Glucose Transport

The mammary gland undergoes dramatic functional and metabolic changes during the transition from late pregnancy to lactation. To better understand the molecular events underlying these changes, we analyzed expression profiles of approximately 23,000 gene transcripts in bovine mammary tissue about day 5 before parturition and day 10 after parturition. At the cutoff criteria of the signed fold change ≥2 or ≤-2 and false discovery rate (FDR) ≤0.1, a total of 389 transcripts (1.6%) were significantly differentially expressed at the two stages. Of these transcripts with significant changes, 105 were up-regulated while 284 were down-regulated. Gene ontology analysis showed that the main up-regulated genes were those associated with transport activity (amino acid, glucose, and ion transporters), lipid and carbohydrate metabolism (lipoprotein lipase, acetyl-Coenzyme A synthetases, 6-phosphofructo-2kinase, etc.), and cell signaling factors (protein p8, Rab18, etc.). The main down-regulated genes were associated with cell cycle and proliferation (cyclins, cell division cycle associated proteins, etc.), DNA replication and chromosome organization (centromere proteins, minichromosome maintenance proteins, histone, etc.), microtubule-based processes (microtubule associated protein tau, kinesin, tubulins, etc.), and protein and RNA degradation (proteasome, proteasome activator, RNA binding motif protein, etc.). The increased expression of glucose transporter GLUT1 mRNA during lactation was verified by quantitative reverse transcription/polymerase chain reactin (PCR) (P < 0.05). GLUT1 protein also increased twofold during lactation (P < 0.05). Furthermore, GLUT1 protein was primarily localized in mammary ductal epithelia and blood vessel endothelia before parturition, but was predominantly localized in the basolateral and apical membranes of mammary alveolar epithelial cells during lactation. Our microarray data provide insight into the molecular events in the mammary gland at the onset of lactation, indicating the up-regulation of genes involved in milk synthesis concomitant with the inhibition of those related to cell proliferation.

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/ near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.

Localized 1H nuclear magnetic resonance spectroscopy has been applied to determine human brain gray matter and white matter glucose transport kinetics by measuring the steady-state glucose concentration under normoglycemia and two levels of hyperglycemia. Nuclear magnetic resonance spectroscopic measurements were simultaneously performed on three 12-mL volumes, containing predominantly gray or white matter. The exact volume compositions were determined from quantitative T1 relaxation magnetic resonance images. The absolute brain glucose concentration as a function of the plasma glucose level was fitted with two kinetic transport models, based on standard (irreversible) or reversible Michaelis-Menten kinetics. The steady-state brain glucose levels were similar for cerebral gray and white matter, although the white matter levels were consistently 15% to 20% higher. The ratio of the maximum glucose transport rate, Vmax, to the cerebral metabolic utilization rate of glucose, CMRGlc, was...

A c c e p t e d M a n u s c r i p t Trehalose activates hepatic fasting and thermogenesis Abbreviations: AAV8 -adeno-associated virus serotype 8; ASO -antisense oligonucleotide; ATG16L1 -autophagy related 16-like 1 (S. cerevisiae); BECN1 -beclin 1, autophagy related; ECAR -extracellular acidification rate, EPG5 -ectopic P-granules autophagy protein 5 homolog (C. elegans); eWAT -epididymal white adipose tissue; FCCP -carbonyl cyanide-ptrifluoromethoxyphenylhydrazone; FGF21 -fibroblast growth factor 21; FGFR -fibroblast growth factor receptor; iBAT -interscapular brown adipose tissue; LEP -leptin; MTORmechanistic target of rapamycin kinase; OCR -oxygen consumption rate; PPARGC1Aperoxisome proliferative activated receptor, gamma, coactivator 1 alpha; PRKAA1/AMPKprotein kinase, AMP-activated, alpha 1 catalytic subunit; SLC2A -solute carrier family 2 (facilitated glucose transporter); TFEB -transcription factor EB; UCP1 -uncoupling protein 1

Various biochemical and genomic mechanisms are considered to be hallmark of metabolic remodeling in the stressed heart, including in the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5&#39; flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affin...

catalytic site. The PsDef5.1-AmyIt complex has the largest interfacial contact area, 2328 Å 2 , in comparison with two other defensins, which correlates well with the inhibitory activity of defensins in this study. Conclusion. Thus, we have identified α-amylase activity in I. acuminatus and demonstrated the ability of Scots pine defensins to inhibit it, sugges ting that they play a role in pine defenses against this pest.

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyi glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric Immediately after the ion-exchange purification: the Mr of the Glut 1 polypeptide was estimated to be 49000 + 6000 by TSKgel G3000SW chromatography monitored by low.angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut !, which was measured to be 1.13 + 0.03 ml mg -t cm-t at 280 nm, referring to the polypeptide concentration. The M r value is consistent with the cDNA-deduced M r 54117 of the very similar HepG2 glucose transporter polypeptide. At 2°C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane llpids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluenl lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyi glncoside and PC was nearly as effective as PS.

