Charles F. Burant

Molecular Biology of Mammalian Glucose Transporters

The oxidation of glucose represents a major source of metabolic energy for mammaliancells. However, because the plasma membrane is impermeable to polar molecules such as glucose, the cellular uptake of this important nutrient is accomplished by membrane-associated carrier proteins that bind and transfer it across the lipid bilayer. Two classes of glucose carriers have been described in mammalian cells: the Na+-glucose cotransporter and the facilitative glucose transporter. The Na+-glucose cotransporter transports glucose against its concentration gradient by coupling its uptake with the uptake of Na+ that is being transported down its concentration gradient. Facilitative glucose c rriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport. cDNAs have been isolated from human tissues encoding a Na+-glucose-cotransporter protein and five functional facilitative glucosetransporter isoforms. The Na+-glucose cotransporter is expressed by absorptive epithelial cells of the small intestine and is involved in the dietary uptake of glucose. The same or a related protein may be responsible for the reabsorption of glucose by the kidney. Facilitative glucose carriers are expressed by most if not all cells. The facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions. The GLUT1 (erythrocyte) and GLUT3 (brain) facilitative glucose-transporter isoforms may be responsible for basal or constitutive glucose uptake. The GLUT2 (liver) isoform mediates the bidirectional transport of glucose by the hepatocyte and is responsible, at least in part, for the movement of glucose out of absorptive epithelial cells into the circulation in the small intestine and kidney. This isoform may also comprise part of the glucosesensing mechanism of the insulin-producing β-cell. The subcellular localization of the GLUT4 (muscle/fat) isoform changes in response to insulin, and this isoform is responsible for most of the insulin-stimulated uptake of glucose that occurs in muscle and adipose tissue. The GLLJT5 (small intestine) facilitative glucose-transporter isoform is expressed at highest levels in the small intestine and may be involved in the transcellular transport of glucose by absorptive epithelial cells. The exon-intron organizations of the human GLUT1, GLUT2, and GLUT4 genes have been determined. In addition, the chromosomal locations of the genes encoding the Na+-dependent and facilitative glucose carriers have been determined. Restriction-fragment-length polymorphisms have also been identified at several of these loci. The isolation and characterization of cDNAs and genes for these glucose transporters will facilitate studies of their role in the pathogenesis of disorders characterized by abnormal glucose transport, including diabetes mellitus, the glucose-galactose malabsorption syndrome, and benign renal glycosuria.

Troglitazone action is independent of adipose tissue.

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.

Glucose Transporter Isoforms GLUT1 and GLUT3 Transport Dehydroascorbic Acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1–5 and SGLT1. The apparentK m of DHA transport via GLUT1 and GLUT3 was 1.1 ± 0.2 and 1.7 ± 0.3 mm, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2–4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, d-glucose, and 3-O-methylglucose among other hexoses while fructose and l-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-α-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2–8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

Metabolic Effects of Troglitazone Monotherapy in Type 2 Diabetes Mellitus: A Randomized, Double-Blind, Placebo-Controlled Trial

