγ protein expression. Moreover, chronic activation of the PPARγ pathway in 3T3-L1 adipocytes with the PPARγ agonist and anti-diabetic agent, rosiglitazone (BRL 49653), enhances FGF-21 action to induce tyrosine phosphorylation of FGF receptor-2. Strikingly, treatment of cells with FGF-21 and rosiglitazone in combination leads to a pronounced increase in expression of the GLUT1 glucose transporter and a marked synergy in stimulation of glucose transport. Together these results reveal a novel synergy between two regulators of glucose homeostasis, FGF-21 and PPARγ, and further define FGF-21 mechanism of action. J. Cell. Physiol. 210: 1–6, 2007. © 2006 Wiley-Liss, Inc.

The tremendous increase in the global prevalence of Type 2 diabetes (T2D) and its conglomeration of metabolic disorders has dramatically intensified the search for innovative therapies to fight this emerging epidemic. Over the last decade, the family of nuclear receptors, especially the peroxisome proliferator-activated receptors (PPARs), has emerged as one of the most important drug targets aimed at combating the metabolic syndrome. Consequently, compounds that activate the PPARs have served as potential therapeutics for the treatment of T2D and the metabolic anomalies associated with this disorder. This review focuses on the currently marketed compounds and also describes the discovery and development of the next generation of PPAR ligands that are under investigation for the potential treatment of T2D and the metabolic syndrome.

PPAR ligands with varied subtype selectivity have been synthesized using an achiral aminomethyl dihydrocinnamate template. Several compounds in this series have demonstrated potent plasma glucose and triglyceride lowering capability in rodent models of type 2 diabetes.

Specific retinoid X receptor (RXR) agonists, such as LG100268 (LG268), and the thiazolidinedione (TZD) PPARgamma agonists, such as rosiglitazone, produce insulin sensitization in rodent models of insulin resistance and type 2 diabetes. In sharp contrast to the TZDs that produce significant increases in body weight gain, RXR agonists reduce body weight gain and food consumption. Unfortunately, RXR agonists also suppress the thyroid hormone axis and generally produce hypertriglyceridemia. Heterodimer-selective RXR modulators have been identified that, in rodents, retain the metabolic benefits of RXR agonists with reduced side effects. These modulators bind specifically to RXR with high affinity and are RXR homodimer partial agonists. Although RXR agonists activate many heterodimer partners, these modulators selectively activate RXR:PPARalpha and RXR:PPARgamma, but not RXR:RARalpha, RXR:LXRalpha, RXR:LXRbeta, or RXR:FXRalpha. We report the in vivo characterization of one RXR modulator, LG101506 (LG1506). In Zucker fatty (fa/fa) rats, LG1506 is a potent insulin sensitizer that also enhances the insulin-sensitizing activities of rosiglitazone. Administration of LG1506 reduces both body weight gain and food consumption and blocks the TZD-induced weight gain when coadministered with rosiglitazone. LG1506 does not significantly suppress the thyroid hormone axis in rats, nor does it elevate triglycerides in Sprague Dawley rats. However, LG1506 produces a unique pattern of triglycerides elevation in Zucker rats. LG1506 elevates high-density lipoprotein cholesterol in humanized apolipoprotein A-1-transgenic mice. Therefore, selective RXR modulators are a promising approach for developing improved therapies for type 2 diabetes, although additional studies are needed to understand the strain-specific effects on triglycerides.

Benzofused heterocyclic analogs of the RXR selective modulator 1 (LG101506) were synthesized, and tested for their ability to bind RXRalpha and activate RXR homo and heterodimers. Potency and efficacy were observed to be dependent upon the choice of heterocycle as well as the sidechain employed.

New RXR-selective modulators possessing a 6-fluoro trienoic acid moiety (6Z olefin) or a fluorinated/heterocyclic-substituted benzene core ring, were synthesized in an expedient and selective way. A subset of these compounds was evaluated for their metabolic properties (exposure in IRC male mice) and show a dramatic increase of exposure compared to our reference compound, 3 (LG101506).