Tumor necrosis factor ␣ (TNF␣) is a cytokine secreted by macrophages and adipocytes that contributes to the low grade inflammation and insulin resistance observed in obesity. TNF␣ signaling decreases peroxisome proliferator-activated receptor ␥ and glucose transporter isoform 4 (GLUT4) expression in adipocytes, impairing insulin action, and this is mediated in part by the yeast Ste20 protein kinase ortholog Map4k4. Here we show that Map4k4 expression is selectively up-regulated by TNF␣, whereas the expression of the protein kinases JNK1/2, ERK1/2, p38 stress-activated protein kinase, and mitogen-activated protein kinase kinases 4/7 shows little or no response. Furthermore, the cytokines interleukin 1␤ (IL-1␤) and IL-6 as well as lipopolysaccharide fail to increase Map4k4 mRNA levels in cultured adipocytes under conditions where TNF␣ elicits a 3-fold effect. Using agonistic and antagonistic antibodies and small interfering RNA (siRNA) against TNF␣ receptor 1 (TNFR1) and TNF␣ receptor 2 (TNFR2), we show that TNFR1, but not TNFR2, mediates the increase in Map4k4 expression. TNFR1, but not TNFR2, also mediates a potent effect of TNF␣ on the phosphorylation of JNK1/2 and p38 stress-activated protein kinase and their downstream transcription factor substrates c-Jun and activating transcription factor 2 (ATF2). siRNA-based depletion of c-Jun and ATF2 attenuated TNF␣ action on Map4k4 mRNA expression. Consistent with this concept, the phosphorylation of ATF2 along with the expression and phosphorylation of c-Jun by TNF␣ signaling was more robust and prolonged compared with that of IL-1␤, which failed to modulate Map4k4. These data reveal that TNF␣ selectively stimulates the expression of a key component of its own signaling pathway, Map4k4, through a TNFR1-dependent mechanism that targets the transcription factors c-Jun and ATF2.

It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifity week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor β (IRβ) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity.

It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifity week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor β (IRβ) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity.

The Na+,K+-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na+,K+-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na+,K+-ATPase binding partners revealed a direct association between the Na+,K+-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na+,K+-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na+,K+-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na+,K+-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell sur...

Insulin maintains glucose homeostasis by stimulating glucose uptake from extracellular environment to adipose and muscle tissue through glucose transporter (GLUT4). Insulin resistance plays a significant role in pathologies associated with type2 diabetes. It has been previously shown that hyperinsulinemia can lead to insulin resistance. In these studies very high levels of insulin was used to achieve insulin resistance. We hypothesized that one of the causes of type 2 diabetes could be insulin synthesis in the absence of glucose stimulation. We used CHO cell line, stably expressing Myc-GLUT4-GFP along with human insulin receptor to study the effect of hyperinsulinemia in the presence of low glucose (6.5 mM) or high glucose (20 mM). The insulin responsiveness of these cells was assessed by FRAP, FACS and subcellular fractionation. The results suggest that exposure of cells to insulin in low glucose conditions made these cells insulin resistant within 10 passages, while the same level of insulin in the presence of high glucose did not result in insulin resistance. These results clearly suggest that hyperinsulinemia combined with hypoglycaemia may lead to insulin resistance and may be one of the causes for the typ2 diabetes.

In obesity and type 2 diabetes, expression of the GLUT4 glucose transporter is decreased selectively in adipocytes. Adipose-specific Glut4 (also known as Slc2a4) knockout (adipose-Glut4 2 /2 ) mice show insulin resistance secondarily in muscle and liver. Here we show, using DNA arrays, that expression of retinol binding protein-4 (RBP4) is elevated in adipose tissue of adipose-Glut4 2 /2 mice. We show that serum RBP4 levels are elevated in insulin-resistant mice and humans with obesity and type 2 diabetes. RBP4 levels are normalized by rosiglitazone, an insulin-sensitizing drug. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance. Conversely, genetic deletion of Rbp4 enhances insulin sensitivity. Fenretinide, a synthetic retinoid that increases urinary excretion of RBP4, normalizes serum RBP4 levels and improves insulin resistance and glucose intolerance in mice with obesity induced by a high-fat diet. Increasing serum RBP4 induces hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and impairs insulin signalling in muscle. Thus, RBP4 is an adipocytederived 'signal' that may contribute to the pathogenesis of type 2 diabetes. Lowering RBP4 could be a new strategy for treating type 2 diabetes.

O prejuízo no transporte de glicose estimulada por insulina no músculo constitui um defeito crucial para o estabelecimento da intolerância à glicose e do diabetes tipo 2. Por outro lado, é notório o conhecimento de que tanto o exercício aeróbio agudo quanto o crônico podem ter efeitos benéficos na ação da insulina em estados de resistência à insulina. No entanto, pouco se sabe sobre os efeitos moleculares pós-exercício sobre a sinalização da insulina no músculo esquelético. Assim, esta revisão apresenta novos entendimentos sobre os mecanismos por meio dos quais o exercício agudo restaura a sensibilidade à insulina, com destaque ao importante papel que proteínas inflamatórias e a S-nitrosação possuem sobre a regulação de proteínas da via de sinalização da insulina no músculo esquelético.

