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Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment. Preclinical studies show that fluoxazolevir, which inhibits the fusion of hepatitis C virus (HCV) with hepatic cells by binding viral envelope protein 1, could be useful in drug cocktails to treat HCV.

Hepatitis C virus (HCV) is a small, single-stranded, positive-sense RNA virus that infects more than an estimated 70 million people worldwide. Untreated, persistent HCV infection often results in chronic hepatitis, cirrhosis, or liver failure, with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can provide curative treatment; however, due to high costs there remains a need for effective, shorter-duration, and affordable treatments. Recently, we disclosed anti-HCV activity of the cheap antihistamine chlorcyclizine, targeting viral entry. Following our hit-to-lead optimization campaign, we report evaluation of preclinical in vitro absorption, distribution, metabolism, and excretion properties, and in vivo pharmacokinetic profiles of lead compounds. This led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this compound into DAA regimens has the potential to improve treatment efficacy, affordability, and accessibility.

Growth hormone (GH) plays a pivotal role in growth and metabolism, with growth promotion mostly attributed to generation of insulin-like growth factor I (IGF-I) in liver or at local sites of GH action, whereas the metabolic effects of GH are considered to be intrinsic to GH itself. To distinguish the effects of GH from those of IGF-I, we developed a Cre-lox-mediated model of tissue-specific deletion of the growth hormone receptor (GHR). Near total deletion of the GHR in liver (GHRLD) had no effect on total body or bone linear growth despite a >90% suppression of circulating IGF-I; however, total bone density was significantly reduced. Circulating GH was increased 4-fold, and GHRLD displayed insulin resistance, glucose intolerance, and increased circulating free fatty acids. Livers displayed marked steatosis, the result of increased triglyceride synthesis and decreased efflux; reconstitution of hepatic GHR signaling via adenoviral expression of GHR restored triglyceride output to normal, whereas IGF-I infusion did not correct steatosis despite restoration of circulating GH to normal. Thus, with near total absence of circulating IGF-I, GH action at the growth plate, directly and via locally generated IGF-I, can regulate bone growth, but at the expense of diabetogenic, lipolytic, and hepatosteatotic consequences. Our results indicate that IGF-I is essential for bone mineral density, whereas hepatic GH signaling is essential to regulate intrahepatic lipid metabolism. We propose that circulating IGF-I serves to amplify the growth-promoting effects of GH, while simultaneously dampening the catabolic effects of GH.

Most patients with type 2 diabetes mellitus will eventually require insulin therapy to achieve or maintain adequate glycaemic control. The introduction of insulin analogues, with pharmacokinetics that more closely mimic endogenous insulin secretion, has made physiologic insulin replacement easier to achieve for many patients. However, there are also concerns regarding alteration of binding affinities for the insulin receptor (IR) or insulin-like growth factor-1 receptor (IGF-1R) may increase the mitogenic potential of some analogues. Therefore, this article will review the relevant preclinical and clinical data to assess the mitogenic potential of insulin glargine, a basal insulin analogue, compared with regular human insulin (RHI). Searches of the PubMed database were performed using terms that included ‘IR,’ ‘insulin-like growth factor-1,’ ‘IGF-1R,’ ‘type 2 diabetes mellitus,’ and ‘insulin glargine.’ Original articles and reviews of published literature were retrieved and reviewed. Although one study reported increased binding affinity of insulin glargine for the IGF-1R and increased mitogenic potential in cells with excess IGF-1Rs (Saos/B10 osteosarcoma cells), most in vitro binding-affinity and cell-culture studies have demonstrated behaviour of insulin glargine comparable to that of RHI for both IR and IGF-1R binding, insulin signalling, and metabolic and mitogenic potential. Currently published in vivo carcinogenic studies and human clinical trial data have shown that insulin glargine is not associated with increased risk for either cancer or the development or progression of diabetic retinopathy. Copyright © 2007 John Wiley & Sons, Ltd.

