Development of diet-induced fatty liver disease in the aging mouse is suppressed by brief daily exposure to low-magnitude mechanical signals.

The age-induced decline in the body's ability to fight disease is exacerbated by obesity and metabolic disease. Using a mouse model of diet-induced obesity, the combined challenge of a high-fat diet and age on liver morphology and biochemistry was characterized, while evaluating the potential of 15 min per day of high frequency (90 Hz), extremely low-magnitude (0.2 G) mechanical signals (LMMS) to suppress lipid accumulation in the liver. Following a 36-week protocol (animals 43 weeks of age), suppression of hepatomegaly and steatosis was reflected by a 29% lower liver mass in LMMS animals as compared with controls. Average triglyceride content was 101.7+/-19.4 microg mg(-1) tissue in the livers of high-fat diet control (HFD) animals, whereas HFD+LMMS animals realized a 27% reduction to 73.8+/-22.8 microg mg(-1) tissue. In HFD+LMMS animals, liver free fatty acids were also reduced to 0.026+/-0.009 microEq mg(-1) tissue from 0.035+/-0.005 microEq mg(-1) tissue in HFD. Moderate to severe micro- and macrovesicular steatosis in HFD was contrasted to a 49% reduction in area covered by the vacuoles of at least 15 microm(2) in size in HFD+LMMS animals. These data provide preliminary evidence of the ability of LMMS to attenuate the progression of fatty liver disease, most likely achieved indirectly by suppressing adipogenesis and thus the total adipose burden through life, thereby reducing a downstream challenge to liver morphology and function.

Insulin receptor substrate 1 and 2 (IRS1 and IRS2): what a tangled web we weave.

The insulin receptor is a transmembrane tyrosine kinase that is essential for mediating multiple intracellular signalling cascades that lead ultimately to the biological actions of insulin Tyrosine phosphorylation o f the cytosolic proteins insulin receptor substrate 1 and 2 (IRS1 and IRS2) produces protein 'scaffolding' for the assembly of effector proteins containing Src homology 2 (SH2) domains, thereby generating multisubunit signalling complexes. Although IRS1 was originally isolated as a specific insulin receptor substrate, both IRS1 and IRS2 appear to play a broader role, functioning also as proximal substrates in growth hormone and cytokine receptor signalling. Current data establish IRS1 and IRS2 as critical effectors integrating various cell-type-specific signals into distinct, but overlapping, biological responses.

Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

In vivo quantification of subcutaneous and visceral adiposity by micro-computed tomography in a small animal model.

Accurate and precise techniques that identify the quantity and distribution of adipose tissue in vivo are critical for investigations of adipose development, obesity, or diabetes. Here, we tested whether in vivo micro-computed tomography (microCT) can be used to provide information on the distribution of total, subcutaneous and visceral fat volume in the mouse. Ninety C57BL/6J mice (weight range: 15.7-46.5 g) were microCT scanned in vivo at 5 months of age and subsequently sacrificed. Whole body fat volume (base of skull to distal tibia) derived from in vivo microCT was significantly (p<0.001) correlated with the ex vivo tissue weight of discrete perigonadal (R(2)=0.94), and subcutaneous (R(2)=0.91) fat pads. Restricting the analysis of tissue composition to the abdominal mid-section between L1 and L5 lumbar vertebrae did not alter the correlations between total adiposity and explanted fat pad weight. Segmentation allowed for the precise discrimination between visceral and subcutaneous fat as well as the quantification of adipose tissue within specific anatomical regions. Both the correlations between visceral fat pad weight and microCT determined visceral fat volume (R(2)=0.95, p<0.001) as well as subcutaneous fat pad weight and microCT determined subcutaneous fat volume (R(2)=0.91, p<0.001) were excellent. Data from these studies establish in vivo microCT as a non-invasive, quantitative tool that can provide an in vivo surrogate measure of total, visceral, and subcutaneous adiposity during longitudinal studies. Compared to current imaging techniques with similar capabilities, such as microMRI or the combination of DEXA with NMR, it may also be more cost-effective and offer higher spatial resolutions.

