Effects of isoflavones on breast tissue and the thyroid hormone system in humans: a comprehensive safety evaluation.

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection.

Tbc1d1 deletion suppresses obesity in leptin-deficient mice.

BACKGROUND: Variants in the gene TBC1D1 have been previously associated with obesity-related traits in several species, including humans, mice, rabbits and chicken. While in humans variants in TBC1D1 were linked to obesity, disruption of the Tbc1d1 gene reduced body weight in mice. TBC1D1 has been identified as a regulator of insulin-dependent glucose transport in skeletal muscle, however, its role in energy homeostasis in the obese state remains unclear. The impact of TBC1D1 deficiency on energy homeostasis, glucose and lipid metabolism in an established mouse model of obesity was examined. METHODS: Obese leptin (ob/ob)- and Tbc1d1-double-deficient mice (D1KO-ob/ob) were generated by crossing obese B6.V.Lep(ob/ob)-mice with lean Tbc1d1-deficient mice on a C57BL/6J background. Male mice on either standard (SD) or high-fat diet (HFD) were analyzed for body weight, body composition, food intake, voluntary physical activity and energy expenditure by indirect calorimetry. Glucose and insulin tolerance as well as glucose transport and fatty acid oxidation in skeletal muscle were analyzed. RESULTS: In obese mice, Tbc1d1 deficiency resulted in reduced body weight on both SD and HFD. However, food intake was unchanged on SD or even increased in HFD-fed Tbc1d1-deficient mice without alterations in voluntary physical activity. Despite substantially reduced insulin-stimulated glucose transport and increased fatty acid oxidation in intact isolated skeletal muscle, obese Tbc1d1-deficient mice showed no gross changes in glycemia and glucose tolerance compared with obese controls. Indirect calorimetry revealed that obese Tbc1d1-deficient mice had a decreased respiratory quotient together with increased daily energy expenditure. CONCLUSIONS: In obese leptin-deficient mice, lack of TBC1D1 has no impact on feeding behavior or energy intake but results in increased energy expenditure, altered energy substrate preference with increased fatty acid oxidation and suppression of obesity. TBC1D1 may have an evolutionary conserved role in regulating energy homeostasis in vertebrates.

Estrogen deficiency aggravates insulin resistance and induces ß-cell loss and diabetes in female New Zealand obese mice.

In several rodent strains such as the New Zealand Obese (NZO) mouse, the incidence of obesity-associated diabetes mellitus is much higher in males than in females. In the present study, we investigated the effects of ovariectomy on glucose homeostasis in female NZO mice in order to elucidate the mechanism of their diabetes resistance. NZO females were ovariectomized at the age of 4 weeks, received a high-fat diet and body weight, body fat, glucose and insulin tolerance were investigated in comparison to sham-operated mice. In a second experiment, operated mice were fed a carbohydrate-free diet up to the age of 19 weeks before they received the high-fat diet. In comparison with a sham-operated control group, ovariectomized female NZO mice exhibited similar body weights, a reduced glucose tolerance, developed significantly higher blood glucose levels, lost insulin producing ß-cells, which finally resulted in a diabetes prevalence of 73% at the age of 16 weeks vs. 25% in controls. Similar to male NZO mice, ovariectomized females presented a more severe insulin resistance in the insulin tolerance test than sham-operated controls. Furthermore, the more severe insulin resistance in ovariectomized mice preceded the development of diabetes and pancreatic insulin depletion that was caused by a dietary regimen of carbohydrate restriction and subsequent re-exposure. In summary our data demonstrate that estrogen protects NZO females from ß-cell loss and obesity-associated diabetes mellitus. This effect is due to a reduced insulin resistance and possibly also to a reduced sensitivity of ß-cells to glucolipotoxic conditions.

Body iron stores and risk of type 2 diabetes: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study.

