Insulin unmasks a COOH-terminal Glut4 epitope and increases glucose transport across T-tubules in skeletal muscle.

An improved immunogold labeling procedure was used to examine the subcellular distribution of glucose transporters in Lowricryl HM20-embedded skeletal muscle from transgenic mice overexpressing either Glut1 or Glut4. In basal muscle, Glut4 was highly enriched in membranes of the transverse tubules and the terminal cisternae of the triadic junctions. Less than 10% of total muscle Glut4 was present in the vicinity of the sarcolemmal membrane. Insulin treatment increased the number of gold particles associated with the transverse tubules and the sarcolemma by three-fold. However, insulin also increased the total Glut4 immunogold reactivity in muscle ultrathin sections by up to 1.8-fold and dramatically increased the amount of Glut4 in muscle sections as observed by laser confocal immunofluorescence microscopy. The average diameter of transverse tubules observed in longitudinal sections increased by 50% after insulin treatment. Glut1 was highly enriched in the sarcolemma, both in the basal state and after insulin treatment. Disruption of transverse tubule morphology by in vitro glycerol shock completely abolished insulin-stimulated glucose transport in isolated rat epitrochlearis muscles. These data indicate that: (a) Glut1 and Glut4 are targeted to distinct plasma membrane domains in skeletal muscle; (b) Glut1 contributes to basal transport at the sarcolemma and the bulk of insulin-stimulated transport is mediated by Glut4 localized in the transverse tubules; (c) insulin increases the apparent surface area of transverse tubules in skeletal muscle; and (d) insulin causes the unmasking of a COOH-terminal antigenic epitope in skeletal muscle in much the same fashion as it does in rat adipocytes.

Overexpression of Glut4 protein in muscle increases basal and insulin-stimulated whole body glucose disposal in conscious mice.

The effect of increased Glut4 protein expression in muscle and fat on the whole body glucose metabolism has been evaluated by the euglycemic hyperinsulinemic clamp technique in conscious mice. Fed and fasting plasma glucose concentrations were 172 +/- 7 and 78 +/- 7 mg/dl, respectively, in transgenic mice, and were significantly lower than that of nontransgenic littermates (208 +/- 5 mg/dl in fed; 102 +/- 5 mg/dl in fasting state). Plasma lactate concentrations were higher in transgenic mice, (6.5 +/- 0.7 mM in the fed and 5.8 +/- 1.0 mM in fasting state) compared with that of non-transgenic littermates (4.7 +/- 0.3 mM in the fed and 4.2 +/- 0.5 mM in fasting state). In the fed state, the rate of whole body glucose disposal was 70% higher in transgenic mice in the basal state, 81 and 54% higher during submaximal and maximal insulin stimulation. In the fasting state, insulin-stimulated whole body glucose disposal was also higher in the transgenic mice. Hepatic glucose production after an overnight fast was 24.8 +/- 0.7 mg/kg per min in transgenic mice, and 25.4 +/- 2.7 mg/kg per min in nontransgenic mice. Our data demonstrate that overexpression of Glut4 protein in muscle increases basal as well as insulin-stimulated whole body glucose disposal. These results suggest that skeletal muscle glucose transport is rate-limiting for whole body glucose disposal and that the Glut4 protein is a potential target for pharmacological or genetic manipulation for treatment of patients with non-insulin-dependent diabetes mellitus.

Identification of glucagon-like peptide 1 (GLP-1) actions essential for glucose homeostasis in mice with disruption of GLP-1 receptor signaling.

