The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells.

The hypothesis that the glucose transporter GLUT2 can function as a protein mediating transcriptional glucose signaling was addressed. To divert the putative interacting proteins from a glucose signaling pathway, two intracytoplasmic domains of GLUT2, the C terminus and the large loop located between transmembrane domains 6 and 7, were transfected into mhAT3F hepatoma cells. Glucose-induced accumulation of two hepatic gene mRNAs (GLUT2 and L-pyruvate kinase) was specifically inhibited in cells transfected with the GLUT2 loop and not with the GLUT2 C terminus. The dual effects of glucose were dissociated in cells expressing the GLUT2 loop; in fact a normal glucose metabolism into glycogen occurred concomitantly with the inhibition of the glucose-induced transcription. This inhibition by the GLUT2 loop could be due to competitive binding of a protein that normally interacts with endogenous GLUT2. In addition, the GLUT2 loop, tagged with green fluorescent protein (GFP), was located within the nucleus, whereas the GFP and GFP-GLUT2 C-terminal proteins remained in the cytoplasm. In living cells, a fraction (50%) of the expressed GFP-GLUT2 loop translocated rapidly from the cytoplasm to the nucleus in response to high glucose concentration and conversely in the absence of glucose. We conclude that, via protein interactions with its large loop, GLUT2 may transduce a glucose signal from the plasma membrane to the nucleus.

Phenotype of transgenic mice overexpressing GLUT4 and hexokinase II in muscle.

To optimize glucose utilization, double transgenic mice were created by crossing mice overexpressing glucose transporter GLUT4 with mice overexpressing hexokinase (HKII) in muscle. Transgenic mice overexpressing GLUT4 alone have exhibited improvements in glucose tolerance and insulin action. In vitro studies of hexose uptake in soleus muscle from transgenic mice suggested that GLUT4 was limiting the glucose flux except at high glucose concentration, where hexokinase became the limiting step. In vivo, glucose tolerance was similar in GLUT4 and GLUT4/HKII mice, although stimulated plasma insulin values were significantly lower in the latter group. Insulin tolerance tests performed in diabetic GLUT4 vs. diabetic GLUT4/HKII transgenic mice yielded identical results. Again, endogenous insulin in GLUT4/HKII mice during a mild hyperglycemic clamp was stimulated by only two- vs. fourfold in GLUT4 mice. Although the overexpression of HKII alone resulted in increased glucose utilization in several muscles, the overexpression of GLUT4 plus HKII did not augment basal or stimulated in vivo glucose utilization compared to GLUT4 overexpression. In conclusion, GLUT4 is rate limiting for muscle glucose utilization but HKII might be important under hyperglycemia. The addition of HKII to GLUT4 overexpression is not sufficient to further augment glucose tolerance or insulin action. In GLUT4/HKII double transgenic mice, glucose clearance is tempered by a low insulin stimulated level.

Improvement of insulin action in diabetic transgenic mice selectively overexpressing GLUT4 in skeletal muscle.

To investigate the role of glucose transporter expression in whole-body glucose homeostasis, we have created transgenic mice that have a 2.0- to 3.5-fold increase in GLUT4 glucose transporter level in skeletal muscle and heart. This increase is sufficient to significantly improve insulin action and to reduce basal blood glucose levels in transgenic streptozotocin-induced diabetic mice. These results provide the first evidence of a direct causality between skeletal muscle GLUT4 transporter level and overall insulin responsiveness.

Route of nutrient delivery affects insulin sensitivity and liver glucose transporter expression in rat.

To optimize artificial nutrition (AN) techniques for patients suffering from malnutrition or reduced intestinal absorption, utilization of energy fuels, especially glucose, requires better understanding. Because the liver plays a key role in glucose homeostasis, the aim of this study was to assess the effects of continuous intragastric and intravenous nutrition on insulin secretion and several markers of liver glucose metabolism, especially glucose transporter GLUT-2. Wistar male rats underwent catheterization of either stomach (intragastric) or vena cava (intravenous) and received 24 h/day the same all-in-one formula over 7 to 14 days. The metabolic parameters from intragastrically fed rats did not differ significantly from those from orally fed control rats. Intravenous nutrition induced insulin resistance (marked hyperinsulinemia and/or mild hyperglycemia) and reduced liver GLUT-2 protein and mRNA levels. The decrease in liver GLUT-2 gene expression might be mediated either by an inhibitory role of hyperinsulinemia or by the decrease in gut or portal factors. These results suggest that the route of nutrient delivery influences their utilization by the liver.