O prejuízo no transporte de glicose estimulada por insulina no músculo constitui um defeito crucial para o estabelecimento da intolerância à glicose e do diabetes tipo 2. Por outro lado, é notório o conhecimento de que tanto o exercício aeróbio agudo quanto o crônico podem ter efeitos benéficos na ação da insulina em estados de resistência à insulina. No entanto, pouco se sabe sobre os efeitos moleculares pós-exercício sobre a sinalização da insulina no músculo esquelético. Assim, esta revisãoapresenta novos entendimentos sobre os mecanismos por meio dos quais o exercício agudo restaura a sensibilidade à insulina, com destaque ao importante papel que proteínas inflamatórias e a S-nitrosação possuem sobre a regulação de proteínas da via de sinalização da insulina no músculo esquelético.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with more than 50 million people suffer from it. Unfortunately, none of the currently available drugs is able to improve cognitive impairment in AD patients. Urolithin A (UA) is a metabolite obtained from ellagic acid and ellagitannin through the intestinal flora, and it has antioxidant and anti-inflammatory properties. Previous reports found that UA had neuroprotective effects in an AD animal model, but the detailed mechanism still needs to be elucidated. In this study, we performed kinase-profiling to show that dualspecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is the main target of UA. Studies showed that the level of DYRK1A in AD patients' brains was higher than that of healthy people, and it was closely related to the occurrence and progression of AD. Our results revealed that UA significantly reduced the activity of DYRK1A, which led to dephosphorylation of tau and further stabilized microtubule polymerization. UA also provided neuroprotective effects by inhibiting the production of inflammatory cytokines caused by Ab. We further showed that UA significantly improved memory impairment in an AD-like mouse model. In summary, our results indicate that UA is a DYRK1A inhibitor that may provide therapeutic advantages for AD patients.

Age and age-dependent inflammation are two main risk factors for cardiovascular diseases. Aging can also affect clock gene-related impairments such as chronodisruption and has been linked to a decline in melatonin synthesis and aggravation of the NF-κB/NLRP3 innate immune response known as inflammaging. The molecular drivers of these mechanisms remain unknown. This study investigated the impact of aging and NLRP3 expression on the cardiac circadian system, and the actions of melatonin as a potential therapy to restore daily rhythms by mitigating inflammaging. We analyzed the circadian expression and rhythmicity of clock genes in heart tissue of wild-type and NLRP3-knockout mice at 3, 12, and 24 months of age, with and without melatonin treatment. Our results support that aging, NLRP3 inflammasome, and melatonin affected the cardiac clock genes expression, except for Rev-erbα, which was not influenced by genotype. Aging caused small phase changes in Clock, loss of rhythmicity in Per2...

The present study was conducted to correlate the developmental competence of large follicle (LF, &gt;6 mm) and small follicle (SF, &lt;6 mm) derived buffalo oocytes in terms of morphological assessment and molecular characteristics. A pool of culture grade cumulus oocytes complexes (COCs) from both the groups separately were subjected to brilliant cresyl blue (BCB) staining. LF derived oocytes had significantly higher percentage of BCB+ve oocytes and were larger in size and maturation status was also visibly superior. Cleavage and blastocysts rate was also significantly higher in LF group than SF group. Relative mRNA expression analysis revealed that expression of GDF9 was significantly higher in LF group than SF group while expression of BMP15 increased insignificantly. Expression of GREM1, EGFR and PTGS2 was significantly higher in LF group than SF group while expression of HAS2 was insignificantly higher. Similarly, expression of GLUT1, OCT4 and IFN- in in vitro produced blast...

The aim of the study is to evaluate the pattern and level of expression of glucose transporter-1 (GLUT-1) in rectal carcinoma in relation to outcome as a potential surrogate marker of tumour hypoxia. Formalin-fixed tumour sections from 43 patients with rectal carcinoma, who had undergone radical resection with curative intent, were immunohistochemically stained for GLUT-1. A mean of three sections per tumour (range 1 -12) were examined. Each section was semiquantitatively scored; 0, no staining; 1, o10%; 2, 10 -50%; 3, 450% and a score given for the whole section, the superficial (luminal) and deep (mural) part of the tumour. Staining was seen in 70% of tumours. Increased staining was noted adjacent to necrosis and ulceration. A diffuse and patchy pattern of staining, with and without colocalisation to necrosis was seen. Patients with high GLUT-1-expressing tumours (score 3 vs 0 -2) had a significantly poorer overall survival (P ¼ 0.041), which was associated with poorer metastasis-free survival with no difference in local control. No significant correlation was seen with other prognostic factors. There was a strong correlation between the score for the superficial and deep parts of the tumour (r ¼ 0.81), but a significant relationship with outcome was only found in the deep part (P ¼ 0.003 vs P ¼ 0.46). In conclusion, increased GLUT-1 expression in rectal tumours was an adverse prognostic factor and is worth further evaluation as a predictive marker of response to therapy.

Neuronal activity is fueled by glucose metabolism, a phenomenon exploited in basic research and clinical diagnosis using fluorodeoxyglucose positron emission tomography (FDG-PET). According to the current view, glucose transport into the brain is not rate-limiting; thus, it cannot exert control over metabolism. This article challenges such a view by showing that basal transport hovers near its maximum, making metabolic activation unable to increase flux on its own. In the light of recent evidence on the identity of the cell type that preferentially breaks down glucose, we suggest that FDG-PET reports the synergistic activation of glucose transport and metabolism in astrocytes, rather than in neurons.

