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Gastrointestinal mucosal polyamines influence enterocyte proliferation and differentiation during small intestinal maturation in the rat. Studies in postnatal rats have shown that ornithine decarboxylase (ODC) protein and mRNA peak before the maximal expression of brush-border membrane (BBM) sucrase-isomaltase (SI) and the sugar transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). This study was undertaken to test the hypothesis that the oral administration of spermidine in postnatal rats upregulates the expression of ODC, thereby enhancing the expression of SI and SGLT1 in the brush-border membrane as well as basolateral membrane-facilitative GLUT2 and Na+-K+-ATPase. Northern and Western blot analyses were performed with antibodies and cDNA probes specific for SI, SGLT1, GLUT2, α1- and β1-subunits of Na+-K+-ATPase, and ODC. Postnatal rats fed 6 μmol spermidine daily for 3 days from days 7 to 9 were killed either on postnatal day 10 (Sp10) or day 13 following a 3-day washout period (Sp13). Sp10 rats showed a precocious increase in the abundance of mRNAs for SI, SGLT1, and GLUT2 and Na+-K+-ATPase activity and α1- and β1-isoform gene expression compared with controls. ODC activity and protein and mRNA abundance were also increased in Sp10 animals. The increased expression of these genes was not sustained in Sp13 rats, suggesting that these effects were transient. Thus, 3 days of oral polyamine administration induces the precocious maturation of glucose transporters in the postnatal rat small intestine, which may be mediated by alterations in ODC expression.11 Supplemental material for this article is available online at the American Journal of Physiology-Gastrointestinal and Liver Physiology website.

Premature infants receiving chronic total parenteral nutrition (TPN) due to feeding intolerance develop intestinal atrophy and reduced nutrient absorption. Although providing the intestinal trophic hormone glucagon-like peptide-2 (GLP-2) during chronic TPN improves intestinal growth and morphology, it is uncertain whether GLP-2 enhances absorptive function. We placed catheters in the carotid artery, jugular and portal veins, duodenum, and a portal vein flow probe in piglets before providing either enteral formula (ENT), TPN or a coinfusion of TPN plus GLP-2 for 6 days. On postoperative day 7, all piglets were fed enterally and digestive functions were evaluated in vivo using dual infusion of enteral (13C) and intravenous (2H) glucose, in vitro by measuring mucosal lactase activity and rates of apical glucose transport, and by assessing the abundances of sodium glucose transporter-1 (SGLT-1) and glucose transporter-2 (GLUT2). Both ENT and GLP-2 pigs had larger intestine weights, longer villi, and higher lactose digestive capacity and in vivo net glucose and galactose absorption compared with TPN alone. These endpoints were similar in ENT and GLP-2 pigs except for a lower intestinal weight and net glucose absorption in GLP-2 compared with ENT pigs. The enhanced hexose absorption in GLP-2 compared with TPN pigs corresponded with higher lactose digestive and apical glucose transport capacities, increased abundance of SGLT-1, but not GLUT-2, and lower intestinal metabolism of [13C]glucose to [13C]lactate. Our findings indicate that GLP-2 treatment during chronic TPN maintains intestinal structure and lactose digestive and hexose absorptive capacities, reduces intestinal hexose metabolism, and may facilitate the transition to enteral feeding in TPN-fed infants.

Although induction of cytochrome P-450 1A1 (CYP1A1) in the Caco-2 clone TC7 alters glucose utilization and modifies the expression of sucrase-isomaltase (SI) and hexose transporters, nothing is known of the events that control these effects. In this study, we analyzed the effects of β-naphthoflavone (β-NF) and hypoxia on these parameters and expression of key enzymes of glucose metabolism. Both β-NF and hypoxia induce similar changes: 1) induction of CYP1A1 mRNA; 2) increased glucose consumption and lactic acid production and lower glycogen content; 3) downregulation of SI and upregulation of GLUT1 mRNAs; 4) downregulation of fructose-1,6-bisphosphatase and pyruvate kinase mRNAs and upregulation of phospho enolpyruvate carboxykinase, pyruvate dehydrogenase, lactate dehydrogenase, and phosphofructokinase mRNAs; and 5) upregulation of c- fos and c- jun mRNAs. Although addition of inhibitors of CYP1A1 catalytic activity to β-NF-treated cells totally inhibits the enzyme activity, it does not modify CYP1A1 mRNA response and associated effects, thus excluding a direct role for the enzyme per se. These results point to a possible physiological implication of the signal-transduction pathway responsible for CYP1A1 induction.