Purpose of review This review will outline the recent advances in the area of insulin-stimulated skeletal muscle glucose uptake and its effect on whole body glucose homeostasis, using gene-deletion and transgenic mouse models. Recent findings Insulin resistance is often the first abnormality detected in cases of type 2 diabetes, and is seen at the level of the peripheral tissues especially muscle. Both the insulin receptor and the insulin-like growth factor I receptor are capable of stimulating glucose uptake into skeletal muscle. One model involves the gene deletion of muscle glucose transport protein 4, which leads to severe insulin resistance and hyperglycemia, and a second model using a transgenic approach abrogates the function of the insulin-like growth factor I receptor and the insulin receptor resulting in severe insulin resistance and progression to diabetes. Both models demonstrate that abrogation of the insulin-like growth factor I receptor and the insulin receptor or a common signalling pathway must be inhibited to cause sufficient insulin resistance to lead to type 2 diabetes; with either glucotoxicity or lipotoxicity being involved in the progression from severe to resistance to full-blown type 2 diabetes. Summary Thus, abrogation of insulin-stimulated glucose uptake in skeletal muscle, at least in mice, may lead to severe insulin resistance and diabetes.

During the development of skeletal muscle, myoblasts withdraw from the cell cycle and differentiate into myotubes. The insulin-like growth factors IGF-I and IGF-II, through their cognate tyrosine kinase receptor (IGF-I receptor), are known to play a role in this process. After withdrawal of myoblasts from the cell cycle, IGF-I promotes muscle differentiation by inducing the expression or activity of myogenic regulatory factors (MyoD, myogenin) and effectors (p21). However, little is known about the intracellular mechanisms by which the IGF-I system regulates these factors during the process of myogenesis. Here we show that MKR mice, which express a dominant negative IGF-I receptor specifically in skeletal muscle, have marked muscle hypoplasia from birth to 3 weeks of age. This hypoplasia occurs concomitantly with a decrease in ERK immunoreactivity levels and decreases in MyoD and myogenin expression. BrdU immunocytochemistry showed a compensatory hyperplasia as MKR mice grew to adulthood. Interestingly, hyperplasia occurred concomitantly with an increase in p38, MyoD, myogenin, and p21 immunoreactivity levels, as well as a decrease in Twist levels. These findings suggest that regulation of these cellular elements by IGF-I may play a role in the development and differentiation of skeletal muscle in vivo.

Increasing the mechanical load on skeletal muscle results in increased expression of insulin-like growth factor I (IGF-I), which is thought to be a critical step in the induction of muscle hypertrophy. To determine the role of the IGF-I receptor in load-induced skeletal muscle hypertrophy, we utilized a transgenic mouse model (MKR) that expresses a dominant negative IGF-I receptor specifically in skeletal muscle. Skeletal muscle hypertrophy was induced in the plantaris muscle using the functional overload (FO) model, a model which has previously been shown to induce significant elevations of IGF-I expression in skeletal muscle. Adult male wild-type (WT) and MKR mice were subjected to 0, 7 or 35 days of FO. In control or unchallenged animals, the plantaris mass was 11% greater in WT compared to the MKR mice (P0.05). After 7 days of FO, plantaris mass increased significantly by 26% and 62% in WT and MKR mice, respectively (P0.05). After 35 days of FO, WT and MKR mice demonstrated significant increases of 100% and 122%, respectively, in plantaris mass (P0.05). Further, at no time point was the degree of hypertrophy significantly different between the WT and MKR mice. Previous research suggests that IGF-I induces muscle growth through activation of the Akt-mTOR signalling pathway; therefore, we measured the phosphorylation status of Akt and p70(s6k) in the WT and MKR mice after 7 days of FO. Significant increases of approximately 100% and approximately 200% in Akt (Ser-473) and p70(s6k) (Thr-389) phosphorylation were measured in overloaded plantaris from both WT and MKR mice, respectively. Moreover, no differences were detected between the WT and MKR mice. These data suggest that increased mechanical load can induce muscle hypertrophy and activate the Akt and p70(s6k) independent of a functioning IGF-I receptor.