AIMS/HYPOTHESIS: The aim of this study was to prospectively examine the association between body iron stores and risk of type 2 diabetes. METHODS: We designed a case-cohort study among 27,548 individuals within the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study. During 7 years of follow-up, 849 incident cases of type 2 diabetes were identified. Of these, 607 remained for analyses after exclusion of participants with missing data or abnormal glucose levels at baseline. A sub-cohort of 2,500 individuals was randomly selected from the full cohort, comprising 1,969 individuals after applying the same exclusion criteria. RESULTS: After adjustment for age, sex, BMI, waist circumference, sports activity, bicycling, education, occupational activity, smoking habit, alcohol consumption and circulating levels of γ-glutamyltransferase, alanine aminotransferase, fetuin-A, high-sensitivity C-reactive protein, adiponectin, HDL-cholesterol and triacylglycerol, higher serum ferritin concentrations were associated with a higher risk of type 2 diabetes (RR in the highest vs lowest quintile, 1.73; 95% CI 1.15, 2.61; p(trend) = 0.002). No significant association was observed for soluble transferrin receptor (RR 1.33; 95% CI 0.85, 2.09; p(trend) = 0.50). The soluble transferrin receptor-to-ferritin ratio was significantly inversely related to risk (RR 0.61; 95% CI 0.41, 0.91; p(trend) = 0.02). CONCLUSIONS/INTERPRETATION: High ferritin levels are associated with higher risk of type 2 diabetes independently of established diabetes risk factors and a range of diabetes biomarkers whereas soluble transferrin receptor concentrations are not related to risk. These results support the hypothesis that higher iron stores below the level of haemochromatosis are associated with risk of type 2 diabetes.

Dissociation of lipotoxicity and glucotoxicity in a mouse model of obesity associated diabetes: role of forkhead box O1 (FOXO1) in glucose-induced beta cell failure.

AIMS/HYPOTHESIS: Carbohydrate-free diet prevents hyperglycaemia and beta cell destruction in the New Zealand Obese (NZO) mouse model. Here we have used a sequential dietary regimen to dissociate the effects of obesity and hyperglycaemia on beta cell function and integrity, and to study glucose-induced alterations of key transcription factors over 16 days. METHODS: Mice were rendered obese by feeding a carbohydrate-free diet for 18 weeks. Thereafter, a carbohydrate-containing diet was given. Plasma glucose, plasma insulin and total pancreatic insulin were determined, and forkhead box O1 protein (FOXO1) phosphorylation and the transcription factors pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 protein (NKX6.1) and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (avian) (MAFA) were monitored by immunohistochemistry for 16 days. RESULTS: Dietary carbohydrates produced a rapid and continuous increase in plasma glucose in NZO mice between day 2 and 16 after the dietary challenge. Hyperglycaemia caused a dramatic dephosphorylation of FOXO1 at day 2, followed by a progressive depletion of insulin stores. The loss of beta cells was triggered by apoptosis (detectable at day 8), associated with reduction of crucial transcription factors (PDX1, NKX6.1 and MAFA). Incubation of isolated islets from carbohydrate-restricted NZO mice or MIN6 cells with palmitate and glucose for 48 h resulted in a dephosphorylation of FOXO1 and thymoma viral proto-oncogene 1 (AKT) without changing the protein levels of both proteins. CONCLUSIONS/INTERPRETATION: The dietary regimen dissociates the effects of obesity (lipotoxicity) from those of hyperglycaemia (glucotoxicity) in NZO mice. Obese NZO mice are unable to compensate for the carbohydrate challenge by increasing insulin secretion or synthesising adequate amounts of insulin. In response to the hyperglycaemia, FOXO1 is dephosphorylated, leading to reduced levels of beta cell-specific transcription factors and to apoptosis of the cells.

Non-fasting lipids and risk of cardiovascular disease in patients with diabetes mellitus.