Development and regulation of glucose transporter and hexokinase expression in rat.

The ontogenesis of the glucose transporters GLUT-1, GLUT-2, and GLUT-4 and the hexokinases HK-I, HK-II, and HK-IV (glucokinase) was studied in rat tissues. In brown adipose tissue, high levels of GLUT-4 and HK-II were observed during fetal life; both decreased at birth and then increased throughout development. At birth, cold exposure increased GLUT-4 and HK-II expression in brown adipose tissue, whereas fasting decreased it. GLUT-1 and HK-I were present in fetal muscle, but GLUT-4 and HK-II were absent. The coordinate appearance of GLUT-4 and HK-II in skeletal muscle was concomitant with the acquisition of insulin sensitivity after weaning. In the heart, the glucose transporter isoform switched from GLUT-1 to GLUT-4 during the suckling period. The coordinate expression of GLUT-4 and HK-II in heart was observed after weaning. GLUT-2, detected in fetal liver, increased throughout development. GLUT-1 and HK-I were detectable in fetal liver, whereas glucokinase appeared after weaning. Consumption of a high-carbohydrate diet after weaning increased GLUT-4 and HK-II in muscle and GLUT-2 in liver, whereas consumption of a high-fat diet prevented these changes. These results showed that 1) GLUT-1 and HK-I are abundant in most fetal rat tissues, 2) GLUT-4 and HK-II expression is associated with the appearance of tissue insulin sensitivity, and 3) GLUT-2 is expressed early in liver, before the appearance of glucokinase.

Developmental expression of Glut1 glucose transporter and c-fos genes in human placental cells.

Glut1, the brain/erythrocyte glucose transporter is one major isoform of the human placenta and displays an age-specific pattern of expression with mRNA levels five-fold higher in first trimester than in term placenta. By contrast, the mRNA level of the insulin-regulatable glucose transporter Glut4 remains at the limit of detection throughout pregnancy indicating a very low expression of this isoform in the placenta. The nuclear proto-oncogenes c-fos and c-myc were also detectable in the human placenta, but c-fos only exhibited an age-specific pattern of expression with levels higher in third trimester than in term placenta. Primary cultures of human trophoblast cells from term placenta were used to further study the expression and regulation of Glut1 and c-fos genes. Fetal calf serum rapidly and transiently (15 to 60 min) stimulated c-fos and Glut1 gene expression suggesting that both genes share similar growth factor-controlled pathways. Glucose inhibited Glut1, but not c-fos expression. An eight-fold decrease in Glut1 mRNA was observed when glucose concentration in the medium was increased from 0 to 25 mM, whereas c-fos mRNA levels remained very low. These results suggest that in the human placenta, the expression of Glut1 is specifically regulated by glucose concentration. These data demonstrate that (1) Glut1 and c-fos mRNA transcripts are expressed in the human placenta exhibiting an age-specific pattern of expression, (2) In cultured trophoblast cells, both genes are stimulatable by fetal calf serum and in contrast to c-fos, Glut1 is negatively regulated by glucose. This differential regulation of Glut1 and c-fos genes could be relevant to specific metabolic and mitogenic pathways implicated in placental growth and differentiation.

Evidence for a transient inhibitory effect of insulin on GLUT2 expression in the liver: studies in vivo and in vitro.

The glucose transporter GLUT2 is expressed predominantly in the liver. Previous studies have shown that glucose increases GLUT2 mRNA concentration in primary cultures of rat hepatocytes. Since insulin controls the glucose metabolism in the liver, it could be involved in the regulation of GLUT2 gene expression. In vivo, hyperinsulinaemia induced a transient inhibitory effect on liver GLUT2 gene expression, the maximal inhibition of GLUT2 mRNA concentration (93 +/- 6%) being observed after 6 h. When hyperglycaemia was associated with hyperinsulinaemia, the decrease in liver GLUT2 mRNA concentration was partially prevented. The respective effects of glucose and insulin were studied in vitro by primary culture of rat hepatocytes. Insulin alone exerted a transient inhibitory effect on GLUT2 mRNA concentration. When insulin and glucose (10-20 mM) were associated, the stimulatory effect of glucose on GLUT2 gene expression was predominant. In conclusion, the present study shows that GLUT2 mRNA concentration was conversely regulated by insulin and glucose, both in vitro and in vivo.