Nuclear receptor coactivator PBP (peroxisome proliferator-activated receptor (PPAR)-binding protein) functions as a coactivator for PPARs and other nuclear receptors. PBP serves as an anchor for TRAP (thyroid hormone receptor-associated proteins)/mediator multisubunit cofactor transcription complex. Disruption of the PBP/TRAP220 gene results in embryonic lethality around embryonic day 11.5 by affecting placental, cardiac, hepatic, and bone marrow development. Because PPAR isoforms alpha, gamma, and beta/delta function as important regulators of lipid homeostasis in mammals, it becomes important to assess the requirement of coactivator PBP in the regulation of PPAR functions in vivo. Sustained activation of PPARalpha by structurally diverse classes of chemicals of biological importance, designated peroxisome proliferators, leads to proliferation of peroxisomes in liver, induction of PPARalpha target genes including those involved in fatty acid oxidation, and the eventual development of liver tumors. Here, we show that targeted deletion of PBP in liver parenchymal cells, using the Cre-loxP system, results in the near abrogation of PPARalpha ligand-induced peroxisome proliferation and liver cell proliferation, as well as the induction of PPARalpha-regulated genes in PBP-deficient liver cells. In contrast, scattered PBP(+/+) hepatocytes in these livers showed DNA synthesis and were markedly hypertrophic with peroxisome proliferation in response to PPARalpha ligands. Chromatin immunoprecipitation data suggest that in PBP conditional null livers, there appears to be reduced association of cofactors, especially of CBP and TRAP150, to the mouse enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase gene promoter. These observations suggest that PBP is required for the stabilization of multiprotein cofactor complexes. In essence, the absence of PBP in hepatocytes in vivo appears to mimic the absence of PPARalpha, indicating that coactivator PBP is essential for PPARalpha-regulated gene expression in liver parenchymal cells.

We studied the effects of genetic background on the phenotype of ob/ob mice, a model of severe obesity, insulin resistance, and diabetes caused by leptin deficiency. Despite a comparable degree of obesity and hyperinsulinemia, C57BL/6J ob/ob mice had much milder hyperglycemia and, surprisingly, normal circulating adiponectin levels despite still-prominent signs of insulin resistance. Hyperinsulinemic-euglycemic clamp revealed relatively less whole-body and muscle insulin resistance in C57BL/6J ob/ob mice, whereas liver insulin resistance tended to be more severe than in FVB/N ob/ob mice. C57BL/6J ob/ob mice had also more rapid clearance of circulating triglycerides and more severe hepatic steatosis. We suggest that strain-related distinction in lipid handling is the most important player in the differences in diabetic phenotype and insulin sensitivity, whereas the impact of circulating adiponectin levels on the overall phenotype of ob/ob mice is less important.

PTEN is a dual protein and lipid phosphatase that dephosphorylates PIP3 at the 3' position, thereby antagonizing PI3-kinase activity. A reduction in PI3' kinase activity enhances the susceptibility of cells to apoptosis. By stably transfecting PC12 cells with an antisense PTEN construct, endogenous PTEN protein levels were reduced by approximately 50% and etoposide-induced apoptosis was markedly decreased. Furthermore, IGF-I receptor abrogation of this apoptotic effect was inhibited by both PI3' kinase and by specific inhibitors of p38 MAP kinase. Thus, we show for the first time that p38 MAP kinase is involved in this process.