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

Glutamate triggers an acute stimulation of the glucose transporter GLUT1 in cultured astrocytes, a phenomenon thought to facilitate energy delivery to active areas in the brain. Here we have explored the cell signaling mechanisms involved in this response. Half-stimulation of GLUT1 occurred at low micromolar glutamate, thus within the physiological range estimated in brain interstitium. The effect was mimicked by D-aspartate and inhibited by Lthreo-beta-benzyloxyaspartate or Na 1 replacement with NMDG 1 , showing the participation of the Na 1 -glutamate co-transporter. AMPA and the mGLURI agonist DHPG had no effect. The stimulation of GLUT1 was fully inhibited by ouabain, but independent activation of the Na 1 /K 1 ATPase pump with gramicidin did not affect glucose transport. Simultaneous with the Na 1 rise, glutamate and D-aspartate triggered a Ca 21 signal, whose inhibition with BAPTA prevented the stimulation of GLUT1. However, an isolated Ca 21 signal, triggered with endothelin 1, ATP or DHPG, did not affect glucose transport. The stimulation of GLUT1 could finally be mimicked by simultaneous induction of Na 1 and Ca 21 signals. The requirement for both cations in the stimulation of the astrocytic glucose transporter, may help to explain how glucose metabolism in the brain is strongly activated by glutamate, but not by GABA or by inter-astrocytic signaling. V

The uptake of glucose into mammalian cells, catalysed by members of the GLUT family of glucose transporters, is regulated by a variety of hormones, growth factors and other agents. In adipocytes, skeletal muscle and heart the principal regulator is the hormone insulin, which rapidly stimulates glucose uptake by bringing about the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular compartment to the cell surface. Recent studies have implicated the C-terminal hydrophilic region of this protein as being primarily responsible for its insulin-regulated trafficking. In an attempt to identify the protein machinery involved in this trafficking, we have used glutathione S-transferase fusion proteins bearing hydrophilic domains of various GLUT transporters in affinity purification experiments on detergent-solubilized extracts of 3T3-L1 adipocyte intracellular membranes. The C-terminal region of GLUT4 was found specifically to bind a number of polypeptides i...

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role of PI-3-kinase and small GTP-binding proteins has been proposed. In previous studies we, among many others, excluded a role for the ras/MAP kinase pathway in insulin-mediated glucose transport. In this study we examined a possible role of the small GTP-binding protein rho in this process. Pretreatment of 3T3-L1 adipocytes with botulinum C3 exoenzyme (C3), which is known to ADP-ribosylate and inactivate rho, potently stimulated glucose uptake to a level similar to insulin. Interestingly, glycogen synthesis was not affected by C3 treatment. Insulin stimulates glucose uptake by triggering the translocation of GLUT4, the insulin-sensitive glucose transporter isotype, from an intracellular compartment to the plasma membrane. Similarly, C3-induced glucose uptake was paralleled by GLUT4 translocation. These data point to an important and novel role of the target of C3 (likely rho) in the regulation of GLUT4-mediated glucose transport. Our data suggest that insulin might stimulate glucose uptake through inactivation of rho.

A major portion of insulin-mediated glucose uptake occurs via the translocation of GLUT 4 glucose transporter proteins from an intracellular depot to the plasma membrane. We have exam- ined gene expression for the GLUT 4 transporter isoform in subcutaneous adipocytes, a classic insulin target cell, to better understand molecular mechanisms causing insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM) and obesity. In subgroups of lean (body mass index [BMII = 24±1) and obese (BMI = 32±2) controls and in obese NIDDM (BMI = 35±2) patients, the number of GLUT 4 glucose transporters was measured in total postnuclear and subcellular membrane fractions using specific antibodies on Western blots. Relative to lean controls, the cellular content of GLUT 4 was decreased 40% in obesity and 85% in NIDDM in total cellular mem- branes. In obesity, cellular depletion of GLUT 4 primarily in- volved low density microsomes (LDM), leaving fewer transport- ers available for insulin-mediated recruitment to the plasma membrane (PM). In NIDDM, loss of GLUT 4 was profound in all membrane subfractions, PM, LDM, as well as high density microsomes. These observations corresponded with decre- ments in maximally stimulated glucose transport rates in intact cells. To assess mechanisms responsible for depletion of GLUT 4, we quantitated levels of mRNA specifically hybridizing with human GLUT 4 cDNA on Northern blots. In obesity, GLUT 4 mRNA was decreased 36% compared with lean con- trols, and the level was well correlated (r = +0.77) with the cellular content of GLUT 4 protein over a wide spectrum of body weight. GLUT 4 mRNA in adipocytes from NIDDM pa- tients was profoundly reduced by 86% compared with lean con- trols and by 78% relative to their weight-matched nondiabetic counterparts (whether expressed per RNA, per cell, or for the amount of CHO-B mRNA). Interestingly, GLUT 4 mRNA lev- els in patients with impaired glucose tolerance (BMI = 34±4) were decreased to the same level as in overt NIDDM. We conclude that, in obesity, insulin resistance in adipo- cytes is due to depletion of GLUT 4 glucose transporters, and

