Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.     

Comparative expression of hexose transporters (SGLT1, GLUT1, GLUT2 and GLUT5) throughout the mouse gastrointestinal tract

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.     

The effect of age on glucose uptake and GLUT1 and GLUT4 expression in rat skeletal muscle

During the life span, phenotypic and structural modifications on skeletal muscle contribute to a reduction on glucose uptake either in basal state or triggered by insulin, but the underlying mechanisms for this decline are not entirely identified. A reduction in the expression of skeletal muscle glucose transporters (GLUTs), glucose transporter type 1 (GLUT1) and glucose transporter type 4 (GLUT4), has been associated to such phenomena, but unlike the case of insulin, only few studies have addressed the effect of age on muscle-contraction-induced glucose uptake. The aim of the study was to investigate the influence of age on GLUT1 and GLUT4 expression in skeletal muscle and its relation to the glucose uptake induced by muscle contraction. For this purpose, soleus muscle from Wistar rats aged 4, 10, 22 and 42 weeks were isolated and electrically stimulated (30 min, 10 Hz, 20 V, 0.2 ms). After stimulation, glucose uptake and GLUT1 and GLUT4 expression and localisation were evaluated. Muscle contraction caused an increase in glucose uptake in all studied groups. In addition, the absolute rates of glucose uptake were negatively correlated with age. The expression of GLUT4 was lower in older animals, whereas no relation between age and GLUT1 expression was found. Immunohistochemistry confirmed the ontogenic effect on GLUT4 expression and suggested an age-related modification on GLUT1 distribution within the muscle fibres; for instance, this protein seems to be present mainly out of the sarcoplasm. The present findings demonstrate that the ability of muscle contraction to increase glucose uptake is not influenced by age, whereas glucose uptake under basal conditions decreases with age.     

Absence of insulin receptor substrate-1 expression does not alter GLUT1 or GLUT4 abundance or contraction-stimulated glucose uptake by mouse skeletal muscle.

The purpose of this study was to determine the influence of insulin receptor substrate-1 (IRS-1) expression on GLUT1 and GLUT4 glucose transporter protein abundance, contraction-stimulated glucose uptake, and contraction-induced glycogen depletion by skeletal muscle. Mice (6 months old) from three genotypes were studied: wild-type (IRS-1(+/+)), heterozygous (IRS-1(+/-)) for the null allele, and IRS-1 knockouts (IRS-1(-/-)) lacking a functional IRS-1 gene. In situ muscle contraction was induced (electrical stimulation of sciatic nerve) in one hindlimb using contralateral muscles as controls. Soleus and extensor digitorum longus were dissected and 2-deoxyglucose uptake was measured in vitro. 2-Deoxyglucose uptake was higher in basal muscles (no contractions) from IRS-1(-/-) vs. both other genotypes. Contraction-stimulated 2-deoxyglucose uptake and glycogen depletion did not differ among genotypes. Muscle IRS-1 protein was undetectable for IRS-1(-/-) mice, and values were approximately 40 % lower in IRS-1(+/-) than in IRS-1(+/+) mice. No difference was found in IRS-1(+/+) compared to IRS-1(-/-) groups regarding muscle abundance of GLUT1 and GLUT4. Substantial reduction or elimination of IRS-1 did not alter the hallmark effects of contractions on muscle carbohydrate metabolism--activation of glucose uptake and glycogen depletion.     

Divergent kinase signaling mediates agonist-induced phosphorylation of phosphatase inhibitory proteins PHI-1 and CPI-17 in vascular smooth muscle cells

Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation. signal transduction; myosin phosphatase holoenzyme inhibitor 1; protein kinase C     

Control of exercise-stimulated muscle glucose uptake by GLUT4 is dependent on glucose phosphorylation capacity in the conscious mouse

Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during exercise because GLUT4 overexpression does not increase exercise-stimulated MGU. Instead of glucose transport, glucose phosphorylation is a primary limitation of exercise-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair exercise-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity was increased. Thus, C57BL/6J mice with hexokinase II (HKII) overexpression (HK(Tg)), a GLUT4 partial knock-out (G4(+/-)), or both (HK(Tg) + G4(+/-)) and wild-type (WT) littermates were implanted with carotid artery and jugular vein catheters for sampling and infusions at 4 months of age. After a 7-day recovery, 5-h fasted mice remained sedentary or ran on a treadmill at 0.6 mph for 30 min (n = 9-12 per group) and received a bolus of 2-deoxy[3H]glucose to provide an index of MGU (Rg). Arterial blood glucose and plasma insulin concentrations were similar in WT, G4(+/-), HKTg, and HKTg + G4(+/-) mice. Sedentary Rg values were the same in all genotypes in all muscles studied, confirming that glucose transport is a significant barrier to basal glucose uptake. Gastrocnemius and soleus Rg were greater in exercising compared with sedentary mice in all genotypes. During exercise, G4(+/-) mice had a marked increase in blood glucose that was corrected by the addition of HK II overexpression. Exercise Rg (micromol/100g/min) was not different between WT and G4(+/-) mice in the gastrocnemius (24 +/- 5 versus 21 +/- 2) or the soleus (54 +/- 6 versus 70 +/- 7). In contrast, the enhanced exercise Rg observed in HKTg mice compared with that in WT mice was absent in HKTg + G4(+/-) mice in both the gastrocnemius (39 +/- 7 versus 22 +/- 6) and the soleus (98 +/- 13 versus 65 +/- 13). Thus, glucose transport is not a significant barrier to exercise-stimulated MGU despite a 50% reduction in GLUT4 content when glucose phosphorylation capacity is normal. However, when glucose phosphorylation capacity is increased by HK II overexpression, GLUT4 availability becomes a marked limitation to exercise-stimulated MGU.     