The characteristics of the basolateral transport of D-glucose (D-Glc) and D-fructose (D-Fru) have been studied in membrane vesicles from the jejunum of 5- to 6-wk-old chickens. Uptake of hexoses was measured using a rapid filtration method. The maximal rate of transport (Vmax) for D-Glc was 2.36 nmol x mg(-1) x s(-1) and for D-Fru was 3.79 nmol x mg(-1) x s(-1). The Michaelis constants were 17.3 mmol/l for D-Glc and 40.4 mmol/l for D-Fru. D-Glc inhibited its own transport (Ki = 17.4 mmol/l) and the transport of D-Fru (Ki = 18.7 mmol/l). D-Fru inhibited its own transport (Ki = 38.1 mmol/l) and the transport of D-Glc (Ki = 40.3 mmol/l). The transport of both hexoses was Na+ independent, theophylline and cytochalasin B sensitive, and showed cis-inhibition by structural analogs. In preloaded vesicles, the uptake of D-Fru was trans-stimulated by D-Glc and 2-deoxy-D-glucose. These properties indicate the presence of a low-affinity high-capacity glucose transporter isoform (GLUT-2)-type carrier in the chicken intestine, responsible for moving both D-Glc and D-Fru across the basolateral membrane.

An in situ dual vascular and luminal perfusion technique was used to study the effect of cholecystokinin octapeptide (CCK-8) on the transport of hexoses by the jejunum of the Sprague-Dawley rat from the lumen to the vascular bed. The lumen of the jejunumwas perfused with hexoses in oxygenated Krebs buffer, while the superior mesenteric artery was infused with Krebs buffer containing Ficoll 70 as a plasma expander. CCK-8 (0.8–8 pM) in the vascular infusate selectively reduced hexose transport in a dose-dependent manner by 20–47%, although having no effect on L-glucose or L-leucine absorption. Vascular tetrodotoxin did not block CCK-8 inhibition, whereas a specific CCK-A receptor antagonist, lorglumide, did. The CCK-B receptor agonist cholecystokinin tetrapeptide had a small effect on hexose absorption, whereas somatostatin-14 and -28 had no effect. These results suggest that cholecystokinin can decrease intestinal absorption of hexoses in the small intestine, acting via CCK-A-type receptors.

The kinetic parameters of hexose uptake by the small and large intestine of the chicken have been determined in vitro. Rates of initial influx of alpha-methyl-D-glucoside and L-glucose were measured in everted sleeves of the duodenum, jejunum, ileum, proximal cecum, and rectum. Results show the following. 1) Maximal transport capacity values for alpha-methyl-D-glucoside show that the jejunum is the segment that is best suited for Na(+)-mediated uptake. 2) The calculated apparent Michaelis constant values were (in mmol/l) 11.6 for duodenum, 7.8 for jejunum, 3.5 for ileum, 2.4 for proximal cecum, and 7.1 for rectum. This suggests that, with the exception of the rectum, the affinity of the carrier for alpha-methyl-D-glucoside progressively increases in the distal direction. 3) Diffusion constant values indicate that influx of hexoses by a passive mechanism in the duodenum and proximal cecum is significantly higher than in the other segments. 4) The sum of passive and mediated mechanisms confers to the duodenum and jejunum a high capacity to absorb hexoses. The ileum, proximal cecum, and rectum have a quantitatively minor role, albeit significant, in completing the absorptive function.

The effect of gastric inhibitory polypeptide (GIP) and the related glucagon-like peptides-1 and -2 (GLP-1 and GLP-2) on jejunal basolateral membrane glucose transport was investigated to determine if the upregulation produced by luminal hexoses could be explained by the release of one or more of these peptides. Luminal perfusion of the rat jejunum for 4 h, under pentobarbital sodium anesthesia, with 100 mM D-glucose produced a significant increase in plasma GIP levels. Vascular infusion of saline containing 100-800 pM GIP also increased the maximal transport rate for carrier-mediated glucose uptake in jejunal basolateral membrane vesicles. The effect of vascular 400 pM GIP was maximal after 1 h and maintained out to 4 h. The effect of luminal glucose could be blocked by preinjection with anti-GIP antibodies, whereas an antineurotensin antibody had no effect. Vascular infusion with 800 pM GLP-1-(7-36) amide had no effect, but GLP-2 (400 and 800 pM) increased the D-glucose maximal transport rate. An anti-GLP antibody was able to block the response to luminal glucose.

Five functional mammalian facilitated hexose carriers (GLUTs) have been characterized by molecular cloning. By functional expression in heterologous systems, their specificity and affinity for different hexoses have been defined. There are three high-affinity transporters (GLUT-1, GLUT-3 and GLUT-4) and one low-affinity transporter (GLUT-2), and GLUT-5 is primarily a fructose carrier. Because their Michaelis constants (Km) are below the normal blood glucose concentration, the high-affinity transporters function at rates close to maximal velocity. Thus their level of cell surface expression greatly influences the rate of glucose uptake into the cells. In contrast, the rate of glucose uptake by GLUT-2 (Km = 17 mM) increases in parallel with the rise in blood glucose over the physiological concentration range. High-affinity transporters are found in almost every tissue, but their expression is higher in cells with high glycolytic activity. Glut-2, however, is found in tissues carrying large glucose fluxes, such as intestine, kidney, and liver. As an adaptive response to variations in metabolic conditions, the expression of these transporters is regulated by glucose and different hormones. Thus, because of their specific characteristics and regulated expression, the facilitated glucose transporters control fundamental aspects of glucose homeostasis. I review data pertaining to the structure and regulated expression of the glucose carriers present in intestine, kidney, and liver and discuss their role in the control of glucose flux into or out of these different tissues.