Retinoid X receptor:peroxisome proliferative-activated receptor (RXR:PPAR) heterodimers play a critical role in the regulation of glucose (RXR/PPARgamma) and lipid metabolism (RXR/PPARalpha). Previously, we described a concise structure-activity relationship study of selective RXR modulators possessing a (2E,4E,6Z)-3-methyl-7-(3,5-dialkyl-6-alkoxyphenyl)-octa-2,4,6-trienoic acid scaffold. These studies were focused on the 2-position alkoxy side chain. We describe here the design and synthesis of a novel series of RXR selective modulators possessing the same aromatic core structure with the addition of a ring locked 6-7-Z-olefin on the trienoic acid moiety. The synthesis and structure-activity relationship studies of these 6,7-locked cyclopentenyl, phenyl, thienyl, furan, and pyridine-trienoic acid derivatives is presented herein.

Fluorinated trienoic acid analogues of the RXR selective modulator 1 (LG101506) were synthesized, and tested for their ability to bind RXRalpha and activate RXR homo and heterodimers. Potency and efficacy were observed to be dependent upon the position of fluorination, and improvement in pharmacological profile was demonstrated in some cases.

Previous data have shown that RXR-selective agonists (e.g., 3 and 4) are insulin sensitizers in rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Unfortunately, they also produce dramatic increases in triglycerides and profound suppression of the thyroid hormone axis. Here we describe the design and synthesis of new RXR modulators that retain the insulin-sensitizing activity of RXR agonists but produce substantially reduced side effects. These molecules bind selectively and with high affinity to RXR and, unlike RXR agonists, do not activate RXR homodimers. To further evaluate the antidiabetic activity of these RXR modulators, we have designed a concise and systematic structure-activity relationship around the 2E,4E,6Z-7-aryl-3-methylocta-2,4,6-trienoic acid scaffold. Selected compounds have been evaluated using insulin-resistant rodents (db/db mice) to characterize effects on glucose homeostasis. Our studies demonstrate the effectiveness of RXR modulators in lowering plasma glucose in the db/db mouse model.

The synthesis and in vitro characterization of novel RXR-selective ligands possessing various substituted 1-benzofuran or 1-benzothiophene moieties are described.

The oxysterol receptors LXR (liver X receptor)-alpha and LXRbeta are nuclear receptors that play a key role in regulation of cholesterol and fatty acid metabolism. We found that LXRs also play a significant role in glucose metabolism. Treatment of diabetic rodents with the LXR agonist, T0901317, resulted in dramatic reduction of plasma glucose. In insulin-resistant Zucker (fa/fa) rats, T0901317 significantly improved insulin sensitivity. Activation of LXR did not induce robust adipogenesis but rather inhibited the expression of several genes involved in hepatic gluconeogenesis, including phosphoenolpyruvate carboxykinase (PEPCK). Hepatic glucose output was dramatically reduced as a result of this regulation. Nuclear run-on studies indicated that transcriptional repression was primarily responsible for the inhibition of PEPCK by the LXR agonist. In addition, we show that the regulation of the liver gluconeogenic pathway by LXR agonists was a direct effect on hepatocytes. These data not only suggest that LXRs are novel targets for diabetes but also reveal an unanticipated role for these receptors, further linking lipid and glucose metabolism.

An in vivo adenoviral gene delivery system was utilized to assess the effect of overexpressing protein kinase C (PKC)-zeta on rat skeletal muscle glucose transport activity. Female lean Zucker rats were injected with adenoviral/human PKC-zeta (hPKC-zeta) and adenoviral/LacZ in opposing tibialis anterior muscles. One week subsequent to adenoviral/gene delivery rats were subjected to hind limb perfusion. The hPKC-zeta protein was expressed at the same level (fast-twitch white) or at approximately 80% of the level (fast-twitch red) of endogenous PKC-zeta, thus approximately doubling the amount of PKC-zeta in tibialis anterior. Basal glucose transport activity was elevated approximately 3.4- and 2-fold, respectively, in fast-twitch white and red hPKC-zeta muscle relative to control. Submaximal insulin-stimulated glucose transport activity, corrected for basal transport, was approximately 90 and 40% over control values, respectively, in fast-twitch white and red hPKC-zeta muscle. The enhancement of glucose transport activity in muscle expressing hPKC-zeta occurred in the absence of any change in GLUT1 or GLUT4 protein levels, suggesting a redistribution of existing transporters to the cell surface. These results demonstrate that an adenoviral vector can be used to deliver expressible hPKC-zeta to adult rat skeletal muscle in vivo and also affirm a role for PKC-zeta in the regulation of glucose transport activity.