Background/Aim: GLUT1 is an erythrocyte-type glucose transporter protein typically expressed in cutaneous proliferating hemangioma. Immunostaining for GLUT1 is becoming valuable for predicting the outcome of vascular skin tumors. Liver vascular tumors (LVTs) are a serious challenge for pediatric surgeons because of their often severe and sometimes unpredictable clinical course. To improve therapeutic strategies, we designed this study in which we tested in pathology specimens of LVT the hypothesis that GLUT1 expression could be useful to understand and classify LVT. Material and Methods: In the last 10 years, we treated 20 children with LVT. Pathology specimens from biopsy, excision, or autopsy were available in 11 of them. The paraffin sections were immunostained for GLUT1 and also for Ki-67 to assess the proportion of proliferating cells. Patients were divided into 2 groups: GLUT1-positive (n = 4) and GLUT1-negative (n = 7) that were compared for age at diagnosis, survival, and proportion of proliferating cells. Nonparametric and v 2 tests were used for statistical analysis as appropriate. Results: Mean age at diagnosis was higher in GLUT1-positive group, although not statistically significant in comparison with GLUT1-negative (308 F 515 vs 70 F 51 days, respectively). Three of 4 children in GLUT1-positive group died versus 1 of 7 in the GLUT1-negative group (not significant). All GLUT1-positive tumors were multicentric hemangiomata without central necrosis and only 1 with large vessels. Among GLUT1-negative tumors, 5 were solitary masses and 2 were multicentric (the value of the last 2 specimens was uncertain), 2 had central necrosis, and 2 had large vessels. Proliferation index was 18% F 1.42% and 1.42% F 0.97%, respectively, in each group ( P b .05). Conclusions: GLUT1-positive tumors have significantly higher proliferation rates than negative ones. Mortality tended to be higher in children with GLUT1-positive tumors. Positive GLUT1 immunostaining is likely specific for proliferating hemangioma, and it predicts the typical course of proliferation followed by involution.

In many patients with human immunodeficiency virus (HIV) treated with HIV protease inhibitors, a complication develops that resembles abdominal obesity syndrome, with insulin resistance and glucose intolerance that, in some cases, progresses to diabetes. In this study, we tested the hypothesis that indinavir, an HIV-protease inhibitor, directly induces insulin resistance of glucose transport in skeletal muscle. Rat epitrochlearis muscles were incubated with a maximally effective insulin concentration (12 nmol/l) and 0, 1, 5, 20, or 40 μmol/l indinavir for 4 h. In control muscles, insulin increased 3-O-[3H]methyl-d-glucose (3MG) transport from 0.15 ± 0.03 to 1.10 ± 0.05 μmol · ml−1 · 10 min−1. Incubation of muscles with 5 μmol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 40%, whereas 20 μmol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 58%. Indinavir induced a similar reduction in maximally insulin-stimulated 3MG transport in the ...

D-It4ClGlucose self exchange and unidirectional efflux from human red blood cells were studied at 20°C (pH 7.2) by means of the Mililpore-Swinnex filtering technique whose time resolution is > I s and the continuous flow-tube method with a time resolution of > 2 ms. The unidirectional efflux data were analyzed using both the method of initial rates and the integrated rate equation. Simple Michaelis-Menten kinetics apply to the results obtained ,ruder both experimental conditions. In self-exchange mode, the half-saturation constant, Kl/2,. ~, was 10 (S.E. :.1-I) raM. In unidirectional efflux mode Ki/z, e was 6.6 (S.E.-I-0.5) mM (initial rates) or by the method of integrated rates 7.7 raM, with a range of 2.7-12.1 raM, Kt/2m increasing with an increased initial intracellular glucose concentration. Our results of KI/2+ ~ oppose previous published values of 32 mM for self exchange (Eilam and Stein (1972) Biochim. Biophys. Acta 266. 161-173) and 25 mM for unidirectional efllux (Kariish et at. (1972) Biochim. Biophys. Acta 255, 126-132) that have be,~.r, used extensively in kinetic considerations of glucose transport models. Under sell-exchange conditions J+~ was 1.8.10 -t° molcm-2s -1, and in unidirectional elflux mode J~'+~ was 8.3.10 -n molcm-Zs -~ (initial rates) and 8.6.10 -u tool cm-2s-i (integrated rates). We suggest that the previous high values of 1~" and in particular Kt/2 are due to the use of methods with insufficient time resolution. Our results indicate that the transport system is less asymmetric than was generally accepted, and that complicated transport models developed to account for the great difference between the determined Kt/z and jma~ values are redundant.

Non-insulin-dependent diabetes mellitus is due to in large part to insulin resistance, a state where the target cells are no longer responding to ordinary levels of circulating insulin. A drug that promotes the initiation of insulin receptor (IR) signaling by enhancing IR phosphorylation should be useful in the treatment of Type 2 diabetes. To investigate a compound that is able to phosphorylate IR, a proanthocyanidin, cinnamtannin B1, has been isolated from the stem bark of Cinnamomum zeylanicum Blume (Lauraceae). The structure of the compound was established by spectroscopic methods. Cinnamtannin B1 (0.11 mM) activates the phosphorylation of insulin receptor β-subunit on 3T3-L1 adipocytes. Like insulin, cinnamtannin B1-stimulated phosphorylation of insulin receptor was inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) and cytochalasin B, an inhibitor of the glucose transporter (GLUT4); otherwise the phosphorylation was enhanced by vanadate, a tyrosine phosphatase inhibitor. These results suggest that the activity of cinnamtannin B1 is specially mediated by phosphorylation of IR through activation of the PI3K.

