Glucose-stimulated insulin secretion, glucose transport, glucose phosphorylation and glucose utilization have been characterized in the insulinoma cell line MIN6, which is derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells. Glucose-stimulated insulin secretion occurred progressively from 5 mmol/l glucose, reached the maximal level approximately seven-fold above the basal level at 25 mmol/l, and remained at this level up to 50 mmol/l. Glucose transport was very rapid with the half-maximal uptake of 3-O-methyl-D-glucose being reached within 15 s at 22 degrees C. Glucose phosphorylating activity in the cell homogenate was due mainly to glucokinase; the Vmax value of glucokinase activity was estimated to be 255 +/- 37 nmol.h-1.mg protein-1, constituting approximately 80% of total phosphorylating activity, whereas hexokinase activity constituted less than 20%. MIN6 cells exhibited mainly the high Km component of glucose utilization with a Vmax of 289 +/- 18 nmol.h-1.mg protein-1. Thus, glucose utilization quantitatively and qualitatively reflected glucose phosphorylation in MIN6 cells. In contrast, MIN7 cells, which exhibited only a small increase in insulin secretion in response to glucose, had 4.7-fold greater hexokinase activity than MIN6 cells with a comparable activity of glucokinase. These characteristics of MIN6 cells are very similar to those of isolated islets, indicating that this cell line is an appropriate model for studying the mechanism of glucose-stimulated insulin secretion in pancreatic beta cells.

The facilitated glucose transporters comprise a structurally related family of proteins predicted to have 12 membrane-spanning domains, with the amino terminus, a relatively large middle loop and the carboxy-terminus all oriented towards the cytoplasm. An alternating conformation model has been proposed to explain the mechanism of facilitated glucose transport. To understand the structure-function relationships, especially the role of the intracellular C-terminal domain, we have modified the rabbit equivalent of the erythroid-type transporter, GLUT1 (ref. 18), using complementary DNA to code for a deletion mutant that lacks most (37 out of 42 amino acids) of the intracellular C-terminal domain. This deletion mutant is expressed at the cell surface of Chinese hamster ovary (CHO) cells, but is functionally inactive, probably because it has lost its capacity to alternate in conformation and so is locked into an inward-facing form.

We have expressed GLUT3 protein, an isoform of a facilitative glucose transporter, in Chinese hamster ovary cells by transfection of its cDNA using an expression vector. The expressed GLUT3 protein was detected by Western-blot analysis as a broad band of 45-65 kDa, indicating intensive glycosylation of the protein. The expressed GLUT3 protein was observed, by immunofluorescence staining, to be located mainly at the plasma membrane, and its expression was associated with a marked increase in glucose-transport activity. Kinetic analysis revealed that the Km value of GLUT3 protein for 3-O-methylglucose uptake was approx. 35% of that of GLUT1 protein, whereas the Km value of GLUT3 protein for 2-deoxy-D-glucose uptake was very similar to that of GLUT1 protein. The Vmax. value of GLUT3 protein for 3-O-methylglucose and 2-deoxyglucose uptake was approx. 20-50% of that of GLUT1 protein. GLUT3 protein was well photolabelled with [3H]cytochalasin B or a mannose derivative, 2-N-4-[3H](1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos -4-yloxy)-2- propylamine. Thus GLUT3 protein has very similar characteristics to GLUT1 protein including its subcellular localization, but exhibits lower Km and Vmax. values for 3-O-methylglucose uptake.

Five patients with sensorineural hearing loss, who harbored a point mutation in the mitochondrial transfer RNA (tRNA) gene tRNA(Leu) (UUR), from five unrelated family pedigrees were examined. In these families diabetes and deafness were maternally inherited. Bilateral hearing was more severely impaired at higher frequencies. Audiometric test results revealed that hearing loss involved the cochlea. Hearing gradually deteriorated; the progression rate ranged from 1.5 to 7.9 dB per year. Proportion of mutant mitochondrial DNAs (mtDNAs) in the leukocytes was not related to the rate or degree of hearing loss, although hearing loss appeared at a younger age in patients with higher heteroplasmy. We speculate that after the proportion of damaged mtDNAs, mostly as a result of mutation, exceeds the expression threshold for deficiencies in mitochondrial protein synthesis and oxygen consumption, a drop in adenosine triphosphate level could lead to an imbalance of ion concentration, resulting in cell death in the cochlea.