Glycemic improvement in diabetic db/db mice by overexpression of the human insulin-regulatable glucose transporter (GLUT4).

The effects of increased GLUT4 (insulin-regulatable muscle/fat glucose transporter) expression on glucose homeostasis in a genetic model of non-insulin-dependent diabetes mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic C57BL/KsJ-db/db mice. A genomic hGLUT4 construct was microinjected directly into pronuclear murine embryos of db/+ matings to maintain the inbred background. Four lines of hGLUT4 transgenic mice were bred to homozygosity at the db locus and all showed a marked reduction of both fasted and fed plasma glucose levels (to approximately 50 and 360 mg/dl, respectively) compared with age-matched nontransgenic db/db mice (approximately 215 and 550 mg/dl, respectively), as well as an enhanced disposal of an oral glucose challenge. In situ immunocytochemical localization of GLUT4 protein in muscle from hGLUT4 db/db mice showed elevated plasma membrane-associated GLUT4 protein in the basal state, which markedly increased after an insulin/glucose injection. In contrast, nontransgenic db/db mice had low levels of plasma membrane-associated GLUT4 protein in the basal state with a relatively small increase after an insulin/glucose challenge. Since the intracellular GLUT4 levels in db/db mice were similar to nontransgenic db/+ mice, the glucose transport defect in db/db mice is at the level of glucose transporter translocation. Together, these data demonstrate that GLUT4 upregulation overcomes the glucose transporter translocation defect and alleviates insulin resistance in genetically diabetic mice, thus resulting in markedly improved glycemic control.

Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance.

To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.

Enhancement or inhibition of insulin signaling by insulin receptor substrate 1 is cell context dependent.

Insulin treatment of Chinese hamster ovary (CHO) cells expressing high levels of the insulin receptor (CHO/IR cells) activates both c-fos serum response element and activator protein 1 (AP-1) reporter genes approximately 10-fold. In contrast, parental CHO cells display only two- to threefold insulin stimulation of reporter gene activity. Transient transfection of parental CHO cells with an insulin receptor substrate 1 (IRS1) expression plasmid enhanced insulin downstream signaling in a biphasic manner, whereas IRS1 transfection of CHO/IR cells inhibited insulin signaling in a dose-dependent fashion. Further, expression of Grb2 in parental CHO cells had no effect on insulin signaling, whereas Grb2 increased insulin activation of reporter gene expression in CHO/IR cells. These data suggest that the expression levels of various effector molecules can either enhance or inhibit insulin downstream signaling events. To assess the relative effects of various insulin receptor, IRS1, and Grb2 levels on insulin signaling, parental CHO cells were transiently transfected with various combinations of expression plasmids encoding these proteins. Although expression of IRS1 resulted in a biphasic increase of insulin signaling in parental CHO cells, coexpression of IRS1 with the insulin receptor resulted in inhibition of signaling. This inhibition of insulin signaling directly correlated with an increased association of Grb2 with IRS1 and a concomitant sequestration of Grb2 away from Shc. Consistent with the Shc-Grb2 pathway as the major route for insulin-stimulated c-Fos and AP-1 transcriptional activation, the IRS1-mediated inhibition was reversed by transfection with an expression plasmid for Grb2. These data demonstrate that the extent of insulin-stimulated downstream signaling was dependent not only on the levels of individual signaling molecules but also on the formation of multiprotein complexes with specific stoichiometries.

Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Insulin-stimulated disassociation of the SOS-Grb2 complex.

Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of whole-cell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the dissociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.

Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.