Dietary enrichment with wild blueberries (Vaccinium angustifolium) affects the vascular reactivity in the aorta of young spontaneously hypertensive rats

We have previously reported on the positive effects of wild blueberries on arterial contractile response to α₁ adrenergic stimuli and on endothelium-mediated vasorelaxation. Our present study was designed to evaluate the effects of the dietary enrichment with wild blueberries on aortic function and reactivity in the developmental phase of essential hypertension in spontaneously hypertensive rats (SHR). We investigated the possible influence blueberries may have on the acetylcholine (Ach)-induced endothelium-dependent vasorelaxation and phenylephrine-induced vasoconstriction in young SHRs, as well as the contribution of the nitric oxide (NO) synthase and cyclooxygenase (COX) pathways in each of the above responses in an animal model with dysfunctional endothelium. Vascular ring studies were conducted in 3-mm isolated rat aortic ring preparations to investigate vasoconstriction induced by l-Phenylephrine (Phe, 10^?8 to 3×10^?6M) and vasorelaxation induced by acetylcholine (Ach, 10^?9 to 3×10^?6M). The major findings of our study were that in Phe-induced vasoconstriction, SHR-BB aortas relaxed to a greater degree in comparison to controls when mefenamic acid (MFA) was present and that the incubation with this COX inhibitor failed to restore — and in fact decreased — the maximum vasodilator response to Ach, in comparison to controls. Our vessel reactivity index (pD₂) observations indicate that blueberries appear to modulate cell membrane–agonist (Ach) interactions primarily in response to Ach in the young SHR model, but not to the α₁ adrenoreceptor agonist. Incorporating wild blueberries in the diet seems to affect the endothelium-dependent vasorelaxation by modulating alternative metabolic pathway(s) (such as affecting the production/activity of COX-derived products) in the young SHR aorta.     

Inhibition of IRS-1 phosphorylation and the alterations of GLUT4 in isolated adipocytes from cachectic tumor-bearing rats

Cellular and molecular mechanisms of insulin resistance in isolated adipocytes from methylcholanthrene-induced sarcoma-bearing rats were investigated by measuring 3-O-[14C]methyl glucose transport activity, glucose transporter-4 (GLUT4) protein in both plasma membrane and low-density microsomes, and insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1). Compared to both pair-fed and freely fed controls, tumor-bearing rats (TBR) had a decreased insulin-stimulated glucose transport activity with a lower Vmax and a higher EC50. GLUT4 protein in low-density microsomes from adipocytes maintained at the basal state was less in TBR than in controls. In insulin-stimulated adipocytes, GLUT4 protein in plasma membranes was also less in tumor-bearing rats than in controls. Insulin-induced tyrosine phosphorylation of IRS-1 was less in TBR than controls, but that of the IR was similar among the three groups. These data suggest that the insulin resistance seen in adipose cells of these tumor-bearing rats was caused in part by a decreased amount of GLUT4 protein in both basal and insulin-stimulated states resulting from the selective inhibition of insulin-stimulated phosphorylation of IRS-1.     

Identification of PECAM-1 association with sphingosine kinase 1 and its regulation by agonist-induced phosphorylation

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator generated from sphingosine by sphingosine kinase (SPHK). S1P acts both extracellularly and intracellularly as a signaling molecule, although its intracellular targets are still undefined. Intracellular level of S1P is under strict regulatory control of SPHK regulation, S1P degradation, and S1P dephosphorylation. Therefore, clarifying the mechanisms regulating SPHK activity may help us understand when and where S1P is generated. In this study, we performed yeast two-hybrid screening to search for SPHK1a-binding molecules that may be involved in the regulation of the kinase localization or activity. Platelet endothelial cell adhesion molecule-1 (PECAM-1) was identified as a protein potentially associating with SPHK1a. Their association was confirmed by co-immunoprecipitation analysis using HEK293 cells overexpressing PECAM-1 and SPHK1a. Moreover, the kinase activity appeared to be reduced in stable PECAM-1-expressing cells. PECAM-1 is expressed on the cell surface of vascular cells, and several stimuli are known to induce phosphorylation of its tyrosine residues. We found that such phosphorylation attenuated its association with SPHK1a. This association/dissociation of SPHK with PECAM-1, regulated by the phosphorylated state of the membrane protein, may be involved in the control of localized kinase activity in certain cell types.     