AIMS/HYPOTHESIS: The aim of this study was to examine the effect of postprandial time on the associations and predictive value of non-fasting lipid levels and cardiovascular disease risk in participants with diabetes. METHODS: This study was conducted among 1,337 participants with diabetes from the Dutch and German (Potsdam) contributions to the European Prospective Investigation into Cancer and Nutrition. At baseline, total cholesterol, LDL- and HDL-cholesterol and triacylglycerol concentrations were measured and the ratio of total cholesterol/HDL-cholesterol was calculated. Participants were followed for incidence of cardiovascular disease. RESULTS: Lipid concentrations changed minimally with increasing postprandial time, except for triacylglycerol which was elevated just after a meal and declined over time (1.86 at 0.1 h to 1.33 at >6 h, p for trend <0.001). During a mean follow-up of 8 years, 116 cardiovascular events were documented. After adjustment for potential confounders, triacylglycerol (HR for third tertile compared with first tertile (HR(t)3(to)1), 1.73 [95% CI 1.04, 2.87]), HDL-cholesterol (HR(t)3(to)1, 0.41 [95% CI 0.23, 0.72]) and total cholesterol/HDL-cholesterol ratio (HR(t)3(to)1, 1.65 [95% CI 0.95, 2.85]) were associated with cardiovascular disease, independent of postprandial time. Cardiovascular disease risk prediction using the UK Prospective Diabetes Study risk engine was not affected by postprandial time. CONCLUSIONS/INTERPRETATION: Postprandial time did not affect associations between lipid concentrations and cardiovascular disease risk in patients with diabetes, nor did it influence prediction of cardiovascular disease. Therefore, it may not be necessary to use fasting blood samples to determine lipid concentrations for cardiovascular disease risk prediction in patients with diabetes.

Nav 1.1 dysfunction in genetic epilepsy with febrile seizures-plus or Dravet syndrome.

Relatively few SCN1A mutations associated with genetic epilepsy with febrile seizures-plus (GEFS+) and Dravet syndrome (DS) have been functionally characterized. In contrast to GEFS+, many mutations detected in DS patients are predicted to have complete loss of function. However, functional consequences are not immediately apparent for DS missense mutations. Therefore, we performed a biophysical analysis of three SCN1A missense mutations (R865G, R946C and R946H) we detected in six patients with DS. Furthermore, we compared the functionality of the R865G DS mutation with that of a R859H mutation detected in a GEFS+ patient; the two mutations reside in the same voltage sensor domain of Na(v) 1.1. The four mutations were co-expressed with ß1 and ß2 subunits in tsA201 cells, and characterized using the whole-cell patch clamp technique. The two DS mutations, R946C and R946H, were nonfunctional. However, the novel voltage sensor mutants R859H (GEFS+) and R865G (DS) produced sodium current densities similar to those in wild-type channels. Both mutants had negative shifts in the voltage dependence of activation, slower recovery from inactivation, and increased persistent current. Only the GEFS+ mutant exhibited a loss of function in voltage-dependent channel availability. Our results suggest that the R859H mutation causes GEFS+ by a mixture of biophysical defects in Na(v) 1.1 gating. Interestingly, while loss of Na(v) 1.1 function is common in DS, the R865G mutation may cause DS by overall gain-of-function defects.

Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome.

