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Biomedical subjects

Jerrold M Olefsky

Publications and source records attributed to Jerrold M Olefsky.

At least 19 recordsLinked to original sources
PubMed2007

Regulation of receptor tyrosine kinase signaling by GRKs and beta-arrestins.

Receptor tyrosine kinases (RTKs) are a unique family of cell surface receptors, each containing a common intracellular domain that has tyrosine kinase activity. However, RTKs share many signaling molecules with another unique family of cell surface receptors, the seven-transmembrane receptors (7TMRs), and these receptor families can activate similar signaling cascades. In this review of RTK signaling, we describe the role of cross talk between RTKs and 7TMRs, focusing specifically on the role played in this process by beta-arrestins and by G proteins.

Animals↗
PubMed2006

Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes.

Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.

3T3-L1 Cells↗
PubMed2006

PPARdelta regulates glucose metabolism and insulin sensitivity.

The metabolic syndrome is a collection of obesity-related disorders. The peroxisome proliferator-activated receptors (PPARs) regulate transcription in response to fatty acids and, as such, are potential therapeutic targets for these diseases. We show that PPARdelta (NR1C2) knockout mice are metabolically less active and glucose-intolerant, whereas receptor activation in db/db mice improves insulin sensitivity. Euglycemic-hyperinsulinemic-clamp experiments further demonstrate that a PPARdelta-specific agonist suppresses hepatic glucose output, increases glucose disposal, and inhibits free fatty acid release from adipocytes. Unexpectedly, gene array and functional analyses suggest that PPARdelta ameliorates hyperglycemia by increasing glucose flux through the pentose phosphate pathway and enhancing fatty acid synthesis. Coupling increased hepatic carbohydrate catabolism with its ability to promote beta-oxidation in muscle allows PPARdelta to regulate metabolic homeostasis and enhance insulin action by complementary effects in distinct tissues. The combined hepatic and peripheral actions of PPARdelta suggest new therapeutic approaches to treat type II diabetes.

Animals↗
PubMed2006

Suppression of Egr-1 transcription through targeting of the serum response factor by oncogenic H-Ras.

The transcription factor Egr-1 functions as a key regulator in cellular growth, differentiation, and apoptosis. The loss of Egr-1 expression is closely associated with tumor development, although the molecular mechanism behind the suppression of Egr-1 is largely unknown. In this report, we show that growth factor-induced transcriptional activation of Egr-1 gene is downregulated by chronic expression of oncogenic H-Ras in NIH3T3 fibroblasts. Our results demonstrate that phosphoinositide 3-kinase (PI3K) signaling is necessary for oncogenic H-Ras-mediated reduction of Egr-1 gene expression. Aberrant activation of PI3K signaling by oncogenic Ras decreased the level of serum response factor (SRF) protein through the acceleration of proteolysis, which resulted in decreased SRF binding to the serum response element (SRE) sites within the Egr-1 promoter, leading to the suppression of Egr-1 transcription. Inhibition of PI3K signaling restored the downregulation of SRF and Egr-1 expression caused by oncogenic Ras. Our findings suggest a novel signaling mechanism by which prolonged activation of oncogenic H-Ras can trigger the loss of tumor suppressor Egr-1 through the PI3K pathway in NIH3T3 fibroblast model cell lines.

3T3 Cells↗
PubMed2006

APPLied mechanics: uncovering how adiponectin modulates insulin action.

Adiponectin, an adipocyte secretory hormone, has been causally linked to insulin resistance in the metabolic syndrome and diabetes. A recent paper (Mao et al., 2006) shows that the APPL1 adaptor protein binds to the intracellular domain of adiponectin receptors and mediates some of adiponectin's actions, identifying a novel mechanism linking adiponectin to insulin sensitization.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Inflamed fat: what starts the fire?

Obesity is associated with increased macrophage infiltration of adipose tissue, and these macrophages may be an important component of the chronic inflammatory response playing a crucial role in the development of insulin resistance. This prompts the question as to how macrophages infiltrate obese adipose tissue. In this issue of the JCI, Weisberg et al. show the importance of C-C motif chemokine receptor 2 (CCR2) in macrophage recruitment to adipose tissue and the development of obesity and its complications.

Adipose Tissue↗
PubMed2006

Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects.