Glucagon-like peptide-1 (GLP-1) acts to control blood glucose via multiple mechanisms, including regulation of insulin and glucagon secretion, gastric emptying, satiety, and peripheral insulin sensitivity. However, the relative importance of these actions for regulation of blood glucose remains unclear. We demonstrate here a gene dosage effect for the incretin action of GLP-1, as heterozygous GLP-1R +/- mice exhibit an abnormal glycemic response to oral glucose challenge in association with reduced circulating levels of glucose-stimulated insulin. In contrast, GLP-1 signaling is not required for normal control of fasting and postabsorptive glucagon levels, and no significant changes were detected in the tissue content of pancreatic and intestinal proglucagon mRNA, glucagon-like immunoreactivity, or GLP-1 in GLP-1R -/- or +/- mice. Despite the demonstration that GLP-1 stimulates proinsulin gene transcription, pancreatic insulin mRNA transcripts were similar in wild-type and GLP-1R -/- mice. Furthermore, despite suggestions that GLP-1 regulates peripheral glucose disposal, whole-body glucose utilization was similar in wild-type and GLP-1R -/- mice under both basal and hyperinsulinemic conditions. These observations demonstrate that of the numerous physiological activities ascribed to GLP-1, only the incretin effect on pancreatic beta-cells appears essential for regulation of glucose homeostasis in vivo.

Interaction between insulin and glucose-delivery route in regulation of net hepatic glucose uptake in conscious dogs.

In the presence of fixed basal levels of insulin, the route of intravenous glucose delivery (protal vs. peripheral) determines whether net hepatic glucose uptake (NHGU) occurs. Our aims were to determine if the route of intravenous glucose delivery also plays a role in regulating NHGU in the presence of hyperinsulinemia and to determine if length of fast (18 vs. 36 h) influences regulation of NHGU. Five conscious dogs fasted 18 h were given somatostatin and replacement insulin (245 +/- 34 microU.kg-1.min-1) and glucagon (0.65 ng.kg-1.min-1) infusions intraportally. After a 40-min control period, the insulin infusion rate was increased fourfold, and glucose was infused for 3 h. Glucose was given either through a peripheral vein or the portal vein for 90 min to double the glucose load reaching the liver. The order of infusions was randomized. NHGU was measured with the arterial - venous difference technique. Insulin and glucagon levels were 12 +/- 2, 35 +/- 6, and 36 +/- 5 microU/ml and 55 +/- 12, 61 +/- 13, and 59 +/- 7 pg/ml during the control, peripheral, and portal infusions, respectively. The glucose infusion rate, the load of glucose reaching the liver, and the arterial-portal plasma glucose gradient were 0, 9.58 +/- 2.28, and 10.44 +/- 2.94 mg.kg-1.min-1; 29.4 +/- 3.6, 56.8 +/- 3.4, and 56.8 +/- 2.8 mg.kg-1.min-1; and 2 +/- 1, 5 +/- 1, and -51 +/- 15 mg/dl during the same periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between decrements in glucose level and metabolic response to hypoglycemia in absence of counterregulatory hormones in the conscious dog.

To determine the relationship between decreases in glucose and metabolic regulation in the absence of counterregulatory hormones, we infused overnight-fasted, conscious, adrenalectomized dogs (lacking cortisol and EPI) with somatostatin (to eliminate glucagon and growth hormone) and intraportal insulin (30 pmol.kg-1.min-1), creating arterial insulin levels of approximately 2000 pM. Glucose was infused during one 120-min period, two 90-min periods, and one 45-min period to establish levels of 5.9 +/- 0.1, 3.4 +/- 0.1, 2.5 +/- 0.1, and 1.7 +/- 0.1 mM, respectively. NE levels were 1.24 +/- 0.23, 1.85 +/- 0.27, 2.04 +/- 0.26, and 2.50 +/- 0.20 nM, respectively. During the euglycemic control period, the liver took up glucose (7.5 +/- 1.9 mumol.kg-1.min-1), but hypoglycemia triggered successively greater rates of net hepatic glucose output (3.0 +/- 0.7, 4.6 +/- 0.9, and 6.9 +/- 1.4 mumol.kg-1.min-1). Total gluconeogenic precursor uptake by the liver increased with hypoglycemia. Intrahepatic gluconeogenic efficiency rose progressively (by 106 +/- 42, 199 +/- 56, and 268 +/- 55%). Both glycerol and NEFA levels rose, indicating lipolysis was enhanced. Net hepatic NEFA uptake and ketone production increased proportionally, but the ketone level rose only with severe hypoglycemia. In conclusion, despite marked hyperinsulinemia and the absence of glucagon, EPI, and cortisol, we observed that lipolysis and glucose and ketone production increase in response to decreases in glucose. This suggests that neural and/or autoregulatory mechanisms can play a role in combating hypoglycemia.