Type 2 diabetes mellitus is characterized by two major pathophysiologic defects: insulin resistance and impaired capacity to secrete insulin [1, 2]. A major component of insulin resistance exists in peripheral tissues, where insulins ability to stimulate glucose uptake from the circulation is blunted. During the past three decades, treatment of hyper-glycemia in patients with type 2 diabetes mellitus who do not respond to such behavioral modifications as diet and exercise has focused on improving the relative insulin deficiency through therapy with sulfonylurea drugs to stimulate endogenous insulin secretion or through administration of insulin itself. Two additional drugs have recently become available: metformin, which seems to exert much of its glucose-lowering effect by suppressing hepatic glucose production [3], and acarbose, which changes the pattern of glucose absorption from the gastrointestinal tract [4]. Thus, no pharmacologic intervention for type 2 diabetes mellitus has had a major effect on improving insulin resistance in peripheral tissues. New compounds, the thiazolidinediones, have recently been developed as glucose-lowering agents. Early studies showed that the glucose-lowering effect of thiazolidinediones was evident in animal models of type 2 diabetes mellitus but not those of type 1 diabetes mellitus [5, 6], suggesting that some endogenous insulin secretion is needed for these agents to act. Troglitazone has been shown to decrease levels of not only plasma glucose and glycosylated hemoglobin [7-13] but also insulin and C-peptide. These observations, coupled with direct measures of whole-body insulin sensitivity in a small number of patients with type 2 diabetes mellitus [7], suggest that troglitazone exerts its major glucose-lowering effect by ameliorating insulin resistance. However, it is not clear whether troglitazone exerts its major insulin-sensitizing effect predominantly in the liver or in peripheral tissues. We studied this issue using detailed metabolic measurements in a large group of patients with type 2 diabetes mellitus. Methods This multicenter study was conducted at six sites: University of Chicago, Chicago, Illinois; University of Southern California, Los Angeles, California; University of Rochester, Rochester, New York; University of Pittsburgh, Pittsburgh, Pennsylvania; University of California, San Diego, San Diego, California; and Yale University, New Haven, Connecticut. Sample size was projected on the basis of study design, major end points, and standard power analysis. Each center enrolled patients while adhering to a common protocol with the same inclusion and exclusion criteria. At each center, patients gave written informed consent to participate in the study, which was approved by the respective university human investigation committees. All patients were studied in a 6-month, randomized, placebo-controlled, double-blind protocol. Patients were randomly assigned to treatment according to a blocked randomization code (block size, five) that was generated by a central computer. In each center, study personnel (executors of treatment assignment) and patients were blinded to the treatment code. Patients were consecutively assigned to treatments; equal numbers of troglitazone or matching placebo tablets were dispensed in a double-blind fashion. Patients Patients had to have type 2 diabetes mellitus according to the criteria of the National Diabetes Data Group [14], HbA1c levels above the upper limit of normal, and fasting C-peptide levels of 0.49 nmol/L or greater. Therapy with oral antidiabetic medication was discontinued before randomization. Patients were excluded if they had clinically symptomatic heart disease, had had a vascular occlusive event in the previous 3 months, had had cancer in the past 5 years, had a serum creatinine level greater than 176.8 mol/L, or had serum amino-transferase levels above the upper limit of normal. Study Design After medical screening, a 2-week wash-out period was allowed for discontinuation of therapy with oral antidiabetic medication in patients who were taking such medication. Metabolic studies were done before patients were randomly assigned to one of five treatment groups: 100, 200, 400, or 600 mg of troglitazone daily or placebo. At 6 months, follow-up metabolic studies were repeated 24 hours after patients received the last troglitazone or placebo tablet. At baseline and 6 months, patients were hospitalized and fasted overnight before a meal tolerance test (day 1) and a euglycemic-hyperinsulinemic clamp procedure (day 2) [15]. During the study, patients were prescribed a diet designed to maintain baseline body weight. Dietary assessment at the time of enrollment determined the patients caloric needs [16]. The prescribed diet consisted of 50% carbohydrates, 34% fat (ratio of saturated fat to polyunsaturated fat, 1:4) and 16% protein. Patients were seen at monthly outpatient visits between the baseline and 6-month metabolic studies so that their clinical condition could be monitored. Meal Tolerance Test At approximately 7:00 a.m., patients were placed on bed rest and an intravenous catheter was inserted into an antecubital vein for blood sampling. A small volume of normal saline (0.9%) was infused to maintain patency. At approximately 8:00 a.m., patients ingested a liquid formula meal (Sustacal-HC [Mead Johnson & Co., Evansville, Indiana], which contained 33% of total daily caloric requirements); this was followed 4 hours later by an identical meal. Fasting blood samples were drawn, and additional samples were obtained every hour thereafter for 8 hours. Samples were processed immediately and stored at 80C for measurement of serum levels of glucose, insulin, free fatty acids, and triglycerides and plasma levels of C-peptide. Fasting blood was also drawn for measurement of HbA1c. After completing the test, patients received an evening meal according to their prescribed diet. They then fasted until the end of the euglycemic-hyperinsulinemic clamp procedure the following day. The intravenous line was left in situ for the clamp procedure. Euglycemic-Hyperinsulinemic Clamp Procedure At 6:00 a.m., a 4-hour primed (corrected for ambient fasting plasma glucose level), continuous (2 mg/m2 body surface area per minute) infusion of [6,6- 2H]-glucose (di-deuterated glucose) isotope into the antecubital vein began. During the third hour of infusion, a retrograde cannula was inserted into a contralateral hand vein. The hand was warmed for sampling of arterialized venous blood. A small volume of normal saline (0.9%) was infused through the sampling cannula to maintain patency. Blood samples were drawn at 10-minute intervals during the final 40 minutes of the fourth hour for measurement of plasma glucose and insulin levels and glucose isotope enrichment. After 4 hours of isotope infusion, a two-step priming dose of insulin was administered (480 mU/m2 per minute followed by 240 mU/m2 per minute; each lasted 5 minutes); this was followed by a continuous infusion of insulin (120 mU/m2 per minute) that lasted 300 minutes (total, 5 hours). The plasma glucose level was allowed to decrease to 5.5 mmol/L; exogenous glucose (dextrose, 20 g/100 mL of water enriched to approximately 2.5% with di-deuterated glucose) was then infused to maintain the plasma glucose level, measured every 5 minutes, at 5.5 mmol/L. The basal isotope infusion was stopped when the exogenous glucose infusion began. Patients also received a continuous infusion of potassium (KCl and KPo 4), 0.105 mmol/L per minute, during the insulin infusion to maintain the serum potassium level between 3.5 and 4.5 mmol/L. During the final hour of the clamp procedure, blood samples were drawn every 10 minutes for measurement of plasma insulin levels and steady-state glucose isotope enrichment. For comparison with diabetic patients, eight persons without diabetes (mean age SD, 46 6 years; mean fasting plasma glucose level, 5.3 0.2 mmol/L; mean body mass index, 29 3 kg/m2) were also studied on one occasion under basal and clamped conditions after an identical hyperinsulinemic clamp protocol. Substrate and Hormone Measurements Serum and plasma samples were shipped frozen to Corning Nichols Institute for chemical analysis and to Yale University for measurement of isotope enrichment. Serum total triglyceride levels (Boehringer Mannheim Diagnostics, Indianapolis, Indiana) and plasma free fatty acid levels (NEFA C-test, Wako Chemicals, Richmond, Virginia) were determined enzymatically; interassay coefficients of variation were 2% and 3.6%, and intraassay coefficients of variation were 1.6% and 1%, respectively. Insulin and C-peptide levels were measured by radioimmunoassay (Corning Nichols Institute); the interassay coefficients of variation were 12.3% and 12.0%, and the intraassay coefficients of variation were 7.4% and 6.5%, respectively. Levels of HbA1c were measured by high-performance liquid chromatography using BioRad (Hercules, California) equipment (Corning Nichols Institute), with a normal reference range of 0.045 to 0.059. At each center, plasma glucose levels were measured at the bedside by using a Beckman glucose analyzer (Fullerton, California). Glucose Isotope Data Gas chromatography mass spectrometer analysis of enrichment of di-deuterated glucose in plasma and infusates was done at one center (Yale Stable Isotope Core Facility, New Haven, Connecticut) by using the penta-acetate derivative of glucose [17]. Calculations Basal hepatic glucose production was calculated as follows: Basal hepatic glucose production = (f/sa) x ([enrichmentinf/enrichmentplasma] 1), where f = basal [6,6- 2H] glucose infusion rate (mg/min), sa = body surface area (m2), enrichmentinf = [6,6- 2H] glucose infusate enrichment (%), and enrichmentplasma = steady-state basal plasma [6,6- 2H] glucose enrichment (%). The term enrichment refers to the fraction of isotope of glucose to naturally occurring (native) glucose,