Increased accumulation of fatty acids and their derivatives can impair insulin-stimulated glucose disposal by skeletal muscle. To characterize the nature of the defects in lipid metabolism and to evaluate the effects of thiazolidinedione treatment, we analyzed the levels of triacylglycerol, long-chain fatty acyl-coA, malonyl-CoA, fatty acid oxidation, AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), malonyl-CoA decarboxylase, and fatty acid transport proteins in muscle biopsies from nondiabetic lean, obese, and type 2 subjects before and after an euglycemic-hyperinsulinemic clamp as well as pre-and post-3-month rosiglitazone treatment. We observed that low AMPK and high ACC activities resulted in elevation of malonyl-CoA levels and lower fatty acid oxidation rates. These conditions, along with the basal higher expression levels of fatty acid transporters, led accumulation of long-chain fatty acyl-coA and triacylglycerol in insulin-resistant muscle. During the insulin infusion, muscle fatty acid oxidation was reduced to a greater extent in the lean compared with the insulin-resistant subjects. In contrast, isolated muscle mitochondria from the type 2 subjects exhibited a greater rate of fatty acid oxidation compared with the lean group. All of these abnormalities in the type 2 diabetic group were reversed by rosiglitazone treatment. In conclusion, these studies have shown that elevated malonyl-CoA levels and decreased fatty acid oxidation are key abnormalities in insulin-resistant muscle, and, in type 2 diabetic patients, thiazolidinedione treatment can reverse these abnormalities.

AMP-Activated Protein Kinases↗
PubMed2005

Disruption of microtubules ablates the specificity of insulin signaling to GLUT4 translocation in 3T3-L1 adipocytes.

Although the cytoskeletal network is important for insulin-induced glucose uptake, several studies have assessed the effects of microtubule disruption on glucose transport with divergent results. Here, we investigated the effects of microtubule-depolymerizing reagent, nocodazole and colchicine, on GLUT4 translocation in 3T3-L1 adipocytes. After nocodazole treatment to disrupt microtubules, GLUT4 vesicles were dispersed from the perinuclear region in the basal state, and insulin-induced GLUT4 translocation was partially inhibited by 20-30%, consistent with other reports. We found that platelet-derived growth factor (PDGF), which did not stimulate GLUT4 translocation in intact cells, was surprisingly able to enhance GLUT4 translocation to approximately 50% of the maximal insulin response, in nocodazole-treated cells with disrupted microtubules. This effect of PDGF was blocked by pretreatment with wortmannin and attenuated in cells pretreated with cytochalasin D. Using confocal microscopy, we found an increased co-localization of GLUT4 and F-actin in nocodazole-treated cells upon PDGF stimulation compared with control cells. Furthermore, microinjection of small interfering RNA targeting the actin-based motor Myo1c, but not the microtubule-based motor KIF3, significantly inhibited both insulin- and PDGF-stimulated GLUT4 translocation after nocodazole treatment. In summary, our data suggest that 1) proper perinuclear localization of GLUT4 vesicles is a requirement for insulin-specific stimulation of GLUT4 translocation, and 2) nocodazole treatment disperses GLUT4 vesicles from the perinuclear region allowing them to engage insulin and PDGF-sensitive actin filaments, which can participate in GLUT4 translocation in a phosphatidylinositol 3-kinase-dependent manner.

3T3-L1 Cells↗
PubMed2005

Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes.

Hormones mobilize intracellular second messengers and initiate signalling cascades involving protein kinases and phosphatases, which are often spatially compartmentalized by anchoring proteins to increase signalling specificity. These scaffold proteins may themselves be modulated by hormones. In adipocytes, stimulation of beta-adrenergic receptors increases cyclic AMP levels and activates protein kinase A (PKA), which stimulates lipolysis by phosphorylating hormone-sensitive lipase and perilipin. Acute insulin treatment activates phosphodiesterase 3B, reduces cAMP levels and quenches beta-adrenergic receptor signalling. In contrast, chronic hyperinsulinaemic conditions (typical of type 2 diabetes) enhance beta-adrenergic receptor-mediated cAMP production. This amplification of cAMP signalling is paradoxical because it should enhance lipolysis, the opposite of the known short-term effect of hyperinsulinaemia. Here we show that in adipocytes, chronically high insulin levels inhibit beta-adrenergic receptors (but not other cAMP-elevating stimuli) from activating PKA. We measured this using an improved fluorescent reporter and by phosphorylation of endogenous cAMP-response-element binding protein (CREB). Disruption of PKA scaffolding mimics the interference of insulin with beta-adrenergic receptor signalling. Chronically high insulin levels may disrupt the close apposition of beta-adrenergic receptors and PKA, identifying a new mechanism for crosstalk between heterologous signal transduction pathways.

3T3-L1 Cells↗
PubMed2005

Synthesis and evaluation of photolabile insulin prodrugs.