Hydrogen peroxide (H2O2) may incite cardiac ischemia-reperfusion injury. We evaluate herein the influence of H2O2-induced oxidative stress on heart muscle hexose metabolism in cultured neonatal rat cardiomyocytes, which have a substrate preference for carbohydrate. Cardiomyocyte exposure to 50 microM-1.0 mM bolus H2O2 transiently activated the pentose phosphate cycle and thereafter inhibited cellular glucose oxidation and glycolysis. These metabolic derangements were nonperoxidative in nature (as assessed in alpha-tocopherol-loaded cells) and occurred without acute change in cardiomyocyte hexose transport or glucose/glycogen reserves. Glycolytic inhibition was supported by the rapid, specific inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The degree of GAPDH inhibition correlated directly with the magnitude of the oxidative insult and was independent of both metal-catalyzed H2O2 reduction to free radicals and lipid peroxidation. Severe GAPDH inhibition was required for a rate-limiting effect on glycolytic flux. Cardiomyocyte pyruvate dehydrogenase was also inhibited by H2O2 overload, but to a lesser degree than GAPDH such that entry of hexose-derived acetyl units into the tricarboxylic acid cycle was not as restrictive as GAPDH inactivation to glycolytic ATP production. An increase in phosphofructokinase activity accompanied GAPDH inactivation, leading to the production and accumulation of glycolytic sugar phosphates at the expense of ATP equivalents. Cardiomyocyte treatment with iodoacetate or 2-deoxyglucose indicated that GAPDH inactivation/glycolytic blockade could account for approximately 50% of the maximal ATP loss following H2O2 overload. Partial restoration of GAPDH activity after a brief H2O2 "pulse" afforded some ATP recovery. These data establish that specific aspects of heart muscle hexose catabolism are H2O2-sensitive injury targets. The biochemical pathology of H2O2 overload on cardiomyocyte carbohydrate metabolism has implications for post-ischemic cardiac bioenergetics and function.

The human colon carcinoma cell line Caco-2 was used as an enterocyte model to study the expression of the facilitative glucose transporters GLUT-1 and GLUT-2, and of the putative hexose transporter GLUT-5, which are expressed specifically in the gut. Northern blots indicate that Caco-2 cells express GLUT-1 and GLUT-5 mRNAs but not the mRNA coding for the basolateral glucose transporter GLUT-2. The level of GLUT-5 mRNA is growth dependent, being detectable only in postconfluent differentiated cells. In addition, the expression of GLUT-5 increases with the number of cell passages and is approximately 10 times higher in later passages (passage 184) than in early ones (passage 26). With the use of polyclonal antibodies directed against the COOH-terminus of GLUT-5, indirect immunofluorescence and Western blotting indicate that GLUT-5 is mainly localized to the brush border of Caco-2 cells. GLUT-5 is also found to be associated with the brush border of epithelial cells from fetal and normal adult human small intestine, but is absent from the colon.

Piglets in three age groups (1-3, 9-11, and 16-25 days after birth) were used for in vivo colonic perfusions. Studies compared an isosmolar (312 mosM) with a high osmolar (551 mosM) solution and two equimolar substrates (with hexose concentrations of 73.1 mM), lactose and glucose-galactose. From the isosmolar perfusates, lactose absorption was 0.43 +/- 0.04 in the 18-20 day olds and 1.04 +/- 0.2 mumol.cm-1.min-1 in the 1-3 day olds; absorption from the glucose-galactose solution was negligible in all age groups (less than 0.05 +/- 0.05 mumol.cm-1.min-1). From the high osmolar perfusate, lactose absorption also exceeded that of glucose and galactose. In a third set of perfusion studies, the concentration of lactose was varied between 15 and 240 mM perfusate. Five-day-old animals absorbed 67% more lactose than 18-day-old animals; the right colon absorbed 57% more than the left. Lactose absorption, correlated with its concentration in the perfusate (r = 0.99), was nonsaturable at concentrations up to 240 mM, and was correlated with the uptake both of sodium (r2 = 0.59 for young and 0.64 for older neonates) and of chloride (r2 = 0.55 for young and 0.31 for older neonates). The results suggest that lactose may be removed from the colon without apparent cleavage by beta-galactosidase.

We measured brush-border uptakes of seven sugars and amino acids by rabbit intestine as a function of age from the day of birth to adulthood. Gut dimensions, especially those of the colon and cecum, increase more rapidly with body weight than would be true if rabbits maintained identical proportions as they grew. However, nominal small intestinal area increases in approximately direct proportion to the animal's basal metabolic rate. For all solutes except fructose, uptake per milligram of intestinal tissue is maximal at or near birth and declines to a level 2.5-5 times lower in the adult. Because of small intestinal growth, though, the total uptake capacity of the whole length of the small intestine increases in approximately direct proportion to metabolic rate. Fructose uptake per milligram is unique in increasing steeply at the time of weaning, correlated with the post-weaning first appearance of fructose in the natural diet. Age-related changes in uptake ratios among aldohexoses or amino acids suggest developmental sequences of related transporters. Correlated with the very high protein content of rabbit milk, the proline-to-glucose uptake ratio is higher in suckling rabbits than in other sucking mammals. Remarkably, the ratio for adult rabbits is higher than in other monogastric herbivores and is instead similar to values for carnivores. In explanation, although the transport capacity of the small intestine appears to account for proline absorption in rabbits of all ages and for sugar absorption in suckling rabbits, the hindgut may be a major site of carbohydrate digestion in adult rabbits.