AIMS/HYPOTHESIS: Numerous new genes have recently been identified in genome-wide association studies for type 2 diabetes. Most are highly expressed in beta cells and presumably play important roles in their function. However, these genes account for only a small proportion of total risk and there are likely to be additional candidate genes not detected by current methodology. We therefore investigated islets from the polygenic New Zealand mouse (NZL) model of diet-induced beta cell dysfunction to identify novel genes and pathways that may play a role in the pathogenesis of diabetes. METHODS: NZL mice were fed a diabetogenic high-fat diet (HF) or a diabetes-protective carbohydrate-free HF diet (CHF). Pancreatic islets were isolated by laser capture microdissection (LCM) and subjected to genome-wide transcriptome analyses. RESULTS: In the prediabetic state, 2,109 islet transcripts were differentially regulated (>1.5-fold) between HF and CHF diets. Of the genes identified, 39 (e.g. Cacna1d, Chd2, Clip2, Igf2bp2, Dach1, Tspan8) correlated with data from the Diabetes Genetics Initiative and Wellcome Trust Case Control Consortium genome-wide scans for type 2 diabetes, thus validating our approach. HF diet induced early changes in gene expression associated with increased cell-cycle progression, proliferation and differentiation of islet cells, and oxidative stress (e.g. Cdkn1b, Tmem27, Pax6, Cat, Prdx4 and Txnip). In addition, pathway analysis identified oxidative phosphorylation as the predominant gene-set that was significantly upregulated in response to the diabetogenic HF diet. CONCLUSIONS/INTERPRETATION: We demonstrated that LCM of pancreatic islet cells in combination with transcriptional profiling can be successfully used to identify novel candidate genes for diabetes. Our data strongly implicate glucose-induced oxidative stress in disease progression.

Fasting plasma glucose and Type 2 diabetes risk: a non-linear relationship.

AIMS: The current definition of impaired fasting glucose (IFG, >or=100 mg/dl) has been criticized as being too low for selective identification of individuals at risk for Type 2 diabetes. Furthermore, it is unclear whether any cut-off is justifiable from the shape of association between fasting plasma glucose (FPG) and diabetes. We therefore evaluated the association between FPG and incidence of Type 2 diabetes in a prospective, population-based study. METHODS: A case-cohort study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort study involved 589 fasted participants in a randomly selected subcohort and 153 incident cases. Restricted cubic spline regression was used to examine non-linearity of associations and we calculated pairs of sensitivities and specificities for different cut-offs of FPG. RESULTS: Spline regression with adjustment for age, sex, body mass index, waist circumference, education, physical activity, alcohol consumption, and plasma levels of triglycerides, high-density lipoprotein cholesterol and gamma-glutamyltransferase indicated that FPG was associated with risk in a non-linear fashion. Risk with higher FPG increased only above approximately 84 mg/dl. FPG>or=84 mg/dl yielded a sensitivity of 95.4% at a false-positive rate of 86.8%. In comparison, FPG>or=100 and>or=110 mg/dl yielded sensitivities of 78.4 and 42.5% and false-positive rates of 27.8 and 6.8%, respectively. The optimal cut-off of FPG was at approximately 102 mg/dl (sensitivity: 75.8%, false-positive rate: 20.7%). CONCLUSIONS: Although our study suggests a threshold for increasing diabetes risk at 84 mg/dl, this cut-off would classify the vast majority of the population as at risk. The statistically optimal cut-off supports the current definition of IFG (>or=100 mg/dl).

Physiology: does gut hormone PYY3-36 decrease food intake in rodents?

Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.

Embryonic lethality caused by apoptosis during gastrulation in mice lacking the gene of the ADP-ribosylation factor-related protein 1.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a membrane-associated GTPase with significant similarity to the family of ARFs. We have recently shown that ARFRP1 interacts with the Sec7 domain of the ARF-specific guanine nucleotide exchange factor Sec7-1/cytohesin and inhibits the ARF/Sec7-dependent activation of phospholipase D in a GTP-dependent manner. In order to further analyze the function of ARFRP1, we cloned the mouse Arfrp1 gene and generated Arfrp1 null-mutant mice by gene targeting in embryonic stem cells. Heterozygous Arfrp1 mutants developed normally, whereas homozygosity for the mutant allele led to embryonic lethality. Cultured homozygous Arfrp1 null-mutant blastocysts were indistinguishable from wild-type blastocysts. In vivo, they implanted and formed egg cylinder stage embryos that appeared normal until day 5. Between embryonic days 6 and 7, however, apoptotic cell death of epiblast cells occurred in the embryonic ectoderm during gastrulation, as was shown by histological analysis combined with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Epiblast cells that would normally differentiate to mesodermal cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. In contrast, the development of extraembryonic structures appeared unaffected. Our results demonstrate that ARFRP1 is necessary for early embryonic development during gastrulation.

Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene.

The ADP-ribosylation factor-like protein 4 (ARL4) is a 22-kDa GTP-binding protein which is abundant in testes of pubertal and adult rodents but absent in testes from prepubertal animals. During testis development, ARL4 expression starts at day 16 when the spermatogenesis proceeds to the late pachytene. In the adult testis, the ARL4 protein was detected in pre- and postmeiotic cells, spermatocytes, and spermatides, but not in spermatogonia and mature spermatozoa. Mouse Arl4-null mutants generated by targeted disruption of the Arl4 gene were viable and grew normally; male as well as female Arl4(-/-) mice were fertile. However, inactivation of the Arl4 gene resulted in a significant reduction of testis weight and sperm count by 30 and 60%, respectively, without reduction of litter size or frequency. It is suggested that the disruption of Arl4 produces a moderate retardation of germ cell development, possibly at the stage of meiosis.

Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity.

Dyrk-related kinases represent a novel subfamily of protein kinases with unique structural and enzymatic features. Its members have been identified in distantly related organisms. The yeast kinase, Yak1, has been characterized as a negative regulator of growth. Mnb from Drosophila is encoded by the minibrain gene, whose mutation results in specific defects in neurogenesis. Its mammalian homolog, Dyrk1A, is activated by tyrosine phosphorylation in the activation loop between subdomains VII and VIII of the catalytic domain. The human gene for Dyrk1A is located in the "Down syndrome critical region" of chromosome 21 and is therefore a candidate gene for mental retardation in Down syndrome. More recently, six additional mammalian Dyrk-related kinases have been identified (Dyrk1B, Dyrk1C, Dyrk2, Dyrk3, Dyrk4A, and Dyrk4B). All members of the Dyrk family contain in the activation loop the tyrosines that are essential for the full activity of Dyrk1A. Outside their catalytic domains, Dyrk kinases exhibit little sequence similarity except for a small segment immediately preceding the catalytic domain (DH-box, Dyrk homology box). An unusual enzymatic property of Dyrk-related kinases is their ability to catalyze tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates. The exact cellular function of the Dyrk kinases is yet unknown. However, it appears reasonable to assume that they are involved in the regulation of cellular growth and/or development.

Cloning of two splicing variants of the novel Ras-related GTPase Rab29 which is predominantly expressed in kidney.

cDNA of a novel Ras-related GTP-binding protein was isolated from rat tissue by a PCR-based cloning approach, and was designated Rab29 because its deduced amino acid sequence (204 aa) is remotely similar to that of members of the Rab family (30% identity with Rab1). mRNA of Rab29 was found predominately in kidney. Recombinant Rab29 exhibited rapid exchange of bound guanine nucleotides for radiolabeled GTP but lacked a detectable intrinsic GTPase activity. A second cDNA clone was isolated which contained a 287 bp in-frame insertion with characteristics of an intron sequence; this insertion introduces a stop codon after arginine 167. The recombinant protein (Rab29delta37) derived from the cDNA carrying the insertion was loaded with GTP during biosynthesis, but showed almost no exchange of the nucleotide for radiolabeled GTP. Thus, the C-terminus of Rab29 appears to harbor a structural element which is essential for the nucleotide exchange of the protein.

Cloning of a novel member (ARL5) of the ARF-family of Ras-related GTPases.