Quantitation of GLUT1 and GLUT4 mRNA using a solution hybridization assay

The development of a solution hybridization assay for detecting GLUT1 and GLUT4 mRNA is described. The details of this assay are described in which copy RNA is used to quantitate messenger RNA in total RNA samples. This solution hybridization assay is highly specific and reproducible and is significantly more sensitive than Northern blotting. Since GLUT mRNAs can be quantitated in as little as 25 mg tissue, this technique is essential when the supply of tissue is limited. Furthermore, the elimination of gel-based separation techniques allows for mRNA quantitation in several hundred samples within two days following isolation of samples.     

微小RNA在自发性高血压大鼠主动脉的差异表达

微小RNAs(microRNAs,miRNAs)是一类基因组编码、非蛋白质编码的小RNA,在转录后水平负性调节靶基因表达.本研究探讨miRNAs在自发性高血压大(spontaneously hypertensive rats,SHR)主动脉的表达特征及其与高血压的关系.取4、8、16和24周龄雄性SHR大鼠及同龄正常血压对照(Wistar-Kyoto,WKY)大鼠.MiRanda、TargetScan和PicTar用于候选miRNAs及靶基因预测分析.通过实时定量RT-PCR检测大鼠主动脉miR-1、miR-133a、miR-155及miR-208的表达,并进一步通过实时定量RT-PCR检测呈差异表达的miR-155和miR-208的预测靶基因mRNA表达.结果显示,SHR大鼠主动脉miR-155表达在4、8、24周时与同龄WKY大鼠无显著差异,但在16周时明显低于同龄WKY大鼠(P<0.05),且大鼠主动脉miR-155表达量与血压呈负相关(r=-0.525,P<0.05).MiR-208表达在4周龄时最高,随年龄增长明显下降(P<0.05),其表达水平与血压和年龄呈负相关(r=-0.400,P<0.05;r=-0.684,P<0.0001),但在SHR和WKY大鼠之间无显著差异.miR-1和miR-133a在各年龄组SHR和WKY大鼠间未呈现差异表达.MiR-155和miR-208表达与相应预测靶基因mRNA表达无显著负相关性.以上结果表明,miR-155表达在成年SHR大鼠主动脉明显低于WKY,并与血压呈负相关,提示miR-155可能参与高血压的发生发展,主动脉miR-155表达异常可能是SHR大鼠血压升高的原因之一.大鼠主动脉miR-208表达在幼年时最高,随年龄增长而明显下降,提示其可能与血管发育有关.     

GLUT 1-glucose transporter protein in adult and fetal mouse lung

We observed approximately 45-50 kD GLUT 1 protein in mouse lung homogenates and demonstrated a greater abundance in fetus compared to adult. In situ immunohistochemical analysis demonstrated GLUT 1 expression only in the perineural sheath of nerves. While the trapped fetal red blood cells expressed GLUT 1 abundantly, adult red blood cells were devoid of GLUT 1. No GLUT 1 was evident in fetal and adult lung alveolar and bronchiolar epithelial cells, vascular endothelial cells and the lung mesenchymal elements. Thus, GLUT 1 is not the major lung glucose transporter.     

APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes

APS is a Cbl-binding protein that is tyrosine phosphorylated by the insulin receptor kinase. Insulin-stimulated phosphorylation of tyrosine 618 in APS is necessary for its association with c-Cbl and the subsequent tyrosine phosphorylation of Cbl by the insulin receptor in both 3T3-L1 adipocytes and CHO-IR cells. When overexpressed in these cells, wild-type APS but not an APS/Y(618)F mutant facilitated the tyrosine phosphorylation of coexpressed Cbl and its association with Crk upon insulin stimulation. APS-facilitated phosphorylation occurred on tyrosines 371, 700, and 774 in the Cbl protein. APS also interacted directly with the c-Cbl-associated protein (CAP) and colocalized with the protein in cells. The association was dependent on the SH3 domains of CAP and was independent of insulin treatment. Overexpression of the APS/Y(618)F mutant in 3T3-L1 adipocytes blocked the insulin-stimulated tyrosine phosphorylation of endogenous Cbl and binding to Crk. Moreover, the translocation of GLUT4 from intracellular vesicles to the plasma membrane was also inhibited by overexpression of the APS/Y(618)F mutant. These data suggest that APS serves as an adapter protein linking the CAP/Cbl pathway to the insulin receptor and, further, that APS-facilitated Cbl tyrosine phosphorylation catalyzed by the insulin receptor is a crucial event in the stimulation of glucose transport by insulin.