glucose transport facilitators (GLUT proteins) mediate the transport of glucose and other monosaccharides into and out of mammalian cells and therefore play a pivotal role in maintaining blood glucose concentrations in a normal range between 4.5 and 6 mM ([14][1]). In addition, GLUT proteins are

The regulation of glycogenolysis in human muscle during epinephrine infusion has been investigated. The content of cAMP in resting muscle was 2.7 +/- 0.7 (SD) mumol . kg dry muscle-1 and increased threefold during the first 5 min of infusion. Total glycogen phosphorylase and glycogen synthase activities were unchanged during the infusion. The proportion of phosphorylase in the a form in the basal state was estimated to be at least 22.5% and during infusion 80–90%. During infusion, synthase I activity decreased. The muscle glycogen content was 340 mmol . kg dry wt-1 and decreased during the first 2 min of infusion at a rate of 11.0 mmol glycosyl units . kg dry wt-1 . min-1. Prolonged infusion resulted in a much lower glycogenolytic rate, even though most of the phosphorylase was still in the a form. Accumulation of hexose monophosphates and lactate followed the changes in glycogen. It was concluded that despite the almost total transformation of phosphorylase to the a form, the in vivo activity was maintained at a low level. It is suggested that this may be due to a low concentration of inorganic phosphate at the active site of the enzyme.

The ability of the synthetic glucocorticoid, dexamethasone, to alter 3-O-methylglucose transport was investigated using isolated rat adipocytes. A maximally effective dose of dexamethasone (10(-7) M) inhibited transport up to 80% within 60–90 min. Inhibition of transport was evident as early as 15–30 min after addition of steroid, and was prevented by both actinomycin D and cycloheximide. When added within 45 or 60 min after dexamethasone, actinomycin D interfered with the cells' ability to respond to the steroid but had no effect when added between 60 and 90 min or longer after the steroid. Cycloheximide interfered with steroid-induced inhibition of transport when added at any time before the 15- to 30-min period immediately preceding the transport assay. This interference with hormone action appeared to be independent of the length of time cells were exposed to dexamethasone before addition of cycloheximide. Thus cells that were maximally inhibited by dexamethasone by 90 min became only partially inhibited when cycloheximide was added at 90 or 120 min, and cells were incubated for an additional 60 or 30 min, respectively. These findings are consistent with the following: dexamethasone inhibits glucose oxidation as a result of inhibiting hexose transport; inhibition of transport by dexamethasone requires the synthesis of RNA during the first 45–60 min after steroid addition and requires protein synthesis during the entire incubation period with dexamethasone; and transport is inhibited within minutes after protein synthesis is initiated.

The effects of insulin on 3-O-[14C]methylglucose transport across the blood-brain barrier (BBB) were studied in conscious rats under steady-state normoglycemic conditions. The [14C]methylglucose was infused intravenously at a constant rate, and animals were killed at various times between 5 and 30 min after the initiation of the infusion. The time course of the arterial plasma concentration of [14C]methylglucose was determined in timed arterial blood samples taken during the infusion. Local cerebral tissue concentrations of [14C]methylglucose at the time of killing were determined by quantitative autoradiography of brain sections. The rate constants for inward and outward transport of [14C]methylglucose across the BBB, K1, and k2, respectively, were estimated by a least-squares, best-fit of a kinetic equation to the measured time courses of plasma and tissue concentrations. K1 and k2 were reduced by an average of 24 and 31%, respectively, in gray matter and 7 and 16% in white matter from values estimated similarly in normal insulinemic control rats. The equilibrium distribution ratio, K1/k2, for [14C]methylglucose in brain increased by approximately 10–11% in the hyperinsulinemic animals. Because 3-O-[14C]methylglucose shares the same carrier that transports glucose and other hexoses across the BBB, these results suggest that hyperinsulinemia decreases the rate constants for transport but increases the distribution space for hexoses in brain. These effects are, however, quite small and are probably minor or negligible when compared with the major effects of insulin in other tissues.