Abnormalities of leukocyte histograms resulting from microorganisms.

The authors report a series of 13 patients seen in their laboratory during October 1985 to August 1988 in which the presence of bacterial, fungal, or malarial parasites visible on peripheral smear was correlated with an abnormal leukocyte histogram. Samples submitted for complete blood count and differential counts were analyzed with Coulter S-Plus VI (seven specimens) or S-Plus STKR (six specimens) instrumentation. Organisms visualized on the Wright-stained peripheral smears included Histoplasma capsulatum (two), Candida sp. (four), Plasmodium sp. (three), and Staphylococcus sp. (four). Two patients had a diagnosis of acquired immune deficiency syndrome (AIDS); intravascular catheters were present in five other patients. In all cases the leukocyte histograms were abnormal. The instrument flagged abnormalities of the R1 region in four patients and multiple regions in nine patients. Similar flags were produced by the in vitro addition of bacteria or fungi to whole blood. These studies document that the presence of microorganisms in the peripheral blood can result in spurious white blood cell (WBC) counts or electronic differentials. The authors' findings indicate that the possibility of circulating organisms should be considered when abnormal WBC flags are detected with Coulter instrumentation.

The effects of epinephrine on ketogenesis in the dog after a prolonged fast.

The effects of a selective increase in epinephrine on ketogenesis and lipolysis were determined in the conscious dog following a prolonged fast (7 days). Plasma insulin and glucagon were fixed at basal levels by infusion of somatostatin (0.8 micrograms/kg/min) and basal intraportal replacement amounts of insulin (210 +/- 20 microU/kg/min) and glucagon (0.65 ng/kg/min). Following a 40-minute control period, saline or epinephrine (0.04 microgram/kg/min) was infused for 3 hours. Plasma insulin, glucagon, and norepinephrine levels did not change during saline (6 +/- 1 microU/mL, 83 +/- 17 pg/mL, and 137 +/- 38 pg/mL, respectively) or epinephrine (10 +/- 1 microU/mL, 73 +/- 18 pg/ml, 98 +/- 13 pg/mL, respectively) infusion. Plasma epinephrine levels increased from 80 +/- 26 to 440 +/- 47 pg/mL in response to infusion of the catecholamine, but remained unchanged during saline infusion. Glycerol levels (93 +/- 10 mumol/L) remained unchanged during saline infusion, but increased in response to epinephrine (108 +/- 9 to 170 +/- 18 mumol/L by 30 minutes). The glycerol level had returned to baseline and to the value apparent in saline controls by 60 minutes. The nonesterified fatty acid (NEFA) level declined slowly during the 3-hour saline infusion, but was elevated in response to epinephrine infusion (1.27 +/- 0.16 to 1.97 +/- 0.25 mmol/L at 30 minutes). After the initial epinephrine-induced increase, the NEFA level declined so that by 3 hours it was not significantly different from the basal or saline values.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of small changes in glucagon on glucose production during a euglycemic, hyperinsulinemic clamp.

The aim of this study was to examine the influence of small changes in glucagon on hepatic glucose production during a euglycemic, hyperinsulinemic clamp. During 1.0 mU/kg.min insulin infusion, euglycemia was maintained by glucose infusion and glucagon was infused at various rates so as to cause plasma glucagon levels to increase, decrease, or remain unchanged. Changes in glucagon were found to be positively associated with changes in glucose production and inversely related to the degree of suppression of tracer or arteriovenous difference determined endogenous glucose production. Thus, animals in which the glucagon levels increased, appeared to have decreased hepatic insulin sensitivity, while animals in which glucagon levels decreased, appeared to have increased insulin sensitivity. In conclusion, since glucagon often declines during a euglycemic hyperinsulinemic clamp, and since small changes in glucagon can have marked effects on the suppression of hepatic glucose output even in the presence of high insulin levels, changes in glucagon should be considered when conclusions regarding hepatic insulin sensitivity are being drawn.

