Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver.

The earliest defect in developing type 2 diabetes is insulin resistance, characterized by decreased glucose transport and metabolism in muscle and adipocytes. The glucose transporter GLUT4 mediates insulin-stimulated glucose uptake in adipocytes and muscle by rapidly moving from intracellular storage sites to the plasma membrane. In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression is decreased in adipose tissue but preserved in muscle. Because skeletal muscle is the main site of insulin-stimulated glucose uptake, the role of adipose tissue GLUT4 downregulation in the pathogenesis of insulin resistance and diabetes is unclear. To determine the role of adipose GLUT4 in glucose homeostasis, we used Cre/loxP DNA recombination to generate mice with adipose-selective reduction of GLUT4 (G4A-/-). Here we show that these mice have normal growth and adipose mass despite markedly impaired insulin-stimulated glucose uptake in adipocytes. Although GLUT4 expression is preserved in muscle, these mice develop insulin resistance in muscle and liver, manifested by decreased biological responses and impaired activation of phosphoinositide-3-OH kinase. G4A-/- mice develop glucose intolerance and hyperinsulinaemia. Thus, downregulation of GLUT4 and glucose transport selectively in adipose tissue can cause insulin resistance and thereby increase the risk of developing diabetes.

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart.

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

Evidence for leptin binding to proteins in serum of rodents and humans: modulation with obesity.

Many hormones circulate bound to serum proteins that modulate ligand bioactivity and bioavailability. To understand the biology of leptin action, we investigated the presence of leptin binding proteins in serum. 125I-labeled leptin binds competitively to at least three serum macromolecules with molecular masses of approximately 85, approximately 176, and approximately 240 kDa in rodents and approximately 176 and approximately 240 kDa in humans. The ability to bind appears to involve sulfhydryl/disulfide interactions because it is inhibited under reducing conditions. When serum is added to recombinant 125I-leptin, there is a shift in sedimentation of 125I-leptin as analyzed by sucrose gradient centrifugation from approximately S1.9 to approximately S4.3. This shift is markedly attenuated in serum from obese mice (ob/ob, db/db, brown-fat ablated, gold-thioglucose treated, high-fat fed) compared with that from nonobese controls. The size distribution of endogenous serum leptin as determined by radioimmunoassay (RIA) after sucrose gradient centrifugation is also consistent with saturation of binding in hyperleptinemic obesity. In humans, free leptin increases with BMI. Thus, in lean rodents and humans a large proportion of leptin circulates bound to several serum proteins. Free leptin is increased in serum of obese subjects, which may alter leptin bioactivity, transport, and/or clearance.

Evidence that glucocorticosteroid-mediated immunosuppressive effects do not involve altering second messenger function.

The mechanism by which glucocorticosteroids (GCS) suppress proliferation of human peripheral blood mononuclear leukocytes (PBML) was investigated. Using the proliferative responses to immobilized anti-CD3 mAb or mitogens (PHA + PMA) as biological readouts, dexamethasone (DEX) and 6 alpha-methylprednisolone (6 alpha-MP) were shown to inhibit PBML proliferation in a concentration-dependent fashion. The mechanism by which GCS mediate immunosuppression did not involve interference with Ca2+ fluxes as: (1) DEX failed to block Ca2+ entry into anti-CD3 + PMA stimulated cells; and (2) Ca2+ ionophores (ionomycin and A23187) failed to circumfent DEX-mediated suppression. DEX also had no effect on protein kinase C (PKC) activity as: (1) inhibitors (H-7 and staurosporin) or stimulators (1,2-dihexanoyl-sn-glycerol [DiC6] and 1,2-dioctanoyl-rac-glycerol [DiC8]) of PKC did not prevent DEX-mediated suppression; (2) DEX did not affect the activation-induced upregulation of CD4 and CD8 expression, an indirect index of PKC activity; and (3) DEX did not alter the activation-associated translocation of PKC from cytosolic to membrane-bound compartments. This, in addition to previous results demonstrating that GCS directly inhibit cytokine gene transcription and that rII-1 + rIL-6 + rIFN-gamma completely abrogated GCS-mediated suppressive effects, further supports the notion that GCS exert their immunosuppressive effects through inhibition of cytokine gene expression.

Interleukin 2 counteracts the inhibition of cytotoxic T lymphocytes by cholera toxin in vitro and in vivo.