Adipocytes accumulate 2-deoxy-D-glucose (2-DG) against significant chemical gradients (J. Foley and J. Gliemann. Biochim. Biophys. Acta 648: 100-106, 1981). The specificity of this accumulation process was examined. Isolated rat adipocytes incubated at 37 degrees C with 0.1 mM hexoses accumulated free 2-deoxy-D-galactose (2-DGal) or D-galactose (Gal) against steady-state gradients of 5.5 +/- 1.2 and 2.5 +/- 0.7, respectively. Like 2-DG, both 2-DGal and Gal are substrates for phosphorylation. Insulin and insulin-mimetic agents increased steady-state accumulations of Gal, 2-DGal, and free and phosphorylated 2-DG (100 nM insulin increased free 2-DG from 2.1 +/- 0.2 to 6.6 +/- 0.3 mM; external 2-DG = 0.1 mM). Removal of extracellular calcium or sodium or the presence of A23187 or ouabain failed to inhibit 2-DG accumulation. Plasma membrane permeabilization induced by either digitonin or high-voltage discharge produced a loss of cellular 2-DG and 2-DG-phosphate without affecting 3-O-methyl-D-glucose equilibration. The data indicate that neither transmembrane ionic gradients nor intracellular compartmentation suffice as explanations of the mechanism of the accumulation process. The possible role of phosphorylation in the process of hexose accumulation is discussed.

Tumoral insulin-producing cells of the RINm5F line were exposed at different temperatures, and for various lengths of time to increasing concentrations of 3-O-methyl-D-[U-14C]glucose. The uptake of the hexose represented a temperature-sensitive and saturable process, so that no rapid equilibration of hexose concentrations across the plasma membrane was reached, especially at low temperature and/or high concentrations of 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-[U-14C]glucose was not affected by a prior loading of the cells with the unlabeled hexose and its release from prelabeled cells was observed in the absence of any concentration gradient across the plasma membrane. The uptake of D-[U-14C]glucose and utilization of D-[5-3H]glucose was inhibited by 3-O-methyl-D-glucose, which failed, however, to affect D-[U-14C]glucose oxidation. At variance with the situation found in normal insulin-producing cells, the transport of D-glucose into the tumoral cells may thus play a regulatory role in its metabolism.

The uptake of D-[U-14C]glucose and D-[5-3H]glucose by tumoral insulin-producing cells of the RINm5F line was measured over 3-30 min of incubation at 7-37 degrees C. The apparent distribution space of the hexose ranged from values as low as the L-[1-14C]glucose space to values 10 times higher than the 3H2O space. Although a major fraction of the radioactivity recovered in the cellular pellet corresponded to the metabolites of D-glucose, the results suggested that the transport of D-glucose into the tumoral cells represents a saturable and temperature-dependent process. When D-glucose was measured by an enzymic procedure in cells incubated for 30 min at 37 degrees C in the presence of 50.0 mM D-glucose, the apparent distribution space of the hexose remained lower than the intracellular water space. These results indicate that the RINm5F cells have lost an essential attribute of the glucose-sensing device in normal insulin-producing cells, namely the ability to ensure the equilibration of D-glucose concentrations across the plasma membrane.

Glutamate, beta-alanine, choline, and myoinositol transport was characterized in jejunal slices and brush-border membrane vesicles from 2- and 21-day chicks. Carrier-mediated, i.e., competitor-inhibitable, transport in slices (wet weight basis) and vesicles (protein basis) declined with age for the two amino acids but increased with age for myo-inositol; transport of choline did not change. These findings, along with previous data for the hexose system, cannot be explained solely by nonspecific changes in brush-border membrane architecture or permeability or by changes in the electrochemical sodium gradient that drives cotransport. They indicate that each specific brush-border membrane transport mechanism follows separate developmental time tables.

Amiloride and four analogues of amiloride were shown to inhibit Na+-dependent, phlorizin-sensitive hexose uptake by a clone of pig kidney cells, LLC-PK1/Cl4. The analogues tested were: 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 5-(N-methyl-N-isobutyl)amiloride (MIBA), 3',4'-dichlorobenzamil, and phenamil. The transport substrate was the nonmetabolizable glucose analogue alpha-methyl-D-glucoside. Blockade of Na+-K+ transport at the basolateral membranes or removal of divalent cations from the assay medium had little effect on the initial rate of hexose uptake, whereas MIBA remained an effective inhibitor under both conditions. The inhibitions by EIPA of Na+-H+ exchange and hexose-dependent Na+ uptake could be distinguished by appropriate choice of concentrations of the inhibitor. Hexose transport inhibition does not appear to be secondary to other known effects of the amilorides. Inhibition by all analogues is enhanced when they are tested in low (2 mM) Na+ medium, where they show half-maximal inhibition in the range of 100-300 microM. More detailed kinetic analysis of inhibition by EIPA shows it to be competitive with Na+ with a Ki of 73-107 microM. It is concluded that the amilorides are acting directly on the hexose transporter.

The L6 muscle cell line is proposed as an excellent cell culture system for studying glucose transport and its regulation by serum and insulin throughout myogenesis. The rate of hexose uptake decreased after cell fusion. Fetal calf serum (5 h) stimulated hexose uptake, and serum deprivation depressed it. Both effects were prevented by cycloheximide. Insulin stimulated the uptake of either 2-deoxy-D-glucose or 3-O-methyl-D-glucose provided that the basal rate was previously depressed by serum deprivation. The response to the hormone was significant only after the onset of cell fusion. Stimulation of transport was observed within the first 10 min after insulin administration and was insensitive to cycloheximide. Half-maximal stimulation of uptake was obtained with 5 X 10(-8) M insulin in 1% albumin solutions. Basal and insulin-stimulated uptake showed equal sensitivity to cytochalasin B. However, they differed in their sensitivity to extracellular Ca2+: basal transport was depressed, whereas insulin-stimulated transport was further enhanced in Ca2+-free media. Advantages of the cell culture system are discussed.