Cloning, Tissue Expression, and Chromosomal Localization of SUR2, the Putative Drug-Binding Subunit of Cardiac, Skeletal Muscle, and Vascular KATP Channels

ATP-sensitive inwardly rectifying potassium channels are expressed in a variety of tissues, including heart, skeletal, and smooth muscle, and pancreatic β-cells. Physiological and pharmacological studies suggest the presence of distinct KATP channels in these tissues. Recently, the KATP channel of β-cells has been reconstituted in functional form by coexpression of SUR, the sulfonylurea-binding protein, and the inwardly rectifying K+ channel subunit, KIR6.2. In this article, we describe the isolation of cDNAs encoding SUR-like proteins from mouse, SUR2A and SUR2B. Northern blotting showed that the highest expression of the SUR2 isoforms is in the heart and skeletal muscle, with lower levels in all other tissues. By reverse transcription-polymerase chain reaction, SUR2B is ubiquitously expressed, while the apparently alternatively spliced variant, SUR2A, is expressed exclusively in heart. In situ hybridization shows that the SUR2 isoforms are expressed in the parenchyma of the heart and skeletal muscle and in the vascular structures of other tissues. Human SUR2 was localized to chromosome 12, p12.1 by fluorescent in situ hybridization. The structure of the predicted protein and expression pattern of SUR2 suggests that it is the drug-binding channel-modulating subunit of the extrapancreatic KATP channel. Differences in sequence between SUR and between SUR2 isoforms may underlie the tissue-specific pharmacology of the KATP channel.