Insulin receptor function is preserved in a physiological state of hypoinsulinemia and insulin resistance.

The suckling period in the rat is characterized by a continuously low plasma insulin concentration and a physiological insulin resistance, particularly in the adipose tissue. This insulin resistance disappears after weaning on the high-carbohydrate adult diet. We have studied the number, structure, and function of adipose tissue insulin receptors during the suckling-weaning transition. The insulin receptor number determined either on intact adipocytes or after partial purification was higher during suckling (15 days), whereas the affinity was similar when compared with weaned rats (30 days). The molecular weight of the alpha- and beta-subunits were identical in both groups and, when analyzed in nonreducing conditions, the alpha 2 beta 2-form was the unique detectable form of the receptor. Neither the basal and insulin-stimulated autophosphorylation of the insulin receptor beta-subunit nor the tyrosine kinase activity toward a synthetic substrate was decreased during the suckling period. Thus, in the adipose tissue of the suckling rat, a marked insulin resistance is concomitant with a normal insulin receptor number and function.

Insulin receptor kinase activity in muscles and white adipose tissue during course of VMH obesity.

Early after lesion of the ventromedial hypothalamus nuclei (VMH), insulin-induced glucose utilization is increased in white adipose tissue (WAT), whereas oxidative and glycolytic muscles are, respectively, normoresponsive or resistant to insulin. Five weeks later, all of the muscles are resistant, whereas WAT returns to normal responsiveness. The aim of this study was to characterize the insulin receptor kinase activity in WAT and muscles 1 and 6 wk after lesion. The number and affinity of insulin receptors were not modified in any of the tissues studied. Autophosphorylation and phosphorylation of an exogenous substrate were similar in oxidative and glycolytic muscles of VMH and control rats both 1 and 6 wk after the lesion. Insulin receptors from WAT of 1-wk VMH rats exhibited a 2.5-fold increase in insulin-stimulated autophosphorylation and phosphorylation. Six weeks after the lesion, both autophosphorylation and phosphorylation returned to normal values. This suggests that insulin receptor tyrosine kinase activity does not play a significant role in the insulin resistance of skeletal muscles but has a crucial role in mediating the variations of insulin action on WAT observed during the development of VMH obesity.

Insulin receptor activity and insulin sensitivity in mammary gland of lactating rats.

The mammary gland is a tissue that is extremely sensitive to insulin during lactation; during weaning, the effect of insulin is rapidly abolished. The purpose of this study was to characterize the mammary gland insulin receptors and their kinase activity in lactating and weaned mammary gland. The apparent molecular weight of the alpha-subunit was slightly lower in the mammary gland than in liver and white adipose tissue (127,000 vs. 134,000), but the apparent molecular weight of the beta-subunit was similar in the three tissues (95,000). Insulin induced a 10-fold increase in beta-subunit autophosphorylation, and the half-maximal effect was achieved at 2 nM insulin. After 24 h of weaning, the number of insulin receptors was decreased by 30%, but the kinase activity of the beta-subunit was unchanged. During the euglycemic hyperinsulinemic clamp, insulin entirely activated pyruvate dehydrogenase in lactating rat mammary gland, whereas after 24 h of weaning it was unable to increase the proportion of the enzyme in the active form. These results suggest that the site of alteration in the action of insulin on the mammary gland during weaning is distal to the receptor.

Glucose transporter expression in rat mammary gland.

The expression of different glucose transporter isoforms was measured during the development and differentiation of the rat mammary gland. Before conception, when the mammary gland is mainly composed of adipocytes, Glut 4 and Glut 1 mRNAs and proteins were present. During pregnancy, the expression of Glut 4 decreased progressively, whereas that of Glut 1 increased. In the lactating mammary gland only Glut 1 was present, and was expressed at a high level. The absence of Glut 4 suggests that glucose transport is not regulated by insulin in the lactating rat mammary gland.

Effects of long-term treatment with D-penicillamine on antigen-induced arthritis in rabbits: studies on in vivo articular chondrocyte damage and in vitro collagen biosynthesis by cultured chondrocytes.