It has been proposed that Ca2+ ions mediate the stimulation by insulin of glucose uptake in muscle (Clausen, T., Cell Calcium 1:311–325, 1980). However, absolute measurements of the concentration of cytosolic free Ca2+, [Ca2+]i, during the course of insulin action have not been made. The stimulation of hexose uptake by insulin was studied in an in vitro model system of muscle cells, the L6 cell line. The following evidence suggests that Ca2+ ions are not likely to fulfill the purported role. 1) Insulin in Ca2+-free media induced stimulation of 2-deoxy-D-glucose uptake. 2) Elevation of [Ca2+]i with the ionophore A23187 did not enhance hexose uptake. 3) Insulin action was not diminished when the hormone was added to Ca2+-depleted cells in Ca2+-free media with A23187. 4) Hexose uptake was not affected by a number of agents thought to modify [Ca2+]i including epinephrine, caffeine, 2,4-dinitrophenol, hyperosmolar mannitol, salicylate, vanadate, veratrine, and trypsin. 5) Direct determinations of [Ca2+]i by fluorescence of the novel indicator Quin-2 did not show differences between basal and insulin-stimulated cells; under identical conditions hexose uptake was stimulated by the hormone. 6) Chelation of [Ca2+]i with Quin-2 in Ca2+-free media did not affect the response to insulin. 7) Low concentrations of trypsin (7.5 micrograms/ml) elevated [Ca2+]i but did not increase the rate of hexose uptake.

Intravenous infusions of fructose, a hexose that does not cross the blood-brain barrier, suppressed insulin-induced gastric acid secretion and electromyographic (EMG) activity despite continuing hypoglycemia. Hepatic portal vein infusions of 0.15 M fructose inhibited acid output while the same concentration delivered via the jugular vein did not, suggesting a hepatic site of action of the hexose. Only infusions of fructose that began before onset of the insulin-induced gastric responses were effective, whereas glucose infusions, which elevated plasma glucose levels, readily reversed ongoing gastric activity. The suppressive effects of fructose on gastric activity were prevented by prior section of the hepatic branch of the vagus nerve. In contrast, hepatic vagotomy did not prevent suppression of gastric responses by infusions of glucose, a hexose utilized by both brain and liver. These results suggest that receptors in the brain may initiate and terminate insulin-induced gastric acid secretion and motor activity, whereas sensors in the liver may inhibit these responses.

The regulation of hexose transport by growth hormone (GH) was investigated using isolated rat adipocytes. GH caused a rapid (less than 3 min) rise in rates of 3-O-methylglucose transport that reached a maximum of two to six times the basal rates in 10-30 min. The stimulation of transport was transitory, and rates of transport started to decline 15-30 min after GH was added. Transport stimulation required a period of preincubation; no stimulation was observed in freshly isolated cells. GH stimulated hexose transport between 100 and 5,000 ng/ml, with a 50% effective dose between 200 and 300 ng/ml. Depletion of cellular ATP by 2,4-dinitrophenol blocked the ability of GH to stimulate transport but not the decline of transport rates following stimulation by GH. In contrast, an inhibitor of RNA synthesis, actinomycin D, had no effect on either the initial stimulation by GH or the initial subsequent decline of transport when added simultaneously or 15 min prior to GH. Actinomycin D did, however, cause a second rise in hexose transport at approximately 120 min that was blocked by 2,4-dinitrophenol. These results suggest that changes in glucose transport contribute to the effects of GH on carbohydrate and lipid metabolism in adipose tissue. These changes are rapid, of substantial magnitude, and of a complex nature, suggesting that regulation of glucose transport by GH most likely involves multiple mechanisms.

Hepatocytes obtained from sucrose-fed rats secreted triacylglycerol at a rate that was about twice that of cells from control rats. The increased rate of triacylglycerol secretion by cells from sucrose-fed rats was accompanied by a twofold increase in its rate of synthesis as determined by 3H2O incorporation. In addition, cells from sucrose-fed rats had a two- to threefold increase in apolipoprotein synthesis. Differences between the two groups became even more marked when cells were challenged in vitro with glucose. Double-reciprocal analysis showed that compared with controls, cells from sucrose-fed rats had a fourfold increase in the Vmax that described the glucose stimulation of [3H]triacylglycerol secretion. In contrast to in vivo carbohydrate (sucrose) induction of apolipoprotein synthesis, glucose added in vitro did not affect apolipoprotein synthesis. These data suggest that in vivo induction by dietary carbohydrate requires factors in addition to increased hexose that are not contained within the isolated hepatocyte system. The coinduction by dietary carbohydrate of both lipogenesis and apolipoprotein synthesis is likely to play a role in the increased capacity of cells from sucrose-fed rats to both assemble and secrete triacylglycerol-rich lipoproteins.