Small intestine hexose transport in experimental diabetes. Increased transporter mRNA and protein expression in enterocytes.

The effect of insulinopenic diabetes on the expression of glucose transporters in the small intestine was investigated. Enterocytes were sequentially isolated from jejunum and ileum of normal fed rats, streptozotocin-diabetic rats, and diabetic rats treated with insulin. Facilitative glucose transporter (GLUT) 2, GLUT5, and sodium-dependent glucose transporter 1 protein content was increased from 1.5- to 6-fold in enterocytes isolated from diabetic animals in both jejunum and ileum. Insulin was able to reverse the increase in transporter protein expression seen after induction of diabetes. There was a four- to eightfold increase in the amount of enterocyte glucose transporter mRNA after diabetes with greater changes in sodium-dependent glucose transporter 1 and GLUT2 than in GLUT5 levels. In situ hybridization showed that after the induction of diabetes there was new hybridization in lower villus and crypt enterocytes that was reversed by insulin treatment. Thus, the increase in total hexose transport caused by diabetes is due to a premature expression of hexose transporters by enterocytes along the crypt-villus axis, causing a cumulative increase in enterocyte transporter protein during maturation. These changes are likely to represent an adaptive response by the organism to increase nutrient absorption in a perceived state of tissue starvation. These adaptive changes may lead to exacerbation of hyperglycemia in uncontrolled diabetes.

Alternative splicing of sur2 exon 17 regulates nucleotide sensitivity of the ATP-sensitive potassium channel

ATP-sensitive potassium channels (KATP) are implicated in a diverse array of physiological functions. Previous work has shown that alternative usage of exons 14, 39, and 40 of the muscle-specific KATP channel regulatory subunit, sur2, occurs in tissue-specific patterns. Here, we show that exon 17 of the first nucleotide binding fold of sur2 is also alternatively spliced. RNase protection demonstrates that SUR2(Δ17) predominates in skeletal muscle and gut and is also expressed in bladder, fat, heart, lung, liver, and kidney. Polymerase chain reaction and restriction digest analysis of sur2 cDNA demonstrate the existence of at least five sur2 splice variants as follows: SUR2(39), SUR2(40), SUR2(Δ17/39), SUR2(Δ17/40), and SUR2(Δ14/39). Electrophysiological recordings of excised, inside-out patches from COS cells cotransfected with Kir6.2 and the sur2 variants demonstrated that exon 17 splicing alters KATP sensitivity to ATP block by 2-fold from ≈40 to ≈90 μm for exon 17 and Δ17, respectively. Single channel kinetic analysis of SUR2(39) and SUR2(Δ17/39) demonstrated that both exhibited characteristic KATP kinetics but that SUR2(Δ17/39) exhibited longer mean burst durations and shorter mean interburst dwell times. In sum, alternative splicing of sur2 enhances the observed diversity of KATP and may contribute to tissue-specific modulation of ATP sensitivity.

Effects of troglitazone on substrate storage and utilization in insulin-resistant rats