Introduction of the cytoplasmic domain of syntaxin 4, using either recombinant vaccinia virus or single-cell microinjection, resulted in an inhibition of insulin-stimulated GLUT4 but not GLUT1 translocation to the plasma membrane. This was specific for syntaxin 4, since neither the expression of syntaxin 3 nor the expression of a syntaxin 4 mutant in which the vesicle-associated membrane protein (VAMP) binding site was deleted had any significant effect. Consistent with the requirement for a functional VAMP binding site, expression of the cytoplasmic domains of VAMP2 or VAMP3/cellubrevin also resulted in an inhibition of insulin-stimulated GLUT4 translocation. In addition, immunoprecipitation of the expressed syntaxin 4 cytoplasmic domain resulted in an insulin-stimulated increase in the coimmunoprecipitation of GLUT4-containing vesicles. Together, these data demonstrate that syntaxin 4, VAMP2, and/or VAMP3/cellubrevin can function as target membrane and vesicle SNAP receptors, respectively, for insulin-responsive GLUT4 translocation to the plasma membrane.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles.

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.

Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways.

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.

Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state.

We have previously reported that insulin and osmotic shock stimulate an increase in glucose transport activity and translocation of the insulin-responsive glucose transporter isoform GLUT4 to the plasma membrane through distinct pathways in 3T3L1 adipocytes (D. Chen, J. S. Elmendorf, A. L. Olson, X. Li, H. S. Earp, and J. E. Pessin, J. Biol. Chem. 272:27401-27410, 1997). In investigations of the relationships between these two signaling pathways, we have now observed that these two stimuli are not additive, and, in fact, osmotic shock pretreatment was found to completely prevent any further insulin stimulation of glucose transport activity and GLUT4 protein translocation. In addition, osmotic shock inhibited the insulin stimulation of lipogenesis and glycogen synthesis. This inhibition of insulin-stimulated downstream signaling occurred without any significant effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, there was no effect on either the insulin-stimulated association of the p85 type I phosphatidylinositol (PI) 3-kinase regulatory subunit with IRS1 or phosphotyrosine antibody-immunoprecipitated PI 3-kinase activity. In contrast, osmotic shock pretreatment markedly inhibited the insulin stimulation of protein kinase B (PKB) and p70S6 kinase activities. In addition, the dephosphorylation of PKB was prevented by pretreatment with the phosphatase inhibitors okadaic acid and calyculin A. These data support a model in which osmotic shock-induced insulin resistance of downstream biological responses results from an inhibition of insulin-stimulated PKB activation.

VAMP3 null mice display normal constitutive, insulin- and exercise-regulated vesicle trafficking.

To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.

Effect of membrane phospholipid composition changes on adenylate cyclase activity in normal and rous-sarcoma-transformed chicken embryo fibroblasts.

Adenylate cyclase specific activities in membranes isolated from chicken embryo fibroblasts transformed by Rous sarcoma virus are significantly lower than the specific activity of the enzyme in normal membranes. Since normal and transformed membranes have different phospholipid and fatty acid compositions, adenylate cyclase activities were examined in normal and transformed membranes which had been supplemented with polar head groups or fatty acids. Basal, fluoride, and prostaglandin E1-stimulated activities changed systematically with phospholipid composition. Increases in the primary amino group of the phospholipid polar head groups or the average degree of fatty acid unsaturation both inhibited adenylate cyclase activity. In general, adenylate cyclase activities in normal membranes were more sensitive to phospholipid compositional changes compared to adenylate cyclase in transformed membranes. The data indicate that the lower adenylate cyclase activities in transformed membranes are not solely attributable to phospholipid changes but do suggest that increases in the percentage of phosphatidylethanolamine may contribute to the lower adenylate cyclase activities in transformed membranes.

Human GLUT4/muscle-fat glucose-transporter gene. Characterization and genetic variation.