We examined the effect of hypoxic ischemia and hypoxia vs. normoxia on postnatal murine brain substrate transporter concentrations and function. We detected a transient increase in the neuronal brain glucose transporter isoform (GLUT-3) in response to hypoxic ischemia after 4 h of reoxygenation. This increase was associated with no change in GLUT-1 (blood-brain barrier/glial isoform), monocarboxylate transporter isoforms 1 and 2, synapsin I (neuronal marker), or Bax (proapoptotic protein) but with a modest increase in Bcl-2 (antiapoptotic mitochondrial protein) protein concentrations. At 24 h of reoxygenation, the increase in GLUT-3 disappeared but was associated with a decline in Bcl-2 protein concentrations and the Bcl2:Bax ratio, an increase in caspase-3 enzyme activity (apoptotic effector enzyme), and extensive DNA fragmentation, which persisted later in time (48 h) only in the hippocampus. Hypoxia alone in the absence of ischemia was associated with a transient but modest incre...

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate-(MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2-(by 60%) and 4month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSGmice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by f 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.

A distinct bioenergetic impairment of heart mitochondrial subpopulations in diabetic cardiomyopathy is associated with obesity; however, many type 2 diabetic (T2DM) patients with high-risk for cardiovascular disease are not obese. In the absence of obesity, it is unclear whether bioenergetic function in the subpopulations of mitochondria is affected in heart with T2DM. To address this issue, a rat model of non-obese T2DM was used to study heart mitochondrial energy metabolism, measuring bioenergetics and enzyme activities of the electron transport chain (ETC). Oxidative phosphorylation in the presence of substrates for ETC and ETC activities in both populations of heart mitochondria in T2DM rats were unchanged. Despite the preservation of mitochondrial function, aconitase activity in T2DM heart was reduced, suggesting oxidative stress in mitochondria. Our study indicate that metabolic function of heart mitochondria is unchanged in the face of oxidative stress and point to a critical...

Aims/hypothesis The role of dietary carbohydrate in the pathogenesis of type 2 diabetes is still a subject of controversial debate. Here we analysed the effects of diets with and without carbohydrate on obesity, insulin resistance and development of beta cell failure in the obese, diabetesprone New Zealand Obese (NZO) mouse. Materials and methods NZO mice were kept on a standard diet (4% [w/w] fat, 51% carbohydrate, 19% protein), a high-fat diet (15, 47 and 17%, respectively) and a carbohydrate-free diet in which carbohydrate was ex-changed for fat (68 and 20%, respectively). Body composition and blood glucose were measured over a period of 22 weeks. Glucose tolerance tests and euglycaemichyperinsulinaemic clamps were performed to analyse insulin sensitivity. Islet morphology was assessed by immunohistochemistry. Results Mice on carbohydrate-containing standard or high-fat diets developed severe diabetes (blood glucose >16.6 mmol/l, glucosuria) due to selective destruction of pancreatic beta cells associated with severe loss of immunoreactivity of insulin, glucose transporter 2 (GLUT2) and musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). In contrast, mice on the carbohydrate-free diet remained normoglycaemic and exhibited hyperplastic islets in spite of a morbid obesity associated with severe insulin resistance and a massive accumulation of macrophages in adipose tissue. Conclusions/interpretation These data indicate that the combination of obesity, insulin resistance and the inflammatory response of adipose tissue are insufficient to cause beta cell destruction in the absence of dietary carbohydrate.

Aims/hypothesis: We aimed to characterise the development of autoregulation of glucose transport in vascular endothelial cells and its relationship to 12-lipoxygenase (12-LO) expression. Methods: Bovine aortic endothelial cells were exposed to 5.5 and 23.0 mmol/l glucose for up to 48 h. The rates of glucose transport, GLUT-1 and 12-LO expression and of 12-hydroxyeicosatetraenoic acid (12-HETE) production were determined. Results: We showed high glucose-dependent downregulation of glucose transport and transporter in vascular endothelial cells within 36-48 h. A similar time-dependent increase in the expression of 12-LO and the generation of its product 12-HETE was also observed. This downregulatory process was prevented when lipoxygenase activity was inhibited. Conclusions/interpretation: Vascular endothelial cells, which were previously thought to be "glucose-blind", do in fact downregulate GLUT-1 expression and the rate of glucose transport in response to extended exposure to high glucose concentrations. This slow development of glucose-induced downregulation in vascular endothelial cells is related to the slower basal rate of glucose transport in these cells and the slow induction of 12-LO. These data are interesting in view of current hypotheses that attribute vascular endothelial cell dysfunction in diabetes to the lack of a glucose-induced autoregulatory response.

The water-soluble and cell permeable nitroxide derivative 4-hydroxy tempol (TPL) has been shown to reduce or ameliorate oxidative stress-induced dysfunction and damage in vascular endothelial cells. We studied the effects of TPL on glucose transport and metabolism in bovine aortic endothelial (VEC) and smooth muscle cells (VSMC) under normal and high glucose conditions. Normally, these cells operate an autoregulatory protective mechanism that limits the rate of glucose transport under hyperglycemic conditions by decreasing the cell content of their typical glucose transporter GLUT-1 mRNA and protein as well as its plasma membrane abundance. TPL augmented the rate of glucose transport both under normo-and hyperglycemic conditions by increasing GLUT-1 mRNA and protein content and its plasma membrane abundance in both types of cells, leading to an increased flux of glucose into the cells. These effects were found related to ROS-generating and oxidant activities of TPL and to a decreased rate of mitochondrial ATP production under both normo-and hyperglycemic conditions. Since impaired mitochondrial functions, and in particular decreased rate of ATP production, augment the expression of GLUT-1 protein and glucose transport and metabolism, we suggest that the stimulatory effects of TPL in vascular cells results from its unfavorable interactions in the mitochondrion. It is therefore suggested that effects of TPL in cells of cardiovascular system be evaluated in parallel to its adverse effects on glucose and energy metabolism.

