Developmental programming: exposure to testosterone excess disrupts steroidal and metabolic environment in pregnant sheep.

Gestational exposure to excess T leads to intrauterine growth restriction, low birth weight, and adult metabolic/reproductive disorders in female sheep. We hypothesized that as early mediators of such disruptions, gestational T disrupts steroidal and metabolic homeostasis in both the mother and fetus by both androgenic and metabolic pathways. Maternal blood samples were measured weekly for levels of insulin, glucose, and progesterone from four groups of animals: control; gestational T (twice weekly im injections of 100 mg of T propionate from d 30 to d 90 of gestation); T plus an androgen antagonist, flutamide (15 mg/kg·d oral; T-Flutamide); and T plus the insulin sensitizer, rosiglitazone (0.11 mg/kg·d oral; T-Rosi) (n = 10-12/group). On day 90 of gestation, maternal and umbilical cord samples were collected after a 48-hour fast from a subset (n = 6/group) for the measurement of steroids, free fatty acids, amino acids, and acylcarnitines. Gestational T decreased maternal progesterone levels by 36.5% (P < .05), which was prevented by flutamide showing direct androgenic mediation. Gestational T also augmented maternal insulin levels and decreased medium chained acylcarnitines, suggesting increased mitochondrial fatty acid oxidation. These changes were prevented by rosiglitazone, suggesting alterations in maternal fuel use. Gestational T-induced increases in fetal estradiol were not prevented by either cotreatment. Gestational T disrupted associations of steroids with metabolites and progesterone with acylcarnitines, which was prevented either by androgen antagonist or insulin sensitizer cotreatment. These findings suggest a future combination of these treatments might be required to prevent alteration in maternal/fetal steroidal and metabolic milieu(s).

Efficacy and tolerability of the DPP-4 inhibitor alogliptin combined with pioglitazone, in metformin-treated patients with type 2 diabetes.

CONTEXT: Optimal management of type 2 diabetes remains an elusive goal. Combination therapy addressing the core defects of impaired insulin secretion and insulin resistance shows promise in maintaining glycemic control. OBJECTIVE: The aim of the study was to assess the efficacy and tolerability of alogliptin combined with pioglitazone in metformin-treated type 2 diabetic patients. DESIGN, SETTING, AND PATIENTS: We conducted a multicenter, randomized, double-blind, placebo-controlled, parallel-arm study in patients with type 2 diabetes. INTERVENTIONS: The study consisted of 26-wk treatment with alogliptin (12.5 or 25 mg qd) alone or combined with pioglitazone (15, 30, or 45 mg qd) in 1554 patients on stable-dose metformin monotherapy (≥1500 mg) with inadequate glycemic control. MAIN OUTCOME MEASURE: The primary endpoint was change in glycosylated hemoglobin (HbA(1c)) from baseline to wk 26. Secondary endpoints included changes in fasting plasma glucose and ß-cell function. Primary analyses compared pioglitazone therapy [all doses pooled, pioglitazone alone (Pio alone); n = 387] with alogliptin 12.5 mg plus any dose of pioglitazone (A12.5+P; n = 390) or alogliptin 25 mg plus any dose of pioglitazone (A25+P; n = 390). RESULTS: When added to metformin, the least squares mean change (LSMΔ) from baseline HbA(1c) was -0.9 ± 0.05% in the Pio-alone group and -1.4 ± 0.05% in both the A12.5+P and A25+P groups (P < 0.001 for both comparisons). A12.5+P and A25+P produced greater reductions in fasting plasma glucose (LSMΔ = -2.5 ± 0.1 mmol/liter for both) than Pio alone (LSMΔ = -1.6 ± 0.1 mmol/liter; P < 0.001). A12.5+P and A25+P significantly improved measures of ß-cell function (proinsulin:insulin and homeostasis model assessment of ß-cell function) compared to Pio alone, but had no effect on homeostasis model assessment of insulin resistance. The LSMΔ body weight was 1.8 ± 0.2, 1.9 ± 0.2, and 1.5 ± 0.2 kg in A12.5+P, A25+P, and Pio-alone groups, respectively. Hypoglycemia was reported by 1.0, 1.5, and 2.1% of patients in the A12.5+P, A25+P, and Pio-alone groups, respectively. CONCLUSIONS: In type 2 diabetic patients inadequately controlled by metformin, the reduction in HbA(1c) by alogliptin and pioglitazone was additive. The decreases in HbA(1c) with A12.5+P and A25+P were similar. All treatments were well tolerated.