Elevated serum and tissue lipid stores are associated with skeletal muscle insulin resistance and diminished glucose-stimulated insulin secretion, the hallmarks of type 2 diabetes. We studied the effects of 6-wk treatment with the insulin sensitizer troglitazone on substrate storage and utilization in lean control and Zucker diabetic fatty (ZDF) rats. Troglitazone prevented development of diabetes and lowered serum triglycerides (TG) in ZDF rats. Soleus muscle glycogen and TG content were elevated twofold in untreated ZDF rats, and both were normalized by troglitazone to lean control levels (P < 0.05). Troglitazone also normalized insulin-stimulated glucose uptake as well as basal and insulin-stimulated glycogen synthesis, implying increased skeletal muscle glycogen turnover. The proportion of active pyruvate dehydrogenase (PDH) in soleus muscle was reduced in ZDF relative to lean control rat muscle (16 +/- 2 vs. 21 +/- 2%) but was restored by troglitazone treatment (30 +/- 3%). Increased PDH activation was associated with a 70% increase in glucose oxidation. Muscle lipoprotein lipase activity was decreased by 35% in ZDF compared with lean control rats and was increased twofold by troglitazone. Palmitate oxidation and incorporation into TG were higher in ZDF relative to lean control rats but were unaffected by troglitazone treatment. Troglitazone decreased the incorporation of glucose into the acyl group of TG by 60% in ZDF rats. In summary, ZDF rats demonstrate increased skeletal muscle glycogen and TG stores, both of which were reduced by troglitazone treatment. Troglitazone appears to increase both glycogen and TG turnover in skeletal muscle. Normalization of PDH activity and decreased glucose incorporation into acyl TG may underlie the improvements in intracellular substrate utilization and energy stores, which lead to decreased serum TG and glucose.

The small intestinal fructose transporters: site of dietary perception and evidence for diurnal and fructose sensitive control elements.

To obtain an insight into the mechanisms responsible for GLUT5 diurnality and fructose responsiveness, rats were gavaged at 9:00 AM or 6:00 PM with 1 g of fructose in the presence or absence of cycloheximide. After 4 h of fructose exposure, GLUT5 mRNA and protein levels increased 2-3.5-fold above the natural diurnal levels of expression. In situ hybridization and immunochemical analysis of GLUT5 mRNA and protein demonstrated that both diurnality and fructose responsiveness was confined to mature enterocytes. The protein synthesis inhibitor, cycloheximide, blunted the diurnal and fructose driven increase in GLUT5 mRNA expression in the morning, but had minimal effect on the pattern of expression in the evening. This differential sensitivity of intestinal GLUT5 mRNA to de novo protein synthesis may reflect the increasing presence of diurnal and fructose sensitive control factors during the day. Following vehicle gavage, Cycloheximide was more effective in reducing GLUT5 protein expression levels in the morning when compared to the evening. These data suggest that the turnover of GLUT5 protein may be diurnally influenced.

Insulin regulation of hepatic glucose transporter protein is impaired in chronic pancreatitis

ObjectiveThe effect of chronic pancreatitis and insulin on the expression of the hepatic facilitative glucose transporter protein (GLUT-2) was determined in rats. Summary Background DataChronic pancreatitis is associated with diabetes mellitus or impaired glucose tolerance. Suppression of hepatic glucose production (HGP) by insulin is impaired, although the mechanism is unknown. MethodsNormal rats, rats with chronic pancreatitis induced 12 to 16 weeks earlier by oleic acid injection into the pancreatic ducts, and sham-operated rats were studied. Isolated, single-pass liver perfusion was performed, during which glucagon (1.2 pM) was infused, with or without insulin (0.6 or 1.2 nM). The suppression of HGP production by insulin was compared with changes in GLUT-2 in the membrane fraction of liver biopsies obtained before and after hormone perfusion. ResultsGlycogen-rich (fed) livers of normal rats (n = 16) demonstrated a dose-dependent suppression of hepatic glucose production by insulin (50 ± 5% HGP induced by glucagon alone during 1.2-nM insulin perfusion) and a dose-dependent decrease in GLUT-2 (30 ± 13% of basal level during 1.2-nM insulin perfusion). Sham-operated rats (n = 6) also showed reductions in HGP (51 ± 4%) and GLUT-2 (14 ± 10%) during 1.2-nM insulin perfusion. In contrast, rats with chronic pancreatitis (n = 6) showed no suppression of HGP during 1.2-nM Insulin perfusion, and an increase in GLUT-2 (+20 ± 6%) after insulin perfusion (p < 0.02 vs. sham). ConclusionsInsulin suppresses glucagon-stimulated HGP in normal and sham-operated rats, and this reduction in HGP is associated with a decrease in the membrane-bound quantity of GLUT-2. In chronic pancreatitis, insulin suppression of HGP is absent, and this is accompanied by an increase in GLUT-2 in the hepatocyte membrane. The authors conclude that the insulin-mediated change in the level of hepatocyte GLUT-2 is impaired in chronic pancreatitis, and may contribute to the altered glucose metabolism observed commonly in this disease.