Four overlapping DNA fragments spanning 32 kb containing the human GLUT4 facilitative glucose-transporter gene were isolated and characterized. The sequence of the GLUT4 gene (approximately 6.3 kb) and 2.0 kb of the promoter region was determined. The sequence of the promoter revealed potential binding sites for transcription factors known to regulate gene expression in muscle cells and adipocytes. However, transfection of constructs including 2 kb of the GLUT4 promoter fused to the bacterial CAT gene into 3T3-L1 adipocytes displayed only weak promoter activity. Because insulin resistance plays a prominent role in the development of NIDDM, genetic variation in the sequence of GLUT4 also was evaluated. Oligonucleotide primer pairs were selected that allowed the protein-coding region of the human GLUT4 gene to be amplified by PCR. The sequence of the protein-coding region of the GLUT4 gene and all intron-exon junctions was determined for a single diabetic Pima Indian and was identical to that of the cloned gene and cDNA. SSCP analysis was used to screen patients with diabetes mellitus and normal, healthy nondiabetic individuals for mutations at the GLUT4 locus. In addition to the silent substitution in the codon for Asn130 (AAC or AAT) and a Val383 (GTC)-->Ile(ATC) replacement described previously, two new variants were identified. One was a T-->A substitution in intron 1 that was found in 1 of 36 NIDDM patients who were typed for this variant. The second was a Ile385(ATT)-->Thr(ACT) replacement that occurred in 1 normal individual and was not found in any of 676 other normal and diabetic subjects. A large and racially diverse group of normal and diabetic individuals also was screened for the Ile383 polymorphism. It occurred in both diabetic and nondiabetic subjects. There is no indication from our data that these polymorphisms are associated with NIDDM.

Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3L1 adipocytes.

It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.

Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin.

Analysis of glucose transporter mRNA levels in adipose tissue from streptozotocin (STZ)-induced diabetic rats demonstrated a specific decrease (10-fold) in adipose tissue GLUT-4 mRNA with no significant effect on GLUT-1 mRNA levels. Treatment of STZ-diabetic rats with twice daily injections of insulin for 1-3 days resulted in a 16-fold increase in the relative amount of GLUT-4 mRNA to levels approximately 2-fold greater than those in control animals. However, after 7 days of insulin therapy the amount of GLUT-4 mRNA decreased approximately 2-fold back to the levels in the control animals. Normalization of the STZ-induced serum hyperglycemia by phlorizin treatment, which inhibits renal tubular reabsorption of glucose, had no effect on GLUT-4 mRNA in the absence of insulin. Similar to STZ-diabetes, fasting for 48 h also reduced adipose GLUT-4 mRNA levels. Parenteral administration of insulin with glucose over 7.5 h, but not glucose alone, increased the levels of the GLUT-4 mRNA 3- to 4-fold. These studies demonstrate that the relative glycemic state does not influence GLUT-4 glucose transporter mRNA expression in vivo and strongly suggests that insulin is a major factor regulating the levels of GLUT-4 mRNA in adipose tissue.

Regulation of c-fos expression in adipose and muscle tissue of diabetic rats.

Insulin treatment of control rats demonstrated a marked 8-fold transient increase (15 min) in c-fos mRNA in white adipose tissue, which returns to basal levels by 5 h. Similarly, insulin treatment resulted in a rapid 9-fold increase in cardiac muscle c-fos mRNA, which also returned to control values by 1 h. By contrast, insulin treatment resulted in only a small increase in c-jun mRNA levels in both adipose tissue and cardiac muscle. Similarly, the expression of c-jun mRNA was only slightly responsive to insulin in these tissues from streptozocin-induced insulin-deficient diabetic rats. However, insulin treatment of insulin-deficient diabetic rats resulted in a prolonged increase in c-fos message levels in adipose tissue without any significant change in the time course of c-fos mRNA induction/repression in cardiac muscle. These data demonstrate that in contrast to c-jun, c-fos is transiently increased in both cardiac muscle and adipose tissue by insulin treatment. Furthermore, transrepression of the c-fos gene is specifically attenuated in adipose tissue of insulin-deficient diabetic rats, but not in cardiac muscle.