To evaluate the role of the histamine H2 receptor C terminus in signaling, desensitization, and agonist-induced internalization, canine H2 receptors with truncated C termini were generated. Wild-type (WT) and truncated receptors were tagged at their N termini with a hemagglutinin (HA) epitope and expressed in COS7 cells. Most of the C-terminal intracellular tail could be truncated (51 of 70 residues, termed T 308 mutant) without loss of functions: cAMP production, tiotidine binding, and plasma membrane targeting. In fact, the T 308 mutant produced more cAMP than the WT when cellsurface expression per cell was equivalent. As observed in a number of guanine nucleotide-binding protein-coupled receptors (GPCRs), 1 cAMP responses occurring via the histamine H2 receptor are rapidly desensitized after agonist stimulation (1-5). The H2 receptor also exhibits an agonist-induced internalization from the cell surface (2, 6). However, the mechanisms underlying these phenomena have yet to be identified. Recently, a number of studies focusing on GPCRs have examined the role of the C terminus in agonistinduced desensitization and internalization, with varying results. For example, C-terminal truncation of the angiotensin II receptor inhibited agonist-induced internalization of this receptor, but not desensitization of the calcium response mediated via the receptor (7). On the other hand, C-terminal truncation of ␤ 2 -adrenergic, ␣ 1B -adrenergic, lutropin/choriogonadotropin, platelet-activating factor, and neurokinin-2 receptors resulted in impairment of homologous desensitization (8 -12). Thus, the C termini of GPCRs have functional importances, which may differ among receptors. The present study was designed to analyze the role of this important portion of the histamine H2 receptor in signaling, desensitization, and agonist-induced internalization. To this end, we constructed H2 receptor cDNAs, devoid of either the 71 or the 51 amino acids at the C terminus, and expressed these cDNAs in COS7 cells. Herein, we present evidence that the 51 C-terminal amino acids of the histamine H2 receptor are important for agonist-induced internalization, but not for either signaling or homologous desensitization of cAMP response, which occurs via this receptor. In addition, these amino acids exert inhibitory effects on cAMP production via the H2 receptor.

Rat GLUT4 (adipocyte/muscle-type glucose transporter) was expressed in two fibroblastic cell lines, Chinese hamster ovary (CHO) cells and 3T3-L1 fibroblasts, under the control of the methallothionein I promoter. Although immunoblotting with a GLUT4-specific anti-peptide antibody demonstrated that the amount of GLUT4 expressed was comparable with that in 3T3-L1 adipocytes and rat adipose tissues, no increase in 2-deoxy-D-glucose uptake was observed in the basal state in fibroblasts. Immunocytochemical studies showed that the expressed GLUT4 appeared to be localized in a specific region in the cytoplasm. These results were in marked contrast to those obtained in CHO cells expressing GLUT1 (HepG2/erythrocyte-type glucose transporter) using the same expression vector. In this case the expressed GLUT1 protein appeared to reside mainly on the plasma membranes, and a significant increase in glucose uptake was observed. Although insulin increased glucose uptake in CHO cells and 3T3-L1 fibroblasts as well as in the cells expressing rat GLUT4, an increment due to insulin above basal values was small, at most 2-fold, and no significant differences were observed in insulin-stimulated glucose uptake between transfected and parental cells. In addition, no apparent differences in the subcellular distribution of expressed GLUT4 were observed between the insulin-stimulated and the basal state. These results indicate that in fibroblastic cell lines GLUT1 and GLUT4 proteins are sorted in a different fashion, and the expression of GLUT4 protein per se is not enough to produce a large insulin-induced increase in glucose transport activity such as that observed in rat adipocytes and 3T3-L1 adipocytes. Thus unidentified aspects of the cellular environment which are present in the adipocytes but not in fibroblastic cell lines may be required for a large insulin-induced increase in glucose transport activity to be observed.

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Aims/Introduction:  Reduced insulin sensitivity and secretion are important in the pathogenesis of type 2 diabetes. Their relationships to prediabetes, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) have been previously studied with the oral glucose tolerance test (OGTT). We investigated whether or not baseline measures of insulin secretion and sensitivity obtained from fasting blood specimens were related to the development of prediabetes and how these measures compared with those based on the OGTT.Materials and Methods:  In 152 Japanese subjects with normal glucose tolerance, we measured baseline plasma glucose and insulin after an overnight fast and during a 75 g OGTT, insulin resistance index (homeostasis model assessment [HOMA‐IR]), and insulin secretion (insulinogenic index [30 min insulin − fasting insulin] ÷ [30 min glucose − fasting glucose] or HOMA‐β).Results:  At a 5–6 year (mean 5.7 years) follow‐up examination, we confirmed 36 cases of prediabetes. After adjusting for age, sex, family history of diabetes, body mass index, and 2‐h plasma glucose, the odds ratio comparing the lowest tertile (≤0.82) of insulinogenic index with the highest tertile (≥1.43) was 6.98 (95% confidence interval, 1.96–24.85) and was 10.72 (2.08–55.3) comparing the lowest tertile (≤76.3) of HOMA‐β with the highest tertile (≥122.1), whereas the respective odds ratios of HOMA‐IR were 3.74 (1.03–13.57) and 10.89 (1.93–61.41) comparing the highest tertile (≥1.95) with the lowest tertile (≤1.25).Conclusions:  Lower insulin secretion and sensitivity are independent risk factors for prediabetes. Clinically practical identification of those at risk for prediabetes is obtainable from HOMA‐β and HOMA‐IR, both of which are measured in fasting state. (J Diabetes Invest, doi: 10.1111.j.2040‐1124.2010.00041.x, 2010)