A novel ras-related GTPase with a unique structure was cloned by PCR-amplification with degenerate primers and screening of a rat fat cell cDNA library. The deduced amino acid sequence of the cDNA comprises all 6 GTP binding motifs which are conserved in Ras-related GTPases. The sequence is similar to that of ADP-ribosylation factors (ARF), and shows several structural features typical for the ARF-family. Because its closest relative is the GTPase ARL1 (49% identical amino acids, 54% identical nucleotides within the coding region), the protein was designated ARL5 (ARF-like protein 5). Low amounts of mRNA were found in most rat tissues examined (heart, skeletal muscle, fat, liver, kidney, lung, spleen, intestine, testis, and thymus) with highest levels in brain, intestine, and thymus.

cDNA cloning and characterization of rat Clk3, a LAMMER kinase predominately expressed in testis.

A cDNA clone of a protein kinase with high similarity to the Clk (Cdc2-like kinases) subfamily was isolated from a rat brain library and characterized. Its deduced amino acid sequence exhibited a 99% identity with human Clk3 and was therefore designated rat Clk3. In addition to the protein kinase domain, the sequence (490 amino acids) comprises an N-terminal domain with a strikingly high portion of basic amino acids. A glutathione S-transferase fusion protein of Clk3 catalyzed autophosphorylation of the kinase but not phosphorylation of the exogenous substrates histone or casein. By Northern blot analysis of different rat tissues, mRNA of Clk3 was detected predominately in testis, suggesting that this kinase regulates a predominately testicular function.

Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells.

In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.

In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin-diabetic rats.

The in vivo glucose uptake and the levels of two glucose transporter proteins (GLUT1 and GLUT4) were measured in heart and in various types of skeletal muscle from streptozotocin-diabetic rats. Diabetes (12-16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI), and the levels of GLUT1 and GLUT4 in heart by 75%, 60% and 70%, respectively. In diaphragm consisting of approximately equal amounts of type I (slow-contracting oxidative), IIa (fast-contracting oxidative) and IIb (fast-contracting glycolytic) fibers, GMI and GLUT4 levels were reduced by 60% and 40%, respectively, with no change in GLUT1 levels. In muscle consisting mainly of type I fibers (e.g., m. soleus), GMI and GLUT4 levels were reduced by 60% and 30%, respectively, whereas GLUT1 levels were unaltered. In mixed-type muscle consisting of type IIa and IIb fibers (e.g., m. plantaris and red part of m. gastrocnemius), GMI and GLUT1 levels were unchanged, whereas GLUT4 levels were decreased by 45%. In contrast, GMI was increased by 100% in type IIb fibers (e.g., the white part of m. gastrocnemius), probably reflecting the 4-fold increase in blood glucose levels, whereas GLUT4 levels were lowered by 55% with no change in GLUT1 levels. These data demonstrate a marked difference in the response of in vivo glucose uptake to long-term hypoinsulinemia between oxidative (type I) and glycolytic (type IIb) fibers. Furthermore, in contrast to the GLUT4, GLUT1 levels are regulated differentially in heart and skeletal muscle in response to streptozotocin-induced diabetes.

Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum.

The binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.

Glucose transport activity and ligand binding (cytochalasin B, IAPS-forskolin) of chimeric constructs of GLUT2 and GLUT4 expressed in COS-7-cells.

Chimeric constructs of glucose transporters GLUT2 and GLUT4 were transiently expressed in COS-7 cells in order to determine regions of the proteins responsible for their differences in activity and ligand binding. Exchange of the C-terminal tail (aa 479-509) of GLUT4 failed to affect glucose transport activity assayed at 1 mM glucose or ligand binding (cytochalasin B, IAPS-forskolin). In contrast, exchange of the C-terminal half of GLUT4 (aa 222-509) for that of GLUT2 markedly reduced ligand binding (Kd of cytochalasin B binding 1.88 +/- 0.2 microM vs. 0.21 +/- 0.06 in the wild-type GLUT4), and moderately (25%) reduced glucose transport activity. These data support the conclusion that the domains determining differences in ligand binding between GLUT4 and GLUT2 are located in the C-terminal half of the glucose transporters.