The cloning of the mouse PPAR alpha gene in 1990 by Issemann and Green [1] stimulated intense interest in this family of nuclear receptors, and research efforts over the next decade established important roles for the PPAR isotypes in glucose and lipoprotein metabolism, inflammation, and atherosclerosis. Though the fibrates (PPAR-α agonists) had been used for the treatment of dyslipidemia for nearly 40 years, the discovery of the insulin-sensitizing effects of PPAR-γ agonists brought about the development and commercialization of the thiazolidinedione (TZD) class of oral anti-hyperglycemic medications: troglitazone, approved by the FDA in 1997, and pioglitazone and rosiglitazone, both approved by the FDA in 1999. The withdrawal of troglitazone from the market in 2000 was an early indicator of the potential safety issues of PPAR drugs. Even so, the clinical use of fibrates and TZDs has allowed for a better understanding of the safety profiles and safety issues of PPAR-α and PPAR-γ agonists [2, 3]. At the beginning of this decade, many pharmaceutical companies had development programs focused on delivering “new and improved” PPAR agonists to the market. For example, several programs were developing PPAR-α/γ dual agonists (i.e., glitazars) for the treatment of type 2 diabetes. Although preliminary data from various PPAR-α/γ dual agonist research programs was promising, nearly all of these research programs were discontinued due to safety issues identified during clinical testing and/or during preclinical testing [4–6]. More recently, the highly publicized (and controversial) meta-analysis of rosiglitazone reported by Nissen and Wolski [7] called into question the safety of the TZDs and prompted changes to the labels of both rosiglitazone and pioglitazone. And yet, the promise of the therapeutic potential of PPAR drugs remains. A PubMed search using the term “peroxisome proliferator-activated receptor” yielded 1578 manuscripts (161 of which were review articles) between January 1 and December 1, 2008. In addition to the roles of the PPAR isotypes in lipid/lipoprotein/glucose metabolism, additional roles in diverse physiological processes and disease states are currently being investigated. As additional functions are identified; the PPARs will continue to be important molecular targets for identifying ligands (drugs) with potential applications to reproduction and fertility, normal development; function of the reproductive, gastrointestinal, respiratory, and central nervous systems; skin biology and wound healing; and cell cycle control and cancer. Reports highlighting both challenges and opportunities in PPAR drug development are included in this special issue. For example, edema, weight gain, and a reduction in bone mass (particularly in women) are challenges limiting the clinical utility of the currently marketed TZDs. The renal and vascular mechanisms of TZD-induced fluid retention are reviewed by Yang and Soodvilai, and the recent clinical data describing the effects of TZDs on bone are reviewed by Schwartz. In contrast, several reports highlight opportunities in PPAR drug development. Deeg and Tan compare the effects of rosiglitazone and pioglitazone on lipids, lipoproteins, and apolipoproteins as reported in head-to-head, randomized clinical studies. Two papers describe the effects of PPARs/PPAR ligands on immune/inflammatory responses; Fernandez reviews the roles of the PPARs in modulating the immune/inflammatory response in atherosclerosis, while Yamashita reviews the receptor-independent effects of PPAR-α and PPAR-γ ligands on cysteinyl leukotriene production in mast cells as it relates to the development of potential anti-asthma medications. Technological and methodological approaches that may prove useful in the identification and assessment of new PPAR drugs are also reported. Clarke et al. describe an approach used to determine the species differences in plasma protein binding to MBX-102, a novel PPAR-γ agonist currently in Phase 2 clinical development, and the corresponding differences in PPAR-γ activation across species. Cho et al. review the role of PPARs in metabolic disorders as well as various strategies and technologies used in the identification and assessment of PPAR drugs. Finally, Miyachi and Hashimoto describe the synthesis and SAR of subtype-specific PPAR agonists derived from a single 3,4-disubstituted phenylpropanoic acid “versatile template” scaffold, and Higgins and Mantzoros review the development and safety profile of INT-131, a potent non-TZD selective PPAR modulator (SPPARM) currently in Phase 2 clinical development. In conclusion, though the full therapeutic potential of PPARs has yet to be realized, and serious safety issues are associated with the currently marketed PPAR drugs (PPAR-α and PPAR-γ), there remains intense interest in exploring new physiological roles of the PPARs and in the identification of new and improved PPAR agonists drugs. Jane A. Pinaire Anne Reifel Miller Francine M. Gregoire

The instant invention provides polypeptide compounds of the formula b-BP-a having significant insulin activity, nucleotide sequences encoding said polypeptides, vectors comprising said nucleotide sequences, and cell lines transformed with said vectors useful for treating diabetes.