Counterregulation by epinephrine and glucagon during insulin-induced hypoglycemia in the conscious dog.

We assessed the combined role of epinephrine and glucagon in regulating gluconeogenic precursor metabolism during insulin-induced hypoglycemia in the overnight-fasted, adrenalectomized, conscious dog. In paired studies (n = 5), insulin was infused intraportally at 5 mU.kg-1.min-1 for 3 h. Epinephrine was infused at a basal rate (B-EPI) or variable rate to simulate the normal epinephrine response to hypoglycemia (H-EPI), whereas in both groups the hypoglycemia-induced rise in cortisol was simulated by cortisol infusion. Plasma glucose fell to approximately 42 mg/dl in both groups. Glucagon failed to rise in B-EPI, but increased normally in H-EPI. Hepatic glucose release fell in B-EPI but increased in H-EPI. In B-EPI, the normal rise in lactate levels and net hepatic lactate uptake was prevented. Alanine and glycerol metabolism were similar in both groups. Since glucagon plays little role in regulating gluconeogenic precursor metabolism during 3 h of insulin-induced hypoglycemia, epinephrine must be responsible for increasing lactate release from muscle, but is minimally involved in the lipolytic response. In conclusion, a normal rise in epinephrine appears to be required to elicit an increase in glucagon during insulin-induced hypoglycemia in the dog. During insulin-induced hypoglycemia, epinephrine plays a major role in maintaining an elevated rate of glucose production, probably via muscle lactate release and hepatic lactate uptake.

The continuing debate on deep molluscan phylogeny: evidence for Serialia (Mollusca, Monoplacophora + Polyplacophora).

Molluscs are a diverse animal phylum with a formidable fossil record. Although there is little doubt about the monophyly of the eight extant classes, relationships between these groups are controversial. We analysed a comprehensive multilocus molecular data set for molluscs, the first to include multiple species from all classes, including five monoplacophorans in both extant families. Our analyses of five markers resolve two major clades: the first includes gastropods and bivalves sister to Serialia (monoplacophorans and chitons), and the second comprises scaphopods sister to aplacophorans and cephalopods. Traditional groupings such as Testaria, Aculifera, and Conchifera are rejected by our data with significant Approximately Unbiased (AU) test values. A new molecular clock indicates that molluscs had a terminal Precambrian origin with rapid divergence of all eight extant classes in the Cambrian. The recovery of Serialia as a derived, Late Cambrian clade is potentially in line with the stratigraphic chronology of morphologically heterogeneous early mollusc fossils. Serialia is in conflict with traditional molluscan classifications and recent phylogenomic data. Yet our hypothesis, as others from molecular data, implies frequent molluscan shell and body transformations by heterochronic shifts in development and multiple convergent adaptations, leading to the variable shells and body plans in extant lineages.

Cenozoic climate change and diversification on the continental shelf and slope: evolution of gastropod diversity in the family Solariellidae (Trochoidea).

Recent expeditions have revealed high levels of biodiversity in the tropical deep-sea, yet little is known about the age or origin of this biodiversity, and large-scale molecular studies are still few in number. In this study, we had access to the largest number of solariellid gastropods ever collected for molecular studies, including many rare and unusual taxa. We used a Bayesian chronogram of these deep-sea gastropods (1) to test the hypothesis that deep-water communities arose onshore, (2) to determine whether Antarctica acted as a source of diversity for deep-water communities elsewhere and (3) to determine how factors like global climate change have affected evolution on the continental slope. We show that although fossil data suggest that solariellid gastropods likely arose in a shallow, tropical environment, interpretation of the molecular data is equivocal with respect to the origin of the group. On the other hand, the molecular data clearly show that Antarctic species sampled represent a recent invasion, rather than a relictual ancestral lineage. We also show that an abrupt period of global warming during the Palaeocene Eocene Thermal Maximum (PETM) leaves no molecular record of change in diversification rate in solariellids and that the group radiated before the PETM. Conversely, there is a substantial, although not significant increase in the rate of diversification of a major clade approximately 33.7 Mya, coinciding with a period of global cooling at the Eocene-Oligocene transition. Increased nutrients made available by contemporaneous changes to erosion, ocean circulation, tectonic events and upwelling may explain increased diversification, suggesting that food availability may have been a factor limiting exploitation of deep-sea habitats. Tectonic events that shaped diversification in reef-associated taxa and deep-water squat lobsters in central Indo-West Pacific were also probably important in the evolution of solariellids during the Oligo-Miocene.