Diabetes-related changes in cAMP response element-binding protein content enhance smooth muscle cell proliferation and migration.

We hypothesized that diabetes and glucose-induced reactive oxygen species lead to depletion of cAMP response element-binding protein (CREB) content in the vasculature. In primary cultures of smooth muscle cells (SMC) high medium glucose decreased CREB function but increased SMC chemokinesis and entry into the cell cycle. These effects were blocked by pretreatment with the antioxidants. High glucose increased intracellular reactive oxygen species detected by CM-H(2)DCFA. SMC exposed to oxidative stress (H(2)O(2)) demonstrated a 3.5-fold increase in chemokinesis (p < 0.05) and accelerated entry into cell cycle, accompanied by a significant decrease in CREB content. Chronic oxidative challenge similar to the microenvironment in diabetes (glucose oxidase treatment) decreases CREB content (40-50%). Adenoviral-mediated expression of constitutively active CREB abolished the increase in chemokinesis and cell cycle progression induced by either high glucose or oxidative stress. Analysis of vessels from insulin resistant or diabetic animals indicates that CREB content is decreased in the vascular stroma. Treatment of insulin-resistant animals with the insulin sensitizer rosiglitazone restores vessel wall CREB content toward that observed in normal animals. In summary, high glucose and oxidative stress decrease SMC CREB content increase chemokinesis and entry into the cell cycle, which is blocked by antioxidants or restoration of CREB content. Thus, decreased vascular CREB content could be one of the molecular mechanisms leading to increased atherosclerosis in diabetes.

Disruption of Sur2-containing K(ATP) channels enhances insulin-stimulated glucose uptake in skeletal muscle.

ATP-sensitive potassium channels (K(ATP)) are involved in a diverse array of physiologic functions including protection of tissue against ischemic insult, regulation of vascular tone, and modulation of insulin secretion. To improve our understanding of the role of K(ATP) in these processes, we used a gene-targeting strategy to generate mice with a disruption in the muscle-specific K(ATP) regulatory subunit, SUR2. Insertional mutagenesis of the Sur2 locus generated homozygous null (Sur2(-/-)) mice and heterozygote (Sur2(+/-)) mice that are viable and phenotypically similar to their wild-type littermates to 6 weeks of age despite, respectively, half or no SUR2 mRNA expression or channel activity in skeletal muscle or heart. Sur2(-/-) animals had lower fasting and fed serum glucose, exhibited improved glucose tolerance during a glucose tolerance test, and demonstrated a more rapid and severe hypoglycemia after administration of insulin. Enhanced glucose use was also observed during in vivo hyperinsulinemic euglycemic clamp studies during which Sur2(-/-) mice required a greater glucose infusion rate to maintain a target blood glucose level. Enhanced insulin action was intrinsic to the skeletal muscle, as in vitro insulin-stimulated glucose transport was 1.5-fold greater in Sur2(-/-) muscle than in wild type. Thus, membrane excitability and K(ATP) activity, to our knowledge, seem to be new components of the insulin-stimulated glucose uptake mechanism, suggesting possible future therapeutic approaches for individuals suffering from diabetes mellitus.

Calpains play a role in insulin secretion and action.

Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action.

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

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

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

ATP-sensitive potassium channels (KATP) are implicated in a diverse array of physiological functions. Previous work has shown that alternative usage of exons 14, 39, and 40 of the muscle-specific KATP channel regulatory subunit, sur2, occurs in tissue-specific patterns. Here, we show that exon 17 of the first nucleotide binding fold of sur2 is also alternatively spliced. RNase protection demonstrates that SUR2(Delta17) predominates in skeletal muscle and gut and is also expressed in bladder, fat, heart, lung, liver, and kidney. Polymerase chain reaction and restriction digest analysis of sur2 cDNA demonstrate the existence of at least five sur2 splice variants as follows: SUR2(39), SUR2(40), SUR2(Delta17/39), SUR2(Delta17/40), and SUR2(Delta14/39). Electrophysiological recordings of excised, inside-out patches from COS cells cotransfected with Kir6.2 and the sur2 variants demonstrated that exon 17 splicing alters KATP sensitivity to ATP block by 2-fold from approximately 40 to approximately 90 microM for exon 17 and Delta17, respectively. Single channel kinetic analysis of SUR2(39) and SUR2(Delta17/39) demonstrated that both exhibited characteristic KATP kinetics but that SUR2(Delta17/39) exhibited longer mean burst durations and shorter mean interburst dwell times. In sum, alternative splicing of sur2 enhances the observed diversity of KATP and may contribute to tissue-specific modulation of ATP sensitivity.

