Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes

Insulin and guanosine-5′-O-(3-thiotriphosphate) (GTPγS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (∼30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPγS response was significantly attenuated (∼85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPγS-stimulated GLUT4 translocation by ∼40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPγS-stimulated GLUT4 translocation but inhibited the insulin response by ∼40%. GTPγS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (∼50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPγS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.

Insulin-like growth factor 1 regulates developing brain glucose metabolism

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-d- [1-14C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1−/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-d- [1-14C]glucose is significantly reduced in synaptosomes prepared from Igf1−/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1−/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in Igf1−/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.

Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

The acute effects of contraction and insulin on the glucose transport and GLUT4 glucose transporter translocation were investigated in rat soleus muscles by using a 3-O-methylglucose transport assay and the sensitive exofacial labeling technique with the impermeant photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-y loxy)-2- propylamine (ATB-BMPA), respectively. Addition of wortmannin, which inhibits phosphatidylinositol 3-kinase, reduced insulin-stimulated glucose transport (8.8 +/- 0.5 mumol per ml per h vs. 1.4 +/- 0.1 mumol per ml per h) and GLUT4 translocation [2.79 +/- 0.20 pmol/g (wet muscle weight) vs. 0.49 +/- 0.05 pmol/g (wet muscle weight)]. In contrast, even at a high concentration (1 microM), wortmannin had no effect on contraction-mediated glucose uptake (4.4 +/- 0.1 mumol per ml per h vs. 4.1 +/- 0.2 mumol per ml per h) and GLUT4 cell surface content [1.75 +/- 0.16 pmol/g (wet muscle weight) vs. 1.52 +/- 0.16 pmol/g (wet muscle weight)]. Contraction-mediated translocation of the GLUT4 transporters to the cell surface was closely correlated with the glucose transport activity and could account fully for the increment in glucose uptake after contraction. The combined effects of contraction and maximal insulin stimulation were greater than either stimulation alone on glucose transport activity (11.5 +/- 0.4 mumol per ml per h vs. 5.6 +/- 0.2 mumol per ml per h and 9.0 +/- 0.2 mumol per ml per h) and on GLUT4 translocation [4.10 +/- 0.20 pmol/g (wet muscle weight) vs. 1.75 +/- 0.25 pmol/g (wet muscle weight) and 3.15 +/- 0.18 pmol/g (wet muscle weight)]. The results provide evidence that contraction stimulates translocation of GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which lias distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

Transport of Pirarucu Arapaima gigas juveniles in plastic bag

Este estudo examinou as respostas de estresse de juvenis de pirarucu transportados em sistema fechado. Pirarucu (Arapaima gigas) é um peixe nativo da bacia Amazônica, da família Osteoglossidae que possui respiração aérea obrigatória. Foi realizado um transporte de curta duração (6 h) em sacos de polietileno inflados com ar atmosférico (grupo ar) ou com oxigênio puro (grupo oxi). O oxigênio dissolvido foi o único parâmetro de qualidade da água que apresentou diferença estatística entre os grupos, e como esperado, o oxigênio estava supersaturado para o grupo oxi. Não houve mortalidade após o transporte em ambos os grupos. Os peixes se alimentaram 36 h após o transporte e apresentaram um consumo de ração habitual após 72 h. As respostas fisiológicas foram semelhantes nos dois grupos. O cortisol não apresentou mudança significativa durante o período de amostragem. Ao contrário da maioria das espécies, os valores de cortisol se apresentaram inalterados nos dois grupos durante a amostragem, enquanto a glicose teve um aumento significativo até 12 h após o transporte. Os resultados mostram que o transporte de pirarucu em sacos de polietileno pode ser realizado com ar atmosférico ou oxigênio puro, uma vez que as respostas de estresse, a qualidade da água e o comportamento alimentar após 36 h foram similar entre os grupos.