9 páginas, 5 figuras, 3 tablas -- PAGS nros. 1926-1934, Peripheral insulin resistance and impaired insulin action are the primary characteristics of type 2 diabetes. The first observable defect in this major disorder occurs in muscle, where glucose disposal in response to insulin is impaired. We have developed a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR–IGF-IR) specifically targeted to the skeletal muscle. Expression of KR–IGF-IR resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance. Pancreatic β-cell dysfunction developed at a relative early age, resulting in diabetes. These mice provide an excellent model to study the molecular mechanisms underlying the development of human type 2 diabetes, The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Insulin and insulin-like growth factors (IGFs) mediate a variety of signals involved in mammalian development and metabolism. To study the metabolic consequences of IGF-I deficiency, we used the liver IGF-I–deficient (LID) mouse model. The LID mice show a marked reduction (∼75%) in circulating IGF-I and elevated growth hormone (GH) levels. Interestingly, LID mice show a fourfold increase in serum insulin levels (2.2 vs. 0.6 ng/ml in control mice) and abnormal glucose clearance after insulin injection. Fasting blood glucose levels and those after a glucose tolerance test were similar between the LID mice and their control littermates. Thus, the high levels of circulating insulin enable the LID mice to maintain normoglycemia in the presence of apparent insulin insensitivity. Insulin-induced autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate (IRS)-1 were absent in muscle, but were normal in liver and white adipose tissue of the LID mice. In contrast, IGF-I–induced autophosphorylation of its cognate receptor and phosphorylation of IRS-1 were normal in muscle of LID mice. Thus, the insulin insensitivity seen in the LID mice is muscle specific. Recombinant human IGF-I treatment of the LID mice caused a reduction in insulin levels and an increase in insulin sensitivity. Treatment of the LID mice with GH-releasing hormone antagonist, which reduces GH levels, also increased insulin sensitivity. These data provide evidence of the role of circulating IGF-I as an important component of overall insulin action in peripheral tissues.

Insulin signaling at the target tissue results in a large array of biological outcomes. These events are essential for normal growth and development and for normal homeostasis of glucose, fat, and protein metabolism. Elucidating the intracellular events after activation of the IR has been the primary focus of a large number of investigators for decades, and for excellent reasons. Understanding the signaling pathways involved in insulin action could lead to a better understanding of the pathophysiology of insulin resistance associated with obesity and type 2 diabetes, and identifying key molecules and processes could lead to newer and more effective therapeutic agents for treating these common disorders. This review summarizes our previous understanding of how insulin acts and outlines some recent developments in our understanding of insulin action and insulin resistance at the cellular level, beginning with a discussion on the discovery of evolutionarily conserved molecules of the insulin signaling pathways. This article will also provide a summary of a few in vitro and cellular models of insulin resistance and a description of some new paradigms in the cellular mechanisms of insulin action. This review will not attempt to be all-inclusive; for a more comprehensive understanding, readers are referred to more complete reviews on insulin action (1–5).

S THE introduction of the somatomedin hypothesis by Daughaday et al. (1) almost a half-century ago, our understanding of the insulin-like growth factor (IGF) system has evolved considerably. This evolution, from one of a simple pituitary-dependent growth factor secreted from liver to a ubiquitous and complex family of ligands, receptors, and binding proteins, has been spurred by advances in both protein biochemistry and, more recently, with the advent of the powerful tools of molecular biology. IGF-I and IGF-II are essential for normal growth and development. In tissue and organ systems that have fully differentiated, on the other hand, they play important roles in the homeostatic mechanisms associated with the normal function and maintenance of those tissues (2). Both IGF-I and IGF-II have been implicated in disease progression, either secondarily as the result of altered expression inducing abnormal growth and/or physiology (e.g. renal hypertrophy and deteriorating retinopathy during diabetes) or as one of the primary causative factors of the disease (e.g. overexpression in certain cancers and pediatric overgrowth disorders). This review discusses the normal physiology of the IGF system and describes some examples of pediatric diseases associated with alterations in this system and the possible therapeutic implications due to the availability of recombinant human IGF-I (rhIGF-I).

The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [ 125 I]insulin to cells obtained by mechanicaly dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t 1/2 of association was 14 min and t 1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [ 125 I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the α-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver α-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [ 3 H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [ 3 H]NE uptake was soduim ino-dependent suggesting that 80–98% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (GLU, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the β-subunit of the insulin receptors. These observations corroborate our contentio that insulin exerts neuromodulation effects mediated by the specific insulin receptors in the brain.