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

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

Metabolic effects of troglitazone monotherapy in type 2 diabetes mellitus. A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Troglitazone is a new insulin-sensitizing agent used to treat type 2 diabetes mellitus. The mechanism by which troglitazone exerts its effect on systemic glucose metabolism is unknown. OBJECTIVE: To determine the effects of 6 months of troglitazone monotherapy on glucose metabolism in patients with type 2 diabetes mellitus. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Six general clinical research centers at university hospitals. PATIENTS: 93 patients (mean age, 52 years) with type 2 diabetes mellitus (mean fasting plasma glucose level, 11.2 mmol/L) who were being treated with diet alone or who had discontinued oral antidiabetic medication therapy. INTERVENTION: Patients were randomly assigned to one of five treatment groups (100, 200, 400, or 600 mg of troglitazone daily or placebo) and had metabolic assessment before and after 6 months of treatment. MEASUREMENTS: Plasma glucose and insulin profiles during a meal tolerance test; basal hepatic glucose production and insulin-stimulated glucose disposal rate during a hyperinsulinemic-euglycemic clamp procedure. RESULTS: Troglitazone at 400 and 600 mg/d decreased both fasting (P < 0.001) and postprandial (P = 0.016) plasma glucose levels by approximately 20%. All four troglitazone dosages also decreased fasting (P = 0.012) and postprandial (P < 0.001) triglyceride levels; 600 mg of the drug per day decreased fasting free fatty acid levels (P = 0.018). Plasma insulin levels decreased in the 200-, 400-, and 600-mg/d groups (P < 0.001), and C-peptide levels decreased in all five study groups (P < 0.001). Basal hepatic glucose production was suppressed in the 600-mg/d group compared with the placebo group (P = 0.02). Troglitazone at 400 and 600 mg/d increased glucose disposal rate by approximately 45% above pretreatment levels (P = 0.003). Stepwise regression analysis showed that troglitazone therapy was the strongest predictor of a decrease in fasting (P < 0.001) or postprandial (P = 0.01) glucose levels. Fasting C-peptide level was the next strongest predictor (higher C-peptide level equaled greater glucose-lowering effect). CONCLUSION: Troglitazone monotherapy decreased fasting and postprandial glucose levels in patients with type 2 diabetes, primarily by augmenting insulin-mediated glucose disposal.

Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid.

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

Troglitazone action is independent of adipose tissue.

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

Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels.

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

Prevention of hyperglycemia in the Zucker diabetic fatty rat by treatment with metformin or troglitazone.

To determine whether metformin or troglitazone can delay the onset of diabetes in the Zucker diabetic fatty (ZDF) rat, lean control, fatty, and ZDF rats received metformin, troglitazone, or no treatment from 6 to 12 wk of age. Glucose, insulin, triglyceride (TG), and free fatty acid (FFA) levels and glucose stimulated insulin secretion by the perfused pancreas were measured. Metformin-treated rats gained significantly less weight. Both drugs prevented hyperglycemia by 12 wk in diabetic rats and significantly reduced TG and FFA levels. Insulin secretion at low glucose was elevated in untreated fatty and diabetic animals, and the increment in diabetic animals produced by glucose perfusion was attenuated compared with lean and fatty rats. Both drugs reduced basal insulin secretion in fatty and diabetic rats and improved glucose responsiveness in diabetic rats. Metformin and troglitazone delay the onset of diabetes in the ZDF rat. The significantly improved insulin secretory response of the pancreas undoubtedly contributes to the improved glucose tolerance.

Hexose transporter expression in rat small intestine: effect of diet on diurnal variations.