The beneficial effects of exercise in rodents are preserved after detraining: a phenomenon unrelated to GLUT4 expression

Background: Although exercise training has well-known cardiorespiratory and metabolic benefits, low compliance with exercise training programs is a fact, and the harmful effects of physical detraining regarding these adaptations usually go unnoticed. We investigated the effects of exercise detraining on blood pressure, insulin sensitivity, and GLUT4 expression in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Methods: Studied animals were randomized into sedentary, trained (treadmill running/5 days a week, 60 min/day for 10 weeks), 1 week of detraining, and 2 weeks of detraining. Blood pressure (tail-cuff system), insulin sensitivity (kITT), and GLUT4 (Western blot) in heart, gastrocnemius and white fat tissue were measured. Results: Exercise training reduced blood pressure (19%), improved insulin sensitivity (24%), and increased GLUT4 in the heart (+34%); gastrocnemius (+36%) and fat (+22%) in SHR. In WKY no change in either blood pressure or insulin sensitivity were observed, but there was an increase in GLUT4 in the heart (+25%), gastrocnemius (+45%) and fat (+36%) induced by training. Both periods of detraining did not induce any change in neither blood pressure nor insulin sensitivity in SHR and WKY. One-week detraining reduced GLUT4 in SHR (heart: -28%; fat: -23%) and WKY (heart: -19%; fat: -22%); GLUT4 in the gastrocnemius was reduced after a 2-week detraining (SHR: -35%; WKY: -25%). There was a positive correlation between GLUT4 (gastrocnemius) and the maximal velocity in the exercise test (r = 0.60, p = 0.004). Conclusions: The study findings show that in detraining, despite reversion of the enhanced GLUT4 expression, cardiorespiratory and metabolic beneficial effects of exercise are preserved.

Generation and characterization of human insulin-releasing cell lines

Background: The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results: We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion: We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, som: of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected P-32-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GN in early embryogenesis?

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages, In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose-response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40-50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GK. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription-PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.

Benefits of exercise training in diabetic rats persist after three weeks of detraining

Regarding all benefits of exercise training, a question remains: how long are these benefits kept? This study evaluated the effect of 3-week detraining after 10 weeks of training in STZ-diabetic rats. Male Wistar rats were assigned into: sedentary controls, trained controls, trained-detrained controls. sedentary diabetic, trained diabetic and trained-detrained diabetic. Arterial pressure (AP) and heart rate (HR) were recorded by a data acquisition system. Baroreflex sensitivity (BRS) was evaluated by HR responses to AP changes induced by infusion of vasoactive drugs. Intrinsic heart rate (IHR), sympathetic tonus (ST) and vagal tonus (VT) were evaluated by pharmacological blockade with atenolol and atropine. Spectral analysis of systolic AP and HR variabilities (HRV) was performed to estimate autonomic modulation to the heart and vessels. Diabetes cardiovascular and autonomic dysfunctions were reversed by exercise training and partially maintained in the 3-week detraining period. In controls, training decreased AP and HR and improved BRS. changes that returned to baseline values after detraining. IHR and VT were improved in trained diabetic rats and remained in detrained diabetic ones. LF component of HRV decreased in trained control group. In diabetics. exercise training improved variance, and absolute LF and HF components of HRV. Only HF was maintained in detrained diabetic group. Moreover, there was an inverse relationship between plasma glucose and the absolute HF component of HRV. These changes probably determined the different survival rate of 80% in diabetic detrained and 51% in diabetic sedentary rats. (c) 2008 Elsevier B.V. All rights reserved.

GLUT4 - At the cross roads between membrane trafficking and signal transduction

GLUT4 is a mammalian facilitative glucose transporter that is highly expressed in adipose tissue and striated muscle. In response to insulin, GLUT4 moves from intracellular storage areas to the plasma membrane, thus increasing cellular glucose uptake. While the verification of this 'translocation hypothesis' (Cushman SW. Wardzala LJ. J Biol Chem 1980;255: 4758-4762 and Suzuki K, Kono T. Proc Natl Acad Sci 1980;77: 2542-2545) has increased our understanding of insulin-regulated glucose transport, a number of fundamental questions remain unanswered. Where is GLUT4 stored within the basal cell? How does GLUT4 move to the cell surface and what mechanism does insulin employ to accelerate this process) Ultimately we require a convergence of trafficking studies with research in signal transduction. However, despite more than 30 years of intensive research we have still not reached this point. The problem is complex, involving at least two separate signal transduction pathways which feed into what appears to be a very dynamic sorting process. Below we discuss some of these complexities and highlight new data that are bringing us closer to the resolution of these questions.