We have developed two photolabile insulin prodrugs, insulin-2P and insulin-3P. These prodrugs were synthesized by protecting GlyA1 (N(alphaA1)), and one or both of the PheB1 (N(alphaB1)) and LysB29 (N(epsilonB29)) amino groups in insulin using 5'-(alpha-methyl-nitro-piperonyl)oxy-carbonyl as the protecting group. These insulin prodrugs were efficiently activated by exposure to longwave UV light to produce insulin quantitatively. Using 2-deoxyglucose uptake assays, both di- and tri-protected compounds were less active than native insulin in the protected state, and showed comparable activity to native insulin upon photoactivation.

3T3-L1 Cells↗
PubMed2005

JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes.

Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.

3T3-L1 Cells↗
PubMed2005

Topiramate treatment causes skeletal muscle insulin sensitization and increased Acrp30 secretion in high-fat-fed male Wistar rats.

We show that Topiramate (TPM) treatment normalizes whole body insulin sensitivity in high-fat diet (HFD)-fed male Wistar rats. Thus drug treatment markedly lowered glucose and insulin levels during glucose tolerance tests and caused increased insulin sensitization in adipose and muscle tissues as assessed by euglycemic clamp studies. The insulin-stimulated glucose disposal rate increased twofold (indicating enhanced muscle insulin sensitivity), and suppression of circulating FFAs increased by 200 to 300%, consistent with increased adipose tissue insulin sensitivity. There were no effects of TPM on hepatic insulin sensitivity in these TPM-treated HFD-fed rats. In addition, TPM administration resulted in a three- to fourfold increase in circulating levels of total and high-molecular-weight (HMW) adiponectin (Acrp30). Western blot analysis revealed normal AMPK (Thr(172)) phosphorylation in liver with a twofold increased phospho-AMPK in skeletal muscle in TPM-treated rats. In conclusion, 1) TPM treatment prevents overall insulin resistance in HFD male Wistar rats; 2) drug treatment improved insulin sensitivity in skeletal muscle and adipose tissue associated with enhanced AMPK phosphorylation; and 3) the tissue "specific" effects are associated with increased serum levels of adiponectin, particularly the HMW component.

AMP-Activated Protein Kinases↗
PubMed2005

VAMPIRE microarray suite: a web-based platform for the interpretation of gene expression data.

Microarrays are invaluable high-throughput tools used to snapshot the gene expression profiles of cells and tissues. Among the most basic and fundamental questions asked of microarray data is whether individual genes are significantly activated or repressed by a particular stimulus. We have previously presented two Bayesian statistical methods for this level of analysis, collectively known as variance-modeled posterior inference with regional exponentials (VAMPIRE). These methods each require a sophisticated modeling step followed by integration of a posterior probability density. We present here a publicly available, web-based platform that allows users to easily load data, associate related samples and identify differentially expressed features using the VAMPIRE statistical framework. In addition, this suite of tools seamlessly integrates a novel gene annotation tool, known as GOby, which identifies statistically overrepresented gene groups. Unlike other tools in this genre, GOby can localize enrichment while respecting the hierarchical structure of annotation systems like Gene Ontology (GO). By identifying statistically significant enrichment of GO terms, Kyoto Encyclopedia of Genes and Genomes pathways, and TRANSFAC transcription factor binding sites, users can gain substantial insight into the physiological significance of sets of differentially expressed genes. The VAMPIRE microarray suite can be accessed at http://genome.ucsd.edu/microarray.

Bayes Theorem↗
PubMed2005

G protein-coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes.

G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 alpha-subunit (Galphaq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Galphaq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Galphaq/11 signaling, including tyrosine phosphorylation of Galphaq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Galphaq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.

3T3-L1 Cells↗
PubMed2005

Deletion of Gab1 in the liver leads to enhanced glucose tolerance and improved hepatic insulin action.

Insulin receptor substrate-1 (IRS-1) and IRS-2 are known to transduce and amplify signals emanating from the insulin receptor. Here we show that Grb2-associated binder 1 (Gab1), despite its structural similarity to IRS proteins, is a negative modulator of hepatic insulin action. Liver-specific Gab1 knockout (LGKO) mice showed enhanced hepatic insulin sensitivity with reduced glycemia and improved glucose tolerance. In LGKO liver, basal and insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 was elevated, accompanied by enhanced Akt/PKB activation. Conversely, Erk activation by insulin was suppressed in LGKO liver, leading to defective IRS-1 Ser612 phosphorylation. Thus, Gab1 acts to attenuate, through promotion of the Erk pathway, insulin-elicited signals flowing through IRS and Akt proteins, which represents a novel balancing mechanism for control of insulin signal strength in the liver.

Adaptor Proteins, Signal Transducing↗