Properties of an enzyme isolated from rat intestine and rat kidney and which catalyzes mutarotation of sugars are described. Mutarotation of d-xylose, d-glucose, d-galactose, and l-arabinose was accelerated by the enzyme. Interaction of a number of sugars with the active site was examined by measuring inhibition of enzyme-catalyzed mutarotation of α-d-glucose. Based on this criterion, two sugars with no mutarotable hydroxyl (α-methylglucoside and 1-deoxyglucose) interacted strongly with the enzyme protein. Hexose sugars actively transported by intestine (d-glucose, d-galactose, 6-deoxy-d-galactose, α-methylglucoside, and 1-deoxy-d-glucose) inhibited the enzyme strongly. A number of hexoses and hexitols reportedly not actively transported (d-mannose, d-glucosamine, 2-deoxy-d-glucose, 2-deoxy-d-galactose, d-sorbitol, galactitol, and d-fructose) showed only alight inhibition. Pentoses with the same configuration at carbon 2 as d-glucose (l-arabinose and d-xylose) were good inhibitors of the enzyme; those differing at carbon 2 (d-lyxose and d-arabinose) were not. The enzyme possesses certain of the qualities of the hypothetical carrier or "permease" postulated for sugar transport. Possible significance of some inconsistencies with this hypothesis is discussed.

Immunoreactive serum insulin concentration in 15 wethers fasted 16–24 hr was 126 ± 12 µU/ml. Exogenous glucose produced a tenfold increase in serum insulin within 15 min; concentrations remained high for 1 hr and then declined. Fructose increased both blood glucose and insulin. The maximum response of the latter was delayed, in comparison with that produced by glucose injection, and coincided with a rise of blood glucose derived from fructose conversion. Galactose produced a moderate and immediate rise in insulin associated with elevated blood glucose concentrations. There was a positive relationship between the amount of insulin secreted and the utilization rate of injected glucose. Thus, insulin released by glucose loading in the sheep is both immunologically and biologically active. Prolonged fasting depressed glucose tolerance and the amount of insulin secreted following glucose injection. Certain characteristics of the sheep, such as reduced glucose tolerance and low glucose oxidation, are not due to a relative lack of circulating insulin or to an inability of hyperglycemia to mobilize insulin stores.

The penetration of washed rabbit erythrocytes by deoxy and O-alkyl hexoses present at an initial concentration of 20 mm in a phosphate buffer saline solution was tested in vitro. Equilibration of hexoses with intracellular water was calculated from changes in hexose concentration in the medium during incubation. Analysis of cells plus medium indicated no utilization of deoxy and O-alkyl hexoses by red cells. Alterations in the structure of glucose at individual carbon atoms by the preparation of O-methyl and deoxy derivatives was found to have little effect on the permeability of the rabbit red cells to the molecule. The entrance of such compounds was found to be temperature dependent and all of the sugars tested compete with glucose for entrance. Increasing the chain length of the aliphatic substituent on carbon 3 of glucose from methyl to butyl greatly accelerates the rate of penetration. 3-Hydroxyethyl glucose showed the most rapid rate of equilibration with intracellular water of all of the glucose derivatives tested.

Hexose and/or NaCl solutions were perfused at about 100 cm H2O pressure through small intestinal loops in unanesthetized dogs. The rate of glucose absorption increased in curvilinear fashion as the glucose concentration of the infusate ranged from 1% to 30% and did not differ between jejunal and ileal loops. Net chloride secretion rates were independent of perfusate hexose concentrations and were greater for jejunal than for ileal loops when nonchloride solutions were infused. Net chloride absorption rates increased with infusate concentrations ranging from 0.2% to 1.8% NaCl, and were greater for ileal than for jejunal loops. However, this difference may be due to the greater chloride secretion of jejunal loops. The curvilinear relationship between moles of glucose or chloride absorbed and lumen concentrations of these substances was rectified by plotting absorption rates against log concentration. The resulting Δ mm absorbed per hour/Δ log mm per liter concentration of the perfusate averaged 10.1 and 8.7 for chloride and glucose, respectively, in 6 dogs. Net fluid absorption rate decreased in curvilinear fashion with increasing perfusate osmolar concentration when NaCl or hexose was given. Net fluid absorption at given osmolar perfusate concentrations was greater from NaCl than from glucose or sorbose solutions.

The effect of increasing partial pressure of carbon dioxide (pCO2) on fructose disappearance, lactate accumulation, and CO2 evolution from bicarbonate, after correction for retention, of bovine spermatozoa was studied. In a diluent composed primarily of the sodium salts of citrate and bicarbonate, the metabolism was maximum with a gas phase of N2 and decreased with each increase in pCO2 to a minimum, about 10% of that under N2, with a pCO2 of 100%. The inhibition was reversible upon aeration of the sperm cells. After inhibition under 100% CO2 for 24 hours, the recovery of glycolytic function under 5% CO2, 95% N2 was nearly complete during an additional 4-hour period. In the sodium diluent all the hexose which disappeared was recovered as lactate. However, when the sodium was replaced by potassium, hexose disappearance was inhibited only slightly by CO2 and very little of the hexose was converted to lactate. Hexose disappearance was depressed by sulfanilamide at low pCO2 and acid end-products other than lactic may be formed.