The effects of a long-term (120 days) treatment with D-penicillamine (DP) (50 mg/kg/day; i.v.) on antigen-induced arthritis were studied in rabbit. They were investigated by the terminal histological examination of the joints of different groups of rabbits (unimmunized treated or untreated, immunized treated or untreated) and the study of collagen and non-collagen protein biosynthesis by cultured chondrocytes obtained from articular cartilage of the same groups of animals. Treatment with D-penicillamine diminished the intensity of the erosions of cartilage and subchondral bone, the severity of the inflammatory synovitis, and the loss of chondrocyte clusters found in cartilage sections. In cultures of chondrocytes obtained from immunized treated rabbits, a partial or complete inhibition of the decreased biosynthesis of collagen and non-collagen proteins seen in culture of chondrocytes obtained from immunized untreated animals was observed. These results show that DP could be effective in preventing damage of chondrocytes and inhibition of collagen biosynthesis in them, phenomena important in cartilage destruction induced by a chronic immunological inflammation.

Effect of indomethacin on collagen biosynthesis by rabbit articular chondrocytes in monolayer cultures.

The influence of indomethacin on collagen biosynthesis in rabbit articular chondrocyte monolayer cultures was studied. Two applications within the space of three days of therapeutic doses (10(-5) or 10(-6)M), as well as repeated applications four days running of lower doses (10(-8) or 10(-10)M), increased the biosynthesis of both collagen and non-collagen proteins. Two applications of higher doses (10(-3) or 10(-4M) decreased DNA synthesis and inhibited both collagen and non-collagen protein biosynthesis. These results might well be considered in connection with the adverse reactions observed in some patients with long-term use of indomethacin.

Effect of in vivo treatment by dexamethasone and indometacin on DNA synthesis in rat macrophages in culture stimulated by inflammatory exudate.

The acute inflammatory exudate obtained 4 hr after intrapleural injection of dextran in rats is able to induce DNA synthesis and division of normal rat macrophages in culture. The influence on this phenomenon of two types of pretreatment of rats with dexamethasone or indometacin has been investigated. Exudates of rats treated with dexamethasone decreased the DNA synthesis in control macrophages. On the contrary, exudates of rats treated with indometacin had no effect on this DNA synthesis. These results could be explained by the different actions of the two anti-inflammatory agents on cellular migration in pleural exudates. In macrophages harvested from rats treated with dexamethasone or indometacin the DNA synthesis induced by inflammatory exudates was decreased. This identical response elicited by these two agents could be related to their direct antimitotic activity on macrophages.

[Effect of dexamethasone on DNA synthesis in macrophages stimulated in vitro by an inflammatory exudate]. / Action de la déxaméthasone sur la synthèse d'ADN de macrophages stimulés in vitro par un exsudat inflammatoire.

Rat macrophages in culture are induced to synthesize DNA when incubated with Dextran pleural exudate. Previous or simultaneous in vitro treatment with dexamethasone phosphate used at different concentrations was able to inhibit or decrease that induction. This effect could represent a new aspect of the influence of corticosteroids in the inflammatory process.

[Stimulation of the growth of murine bone marrow colonies in vitro by an acute inflammatory exudate. Comparison with colony stimulating factor CSF)]. / Stimulation de la croissance des colonies de moelle osseuse murine in vitro par un exsudat de réaction inflammatoire aiguë. Comparaison avec le facteur stimulant les colonies (CSF).

Formation of colonies from mice bone marrow progenitors of macrophages and granulocytes in methylcellulose culture was induced by an inflammatory pleural exudate obtained from mice injected with dextran. Mitogenic activity of this acute inflammatory exudate was compared with that of colony stimulating factor (CSF). It was found that colony and cluster counts, during 10 days of culture, were similar with the two types of stimulating factors. When used at the same dose, exudate was less active than CSF. It was concluded that inflammatory exudate showed an activity similar to that of CSF but contained a smaller amount of stimulating factor. Further cytochemical studies are necessary to specify whether or not the two factors induced the same type of colonies (monocytic or granulocytic).

[The effect of mepyramine and 48/80 on the histamine content of pleural exudates in the rat]. / Influence de la mépyramine et du 48/80 sur la teneur en histamine d'exsudats pleuraux chez le rat

The influence of pretreatment with the antihistaminic, mepyramine, or with the histamine liberator 48/80 was studied on the volume and the histamine content of pleural exudates induced in rats by intrapleural injection of turpentine, silver nitrate or carrageenan. The fluorometric determination of histamine was performed in the exudates collected at different times after injection of the irritant. In control animals the histamine levels were different from those previously found by the authors using biological assays. In rats pretreated with mepyramine or compound 48/80, the effects observed varied according to the nature of the irritant used to induce pleurisy.