Differential effects of GLUT-1 or GLUT-4 overexpression on insulin responsiveness in transgenic mice.

The effect of glucose transporter expression on insulin-stimulated whole body glucose disposal was examined in transgenic mice overexpressing GLUT-1 or GLUT-4. Transgenic mice and their control littermates were subjected to a euglycemic hyperinsulinemic clamp under pentobarbital sodium anesthesia using an insulin infusion rate of 20 mU.kg-1.min-1 and a variable glucose infusion rate (GIR). Fasted mice overexpressing GLUT-1 in skeletal muscle exhibited a GIR that was only 54% that of controls (19.3 +/- 1.8 vs. 36.0 +/- 3.9 mg.kg-1.min-1) when blood glucose was clamped at euglycemic values. In contrast, fasted mice overexpressing GLUT-4 in fat and muscle exhibited a GIR that was 40% higher than controls (53.9 +/- 2.3 vs. 39.1 +/- 2.5 mg.kg-1.min-1). At the end of the clamp, beta-hydroxybutyrate levels were 10-fold higher in the GLUT-1 transgenic mice relative to nontransgenic littermates (2.0 +/- 0.6 vs. 0.2 +/- 0.1 mM) but did not differ between the GLUT-4 transgenic mice and their control littermates (0.3 +/- 0.1 vs. 0.3 +/- 0.1 mM). These data demonstrate that the level of expression of a glucose transporter in muscle and fat can have marked effects on whole body glucose homeostasis and fuel metabolism. Insulin responsiveness was enhanced by overexpression of GLUT-4. Strikingly, however, overexpression of GLUT-1 in muscle induced a profound reduction in insulin-stimulated whole body glucose disposal.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective insulin receptors in Rabson-Mendenhall syndrome cause complete peripheral insulin resistance but minimal hepatic insulin response remains.

In Rabson-Mendenhall syndrome, severe insulin resistance is caused by defective insulin receptors. The patient studied lacks insulin receptor binding due to a truncation mutation of one allele and a point mutation of the other allele of the insulin receptor alpha-subunit. He developed pulmonary hypertension and cor pulmonale, and was considered for organ transplantation. A trial of prednisone 1.2 mg/kg/d was initiated to determine if he could tolerate immunosuppressive therapy without deterioration of his pre-existing, difficult to control diabetes mellitus. Insulin responsiveness was measured prior to and after 4 d of glucocorticoid administration ('Before GC' and 'After GC') using the hyperinsulinemic glucose clamp and stable isotope tracer dilution techniques. After a 12-h fast and 24 h of intravenous insulin, a primed continuous infusion of 6,6-(2)H(2)-glucose was administered during a 2-h tracer equilibration period followed by a 2-h insulin-deficient period, and a 2-h hyperinsulinemic glucose clamp period during which insulin was infused at 7 u/kg/h. Blood glucose concentrations during the basal periods, while no insulin was infused, were 245+/-7 and 138+/-8 mg/dL in the studies Before GC and After GC, respectively. During both hyperinsulinemic glucose clamp periods, the blood glucose was 171+/-1 and 167+/-5 mg/dL, respectively. Hepatic glucose production (HGP) was higher during the basal period Before GC than during the same period After GC (7.86+/-0.23 vs. 5.31+/-0.19 mg/kg/min). HGP rate was suppressed by insulin to 1.48+/-0.45 mg/kg/min Before GC, but was not suppressed After GC (4.19+/-0.81 mg/kg/min). The hyperinsulinemic glucose clamp did not increase the glucose utilization rate nor the glucose clearance rate over basal in either Before GC or After GC, indicating complete peripheral insulin resistance. In summary, the liver showed some response to insulin in the absence of insulin receptors but the peripheral tissues had no response to insulin. Glucocorticoids worsened insulin resistance in the liver in this patient.

Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle.

Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.

Transgenic overexpression of hexokinase II in skeletal muscle does not increase glucose disposal in wild-type or Glut1-overexpressing mice.

Glut1 transgenic mice were bred with transgenic mice that overexpress hexokinase II in skeletal muscle in order to determine whether whole-body glucose disposal could be further augmented in mice overexpressing glucose transporters. Overexpression of hexokinase alone in skeletal muscle had no effect on glucose transport or metabolism in isolated muscles, nor did it alter blood glucose levels or the rate of whole-body glucose disposal. Expression of the hexokinase transgene in the context of the Glut1 transgenic background did not alter glucose transport in isolated muscles but did cause additional increases in steady-state glucose 6-phosphate (3.2-fold) and glycogen (7.5-fold) levels compared with muscles that overexpress the Glut1 transporter alone. Surprisingly, however, these increases were not accompanied by a change in basal or insulin-stimulated whole-body glucose disposal in the doubly transgenic mice compared with Glut1 transgenic mice, probably due to an inhibition of de novo glycogen synthesis as a result of the high levels of steady-state glycogen in the muscles of doubly transgenic mice (430 micromol/g versus 10 micromol/g in wild-type mice). We conclude that the hexokinase gene may not be a good target for therapies designed to counteract insulin resistance or hyperglycemia.

Targeted overactivity of beta cell K(ATP) channels induces profound neonatal diabetes.

A paradigm for control of insulin secretion is that glucose metabolism elevates cytoplasmic [ATP]/[ADP] in beta cells, closing K(ATP) channels and causing depolarization, Ca2+ entry, and insulin release. Decreased responsiveness of K(ATP) channels to elevated [ATP]/[ADP] should therefore lead to decreased insulin secretion and diabetes. To test this critical prediction, we generated transgenic mice expressing beta cell K(ATP) channels with reduced ATP sensitivity. Animals develop severe hyperglycemia, hypoinsulinemia, and ketoacidosis within 2 days and typically die within 5. Nevertheless, islet morphology, insulin localization, and alpha and beta cell distributions were normal (before day 3), pointing to reduced insulin secretion as causal. The data indicate that normal K(ATP) channel activity is critical for maintenance of euglycemia and that overactivity can cause diabetes by inhibiting insulin secretion.

Differential effects of GLUT1 or GLUT4 overexpression on hexosamine biosynthesis by muscles of transgenic mice.

Transgenic mice that overexpress GLUT1 or GLUT4 in skeletal muscle were studied; the former but not the latter develop insulin resistance. Because increased glucose flux via the hexosamine biosynthesis pathway has been implicated in glucose-induced insulin resistance, we measured the activity of glutamine:fructose-6-phosphate amidotransferase (GFAT; rate-limiting enzyme) and the concentrations of UDP-N-acetyl hexosamines (major products of the pathway) as well as UDP-hexoses and GDP-mannose in hind limb muscles and liver in both transgenic models and controls. GFAT activity was increased 60-70% in muscles of GLUT1 but not in GLUT4 transgenics. GFAT mRNA abundance was unchanged. The concentrations of all nucleotide-linked sugars were increased 2-3-fold in GLUT1 and were unchanged in GLUT4-overexpressing muscles. Similar results were obtained in fed and fasted mice. GFAT and nucleotide sugars were unchanged in liver, where the transgene is not expressed. We concluded that 1) glucose transport appears to be rate limiting for synthesis of nucleotide sugars; 2) chronically increased glucose flux increases muscle GFAT activity posttranscriptionally; 3) increased UDP-glucose likely accounts for the marked glycogen accumulation in muscles of GLUT1-overexpressing mice; and 4) glucose flux via the hexosamine biosynthetic pathway is increased in muscles of GLUT1-overexpressing but not GLUT4-overexpressing mice; products of the pathway may contribute to insulin resistance in GLUT1 transgenics.

Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes.

The Glut4 glucose transporter is poorly functional compared with other glucose transporter isoforms when expressed in Xenopus oocytes. To investigate the molecular basis for this poor functionality, we compared the biosynthesis and targeting of Glut1 and Glut4 in oocytes after microinjection of the corresponding mRNAs. Both Glut1 and Glut4 were present as lower molecular weight endoglycosidase H-sensitive and higher molecular weight endoglycosidase H-resistant. Subcellular fractionation indicated that Glut1 was targeted to the plasma membrane with a 6.6-fold greater efficiency than was Glut4. Confocal immunofluorescence microscopy confirmed the relative enrichment of Glut1 in the plasma membrane and the efficient intracellular sequestration of Glut4. As in mammalian cells, the endoglycosidase H-resistant form of Glut4 was concentrated in low-density intracellular vesicles, whereas Glut1 was distributed in intracellular vesicles of higher average density. The structural basis for the differential localization of Glut1 and Glut4 was investigated by determining the plasma membrane content of a series of chimeric Glut1/Glut4 molecules. These data indicated that two distinct regions of Glut4, encompassing residues 24-132 and the COOH-terminal cytoplasmic tail, confer intracellular sequestration on the chimeric transporter molecules. At least part of the sequestration effect of the more N-terminal domain was due to the incomplete maturation of chimeras containing this region, resulting in the accumulation of lower molecular weight endoglycosidase H-sensitive and endoglycosidase H-resistant forms, whereas the COOH-terminal cytoplasmic tail conferred sequestration of fully glycosylated chimeras in a low-density intracellular membrane compartment.

Discrete structural domains determine differential endoplasmic reticulum to Golgi transit times for glucose transporter isoforms.

The rate of movement of the glucose transporter isoforms Glut1 and Glut4 from the endoplasmic reticulum (ER) to the Golgi apparatus was investigated by pulse labeling and monitoring endoglycosidase H resistance in mRNA-injected Xenopus oocytes and in 3T3-L1 adipocytes, a cell line that naturally expresses both transporter isoforms. Despite their high degree of sequence identity, Glut1 and Glut4 exhibited dramatically different transit times. The t1/2 values for ER to Golgi transit for Glut1 and Glut4 were < 1 and 24 h, respectively, in oocytes and approximately 5 and 20 min, respectively, in 3T3-L1 adipocytes. Pulse-chase in conjunction with sucrose density gradient analysis revealed that the rate-limiting step in the ER to Golgi processing of Glut4 was exit from the ER and not retention in an early Golgi compartment. We analyzed the biosynthesis of Glut1/Glut4 chimeric transporters in Xenopus oocytes in order to determine whether specific domains in Glut1 and Glut4 were responsible for their distinct transit times. The first exofacial glycosylated loop and the cytoplasmic carboxyl-terminal domain of Glut4 were crucial for its delayed exit from the ER. The first transmembrane, the first exofacial, and the cytoplasmic COOH-terminal domains of Glut1 were largely responsible for Glut1's rapid processing in the ER. Some of the chimeric transporters were not fully processed. Approximately 50% of chimeric molecules containing the cytoplasmic COOH-terminal domain of Glut1 and either the first transmembrane or first exofacial domain of Glut4 were retained in early Golgi compartments and prevented from complete maturation. Normal processing of these chimeras was achieved by replacing the cytoplasmic COOH-terminal domain of Glut1 with that of Glut4. These data suggest that amino acid residues within the glycosylated exofacial loop and the cytoplasmic COOH terminus participate in a rate-limiting step in the folding of both Glut1 and Glut4 or could act as transient ER retention signals. Additionally, these results show that even chimeric molecules constructed from two highly homologous proteins can exhibit aberrant folding and post-translational processing.