The water-soluble and cell permeable nitroxide derivative 4-hydroxy tempol (TPL) has been shown to reduce or ameliorate oxidative stress-induced dysfunction and damage in vascular endothelial cells. We studied the effects of TPL on glucose transport and metabolism in bovine aortic endothelial (VEC) and smooth muscle cells (VSMC) under normal and high glucose conditions. Normally, these cells operate an autoregulatory protective mechanism that limits the rate of glucose transport under hyperglycemic conditions by decreasing the cell content of their typical glucose transporter GLUT-1 mRNA and protein as well as its plasma membrane abundance. TPL augmented the rate of glucose transport both under normo-and hyperglycemic conditions by increasing GLUT-1 mRNA and protein content and its plasma membrane abundance in both types of cells, leading to an increased flux of glucose into the cells. These effects were found related to ROS-generating and oxidant activities of TPL and to a decreased rate of mitochondrial ATP production under both normo-and hyperglycemic conditions. Since impaired mitochondrial functions, and in particular decreased rate of ATP production, augment the expression of GLUT-1 protein and glucose transport and metabolism, we suggest that the stimulatory effects of TPL in vascular cells results from its unfavorable interactions in the mitochondrion. It is therefore suggested that effects of TPL in cells of cardiovascular system be evaluated in parallel to its adverse effects on glucose and energy metabolism.

Lower brain glucose metabolism is present before the onset of clinically-measurable cognitive decline in two groups of people at risk of Alzheimer's disease (AD) -carriers of apoE4, and in those with a maternal family history of AD. Supported by emerging evidence from in vitro and animal studies, these reports suggest that brain hypometabolism may precede and contribute to the neuropathological cascade leading cognitive decline in AD. The reason for brain hypometabolism is unclear but may include defects in glucose transport at the blood-brain barrier, glycolysis, and/ or mitochondrial function. Methodological issues presently preclude knowing with certainty whether or not aging in the absence of cognitive impairment is necessarily associated with lower brain glucose metabolism. Nevertheless, aging appears to increase the risk of deteriorating systemic control of glucose utilization which, in turn, may increase the risk of declining brain glucose uptake, at least in some regions. A contributing role of deteriorating glucose availability to or metabolism by the brain in AD does not exclude the opposite effect, i.e. that neurodegenerative processes in AD further decrease brain glucose metabolism because of reduced synaptic functionality and, hence, reduced energy needs, thereby completing a vicious cycle. Strategies to reduce the risk of AD by breaking this cycle should aim to -(i) improve insulin sensitivity by improving systemic glucose utilization, or (ii) bypass deteriorating brain glucose metabolism using approaches that safely induce mild, sustainable ketonemia.

Chronic hyperinsulinemia is both a marker and a cause for insulin resistance. This study analyzes the effect of long-term exposure to high insulin levels on the insulin-signaling pathway and glucose transport in cultured human myoblasts. Human myoblasts were grown in the presence of low (107 pmol/L, SkMC-L) or high (1430 pmol/L, SkMC-H) insulin concentrations for 3 weeks. Glucose transport, insulin receptor (IR), and IR substrate 1 (IRS1) phosphorylation, phosphatidylinositol 3V-kinase (PI3K) activity, as well as Akt-Ser473 phosphorylation have been investigated at the end of the incubation period and after a further short-term insulin stimulation. At the end of the incubation period, IR, IRS1, p85/PI3K, Akt, and GLUT4 protein expression levels were similar in both culture conditions. Basal glucose transport was similar in SkMC-L and SkMC-H, but after short-term insulin stimulation significantly increased ( P b .01) only in SkMC-L. IR binding was down-regulated in SkMC-H ( P b .01), but IR and IRS1 tyrosine phosphorylation and PI3K activity were significantly higher ( P b .01) in SkMC-H than SkMC-L. Despite increased PI3K activation, Akt-Ser473 phosphorylation was similar in SkMC-L and SkMC-H. After a short-term insulin stimulation (10 nmol/L insulin for 10 minutes), IR and IRS1 tyrosine phosphorylation, PI3K activation, and Akt-Ser473 phosphorylation significantly increased ( P b .01 and P b .05 for Akt) in SkMC-L but not in SkMC-H. Serine phosphorylation of IRS1 was similar in SkMC-L and SkMC-H. Moreover, in the SkMC-H, insulin stimulation was associated with the inhibition of IRS1 tyrosine dephosphorylation ( P b .05). In summary, continuous exposure of cultured myoblasts to high insulin levels induces a persistent upregulation of IR, IRS1, and PI3K activity associated with the demodulation of insulin signaling. Moreover, the impairment of the insulinsignaling steps between PI3K and Akt is concomitant with the desensitization of glucose transport. These alterations may contribute to the derangement insulin-signaling pathway states of hyperinsulinemia such as obesity and type 2 diabetes.