Cholera toxin irreversibly activates a 43-kDa guanosine triphosphate (GTP)-binding protein by adenosine diphosphate ribosylation, resulting in activation of adenylate cyclase and increased intracellular levels of cyclic adenosine monophosphate (cAMP). Because increases in intracellular cAMP inhibit interleukin 2 (IL 2) expression and cytotoxic T lymphocyte (CTL) generation and function in vitro and in vivo, we hypothesized that IL 2 may counteract the inhibition of CTL by cholera toxin. Activated CTL treated with IL 2 were protected from the inhibitory effects of cholera toxin. IL 2 also counteracted the inhibitory effect of cholera toxin on steady-state levels of CTL-specific serine esterase mRNA. Given the putative role of serine esterase for in vitro generated CTL effector activity, these results may account for recovery of CTL activity. Although IL 2 restored CTL function and serine esterase transcription, it did not block cholera toxin-catalyzed ribosylation of the 43-kDa GTP-binding protein, nor did it prevent the accumulation of intracellular levels of cAMP. In vivo, C57BL/6 mice challenged with the allogeneic tumor P815 had suppressed CTL function when cholera toxin was administered. These cholera toxin-treated mice died of tumor overgrowth, whereas untreated mice rejected the allogeneic tumor. Co-treatment of alloimmunized mice with cholera toxin and IL 2 prevented death from tumor overgrowth and restored CTL function; 67% of these mice survived. These data provide evidence that IL 2 acts in CTL through a mechanism independent of cholera toxin-sensitive GTP-binding protein in vitro and in vivo, despite elevated intracellular cAMP levels.

Abrogation of glucocorticoid-mediated inhibition of T cell proliferation by the synergistic action of IL-1, IL-6, and IFN-gamma.

Glucocorticoids (GCS) inhibit the transcription of multiple activation-associated cytokine genes. By Northern blot analysis now we demonstrate that antiproliferative concentrations of dexamethasone and 6 alpha-methylprednisolone block mitogen-induced IL-2 gene expression in human peripheral blood mononuclear leukocytes in a concentration-dependent fashion. In addition, using a mitogen-induced proliferation assay of human peripheral blood mononuclear leukocytes, we show that GCS-mediated anti-proliferative effects are not blocked by rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, rTNF-alpha, rTNF-beta, and rIFN-gamma, individually at 1 to 1000 U/ml, but are totally abrogated, in a concentration-dependent fashion, by the combination of rIL-1, rIL-6, and rIFN-gamma (25 to 50 U/ml for each cytokine). Thus, blockade of cytokine expression is the primary mechanism by which GCS inhibit mitogen-driven and alloantigen-induced T cell proliferation. The immunosuppressive effects of GCS are almost certainly exacted at the level of cytokine gene transcription.

Similar effects of cyclosporine and verapamil on lymphokine, interleukin 2 receptor, and proto-oncogene expression.

Since calcium channel blocking agents and CsA exert an antiproliferative effect upon T cell mitogenesis, we have compared and characterized their immunosuppressive properties at the level of gene activation. Verapamil (greater than or equal to 30 microM), which blocks T cell mitogenesis and a rise in cytosolic calcium, was added to cultures of peripheral blood mononuclear cells stimulated with phytohemagglutinin (5 micrograms/ml) and phorbol myristate acetate (5 ng/ml). Northern blot analysis was performed using cDNA probes for the p55 interleukin 2 receptor (Tac; IL-2R), interleukin 2 and c-myc at 20 hr of culture. Accumulation of IL-2 encoding mRNA within the cytoplasm was completely abrogated by verapamil. However, IL-2R and c-myc encoding mRNA were clearly detectable in verapamil-treated cell cultures. Surface expression of the Tac protein in mitogen-activated T cells was also not blocked by verapamil as shown by FACS analysis. In companion experiments with CsA, verapamil only partially inhibits the intracellular processes leading to T cell activation. A calcium-independent pathway may exist for the expression of IL-2R and c-myc, while an increase of intracellular Ca2+ may provide the additional signal for IL-2 gene expression. Although the in vitro concentrations of verapamil used in these experiments are in excess of common clinically therapeutic levels, the results help clarify the mode of CsA action and may provide a new tool to dissect the early events of T cell activation.