Total protein-bound hexose, hexosamine, and sialic acid were determined in sera of six littermate mongrel pups at monthly intervals from the 4th day after birth up to the age of 7 months. The concentration of the individual constituents fluctuated considerably from month to month, but the values showed neither a definite trend nor a relationship to weight gain. When the carbohydrate moiety was expressed as percentage of total serum protein concentration, the values were high in newborn pups and diminished after the 1st month of life. This was due to a rise in the concentration of total serum protein, brought about by an increase of the albumin fraction which is low in carbohydrate.

The effect of hexose and monoolein on fatty acid esterification was studied in everted rings of rat jejunum. By use of [3H]oleic acid and [U-14C]glucose, esterification via both phosphatidic acid and monoglyceride pathways was quantified. Our results showed that 1) metabolizable sugars stimulate fatty acids esterification regardless of their mode of transport; 2) in the presence of glucose alone, in vitro fatty acid esterification is mediated entirely by the phosphatidic acid pathway; 3) glucose stimulates both esterification pathways in the presence of monoolein; 4) monoolein stimulates only the monoglyceride pathway and has no effect on the phosphatidic acid pathway; and 5) the rate-limiting factor in the presence of monoolein alone is the formation of acyl-CoA, whereas in the presence of glucose alone, the rate-limiting factor is the formation of alpha-glycerophosphate.

The question whether glucose or insulin regulates gluconeogenesis by effecting changes in the fructose-6-phosphate (F-6-P) substrate cycle (phosphofructokinase (PFK), fructose-1,6-diphosphatase (FDPase)) was investigated in vivo in fasted normal rats using [3-3H,U-14C]- or [3-3H,6-14C]glucose. The plasma glucose 3H/14C ratio was used as an index of substrate cycling because 3H loss from the liver hexose phosphate pool is limited by the activities of PFK and FDPase during gluconeogenesis and glycolysis, respectively. The 3H/14C ratio was corrected where necessary for glucose or insulin-induced changes in reincorporation of 14C from C-6 to C-1-3 of plasma glucose. A glucose infusion producing hyperglycemia and insulinemia was accompanied by decreased hepatic glucose production and diminished F-6-P substrate cycling, i.e., decreased FDPase activity. When insulin was infused along with glucose to produce high plasma insulin levels and avoid hypo- or hyperglycemia, the 3H/14C decay rate did not change, suggesting that the hormone does not influence basal rates of gluconeogenesis or PFK or FDPase activities. These in vivo results suggest that increased blood glucose levels inhibit gluconeogenesis and depress F-6-P substrate cycling. Whether these cycle changes constitute primary regulatory actions of glucose or occur secondarily to other metabolic events resulting from excess hexose (e.g., increased glycogen synthetase activity) cannot now be concluded.

The possible role of the hepatic fructose-6-phosphate substrate cycle (phosphofructokinase, fructose-1,6-diphosphatase) in the rapid hormonal regulation of gluconeogenesis was investigated in vivo in fasted normal and adrenalectomized rats after administration of [3-3H, U-14C]- or [3-3H, 6-14C]glucose. The plasma glucose 3H/14C ratio was used as an index of substrate cycling because the amount of 3H loss from liver hexose phosphates is determined by the extent of cycling. PFK and FDPase activities limit 3H loss during gluconeogenesis and glycolysis, respectively. Glucagon-stimulated hepatic glucose production is always accompanied by increased substrate cycling, i.e., increased FDPase and PFK activities. The high PFK activity may be a secondary event due possibly to elevated cellular fructose-6-phosphate levels. Decreased substrate cycling, i.e., lowered FDPase activity, always accompanies the depressed hepatic glucose production that occurs during hyperglycemia. Glucagon has no effect on substrate cycling in adrenalectomized rats that are insensitive to the hormone. The in vivo experiments presented provide evidence, although indirect, that glucagon administration results in changes in the fructose-6-phosphate substrate cycle in a living animal. Whether these changes are primary regulatory events or occur secondarily to hormone actions elsewhere is not known.

The possibility of eliciting changes in the activity of lipomobilizing centers in the rat central nervous system was investigated by injecting glucose or substances that affect glucose metabolism directly into the cerebrospinal fluid of conscious, unrestrained rats. Intracerebroventricular administration of minute amounts of 2-deoxyglucose to fed rats induced a rapid increase in the concentration of plasma free fatty acids (FFA) without affecting plasma insulin levels. Furthermore, small amounts of either glucose or insulin injected intraventricularly reduced the increased plasma FFA levels of normal fasted rats without affecting the glycemia or plasma insulin levels in these animals. In diabetic rats, the increased levels of plasma FFA were not affected by intraventricular administration of glucose, but clearly decreased after intraventricular insulin. The results suggest that the lipomobilizing centers are insulin sensitive and that their activity is modulated by the rate of glucose uptake, rather than by external hexose concentration. The data are also consistent with our previous hypothesis of a direct control of the sympathetic tonus of adipose tissue by these centers.