There is evidence that intracellular insulin may carry out some insulin mediated actions, including glucose transport. As intracellular insulin has never been quantitatively assessed in human cells, we evaluated its concentrations in monocytes from normal subjects (n= 7) and obese patients without (n= 9) and with Type 2 diabetes mellitus (n =10). After the incubation of cells with labeled insulin for 60 min at 37°C, intracellular intact insulin concentrations were measured by HPLC and expressed as pmol . 10 -6 . Insulin concentrations were higher (ANOVA P B 0.01) within cells from obese (115.49 26.4 pmol • 10 -6 /2 • 10 5 cells) and obese diabetic patients (93.2936.3 pmol•10 -6 / 2 • 10 5 cells) compared with normal cells (28.5 9 13.1 pmol • 10 -6 /2 • 10 5 cells). Moreover, after insulin was removed from the incubation medium the decrease of intracellular insulin was significantly lower (PB 0.01) in cells from both obese and obese diabetic patients than in normal subjects. Intracellular undissociated insulin-insulin receptor complexes on average, increased 2-fold (PB0.01) in cells from insulin resistant patients compared with normal cells. Finally, in downregulated cells from obese and obese diabetic patients, the recycling of the internalized insulin receptor was completely disrupted. In conclusion, monocytes from obese patients with and without Type 2 diabetes mellitus, present increased intracellular insulin concentrations and these conditions are associated with a significant impairment of insulin receptor processing. Increased intracellular insulin concentration in cells from these patients may be necessary in order to overcome insulin resistance.

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates‐1 and ‐2 (IRS‐1 and IRS‐2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS‐1 and IRS‐2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate–polyacrylamide gel electrophoresis mobility. IRS‐1/IRS‐2‐associated p85α and phosphatidylinositol (PI) 3‐kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin‐induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin‐induced protein kinase C (PKC) ζ activity. However, activation of Akt/PKB or p70 S6 kinase (p70s6k) by insulin remained unaltered. Our results suggest tha...

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-ras lys12 -transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-␣p85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.

cancer, which may serve as a selective advantage and facilitate the metastatic potential of these cells. The relationship of GLUT isoform expression and breast cancer cell invasiveness has not been adequately addressed. Thus, the purpose of this study was to investigate whether an association exists between GLUT expression and human breast cancer cell invasiveness. Invasiveness of the human breast cancer lines MCF-7, MDA-MB-435 and MDA-MB-231 was measured using an in vitro assay and compared with cellular GLUT isoform expression, assessed by Western blot analysis and verified by immunohis-Key words: breast cancer', invasion; GLUT expression tochemistry in a poorly differentiated human ductal breast cancer. Cell surface GLUT-1 expression was associated with the invasive ability of MCF-7 (2.0 _+ 0.02%), MDA-MB-435 (6.4 _+ 0.4%), and MDA-MB-231 (19.3 _+ 2.0%). However, GLUT-2 and GLUT-5 were inversely associated with invasiveness; GLUT-3 expression was variable; and GLUT-4 was undetected. In a poorly differentiated human ductal breast cancer, in situ GLUT-1 staining was intense. GLUT-1 expression was associated with the in vitro invasive ability of human breast cancer cells which was validated in situ. If this relationship is found to exist in a larger number of human breast cancer tissues, it may be possible to develop diagnostic and therapeutic strategies based on targeted GLUT isoform expression. (Pathology Onco-

Glucose and fructose enter mammalian cells via facilitated diffusion, a process regulated by five glucose transporter isoforms (GLUT1^5) at the plasma membrane. The tissue-specific pattern of GLUT isoform expression likely reflects differing needs for glucose transport by various tissues. Myocytes must respond expeditiously to increased metabolic demand. A basal isoform, GLUT1, and the insulin-regulatable glucose transporter, GLUT4, have been demonstrated in human myocytes. GLUT3 has a high affinity for glucose, but its presence in human myocardium has not been clearly established. The purpose of this study was to determine whether GLUT3 protein is present in human cardiac myocytes. We examined rapidly frozen myocardial tissue from the explanted heart of seven patients undergoing cardiac transplantation, from the heart of a young, previously healthy male organ donor, from the heart of a 67-year-old woman without known cardiac disease who had a fatal stroke, and from the heart of six human fetuses. GLUT3 protein was detected by immunoblots and localized by light and electron microscopy immunohistochemistry. The presence of GLUT3 protein was verified in myocardial tissue by both immunoblots and immunohistochemistry. Light and electron microscopy confirmed that GLUT3 was in cardiac myocytes. GLUT3 was also demonstrated as a 48 kDa protein in fetal myocardium, which was present at 10 weeks, increased at 15 weeks, then decreased at 20 weeks of gestation. GLUT3 is present in human adult and fetal myocardium. Human myocardial GLUT3 regulation and its role in myocardial glucose uptake remain to be elucidated.

Homeostasis of blood glucose by insulin involves the rapid and large stimulation of glucose uptake by muscle and adipose tissue. This important action of the hormone is based on the translocation of the glucose transporter isoform GLUT4 from an intracellular pool to the cell surface . A major aspect of this process that is still to be defined is that of the downstream signalling from the insulin receptor to the GLUT4 compartment and how these signals trigger recruitment of the GLUT4-containing vesicles to the plasma membrane. To explain these processes, several laboratories have isolated the intracellular vesicles containing GLUT4 and characterized their protein composition [3±9]. In addition to GLUT4, three major companion proteins have been identified in GLUT4-containing vesicles including an insulin-reg- Diabetologia (2000) 43: 1518±1527