In rodents, a number of intestinal digestive and absorptive processes demonstrate a diurnal pattern of activity. To investigate if the jejunal hexose transporters are regulated in such a diurnal fashion, the levels for the glucose and fructose transporter mRNA and proteins were determined at 6-h intervals over a 24-h control fed period. SGLT-1, GLUT-2, and GLUT-5 mRNA levels increased between two- and eightfold before the onset of peak feeding. GLUT-5 protein levels also varied in a diurnal fashion but were out of phase with the observed changes in GLUT-5 mRNA levels. In contrast, GLUT-2 protein levels remained relatively constant during the control fed 24-h period. The effect of dietary manipulations on the observed diurnal variation was also investigated. After only 3 h of feeding a 60% fructose-enriched diet, the levels of GLUT-5 mRNA and protein were significantly elevated. GLUT-5 mRNA and protein levels remained elevated relative to the level of control diet-fed animals over the ensuing 24 h and during the 7th day of fructose feeding. Exposure to elevated levels of fructose had no significant effect on the diurnal pattern of GLUT-2 and SGLT-1 mRNA. In contrast, GLUT-2 protein was rapidly downregulated during the length of the fructose feeding study. In conclusion, the data demonstrate a normal daily variation in the level of hexose transporter expression that can be rapidly modulated by diet.

GLUT3 glucose transporter isoform in rat testis: localization, effect of diabetes mellitus, and comparison to human testis.

Facilitative hexose transporter expression was compared in rat and human testes. In rat testis, only GLUT1 and GLUT3 proteins were expressed. By contrast, human testis expressed GLUT1 and GLUT3 in addition to GLUT5. Immunocytochemical studies showed that GLUT3 was expressed in all cells of the seminiferous epithelium of rat testis, including sperm. In human testis, GLUT3 was expressed exclusively in cells juxtaposed to the lumen of the seminiferous tubule and ejaculate sperm, a pattern of expression that was identical to that of GLUT5. Induction of insulinopenic diabetes mellitus in the rat did not alter the levels or the distribution of GLUT3 protein or mRNA in the testis. Moreover insulin treatment of the diabetic rats did not produce changes in GLUT3 mRNA or protein levels. The results show that rat and human testis express the high-affinity glucose transporter GLUT3, which allows for the efficient uptake of glucose. In addition, the testis may be protected from changes in glucose transporter expression in experimental diabetes.

Expression of a thyroid hormone-responsive recombinant gene introduced into adult mice livers by replication-defective adenovirus can be regulated by endogenous thyroid hormone receptor.

We constructed a recombinant adenovirus carrying the firefly luciferase gene driven by the thymidine kinase promoter and controlled by the palindromic thyroid hormone/retinoic acid-responsive element. The same adenovirus vector without the hormone-responsive element was used as control. Regulation of the luciferase gene expression was tested in pituitary-derived GH cells and hepatoma-derived HepG2 cells infected with the recombinant adenoviruses. Administration of triiodothyronine to GH cells and all-trans-retinoic acid to HepG2 cells resulted in 8.0 +/- 0.3-fold and 4.6 +/- 0.5-fold induction of luciferase activity, respectively. No significant increase was observed with the control adenovirus. Hormonal regulation was also examined in adult mice. Mice depleted of thyroid hormone were injected intravenously with the recombinant adenoviruses and given 4 times the replacement dose of triiodothyronine or vehicle only for 4 days. Hormone administration resulted in 4.2-fold increase of luciferase activity in liver homogenates. No significant effect was observed in animals injected with the control adenovirus. This recombinant adenovirus provides a new experimental system in the study of thyroid hormone and retinoic acid action and offers the potential to regulate by physiological means the expression of genes transferred for the purpose of therapy.

Glucocorticoids regulate Na+/H+ exchange expression and activity in region- and tissue-specific manner.

Na+/H+ exchangers (NHEs) are integral membrane proteins that exchange Na+ for H+ across membranes. Four isoforms have been cloned (NHE-1-4). NHE-3 localizes to the apical domain, and its expression is increased in dexamethasone-treated rats by Northern analysis. This stimulatory effect on expression is region and tissue specific, being present in ileum and proximal colon, but not in jejunum, distal colon, or kidney. The increase in transcript expression in ileum correlates with an increase in protein expression by immunoblotting. Changes in apical Na+/H+ exchange activity, as measured by 22Na uptake into brush-border membrane vesicles, correlate with expression differences, with significant increases observed in ileum and proximal colon. In situ hybridization showed NHE-3 mRNA only in villus and absorptive cells of control rats, the pattern not being altered by dexamethasone treatment. This suggests that dexamethasone does not increase expression by inducing crypt cells to express NHE-3 prematurely. We conclude that glucocorticoids selectively increase intestinal NHE-3 activity in a region-specific manner and that this effect also appears to be tissue specific.

Rapid reversible substrate regulation of fructose transporter expression in rat small intestine and kidney.

To understand the regulation of fructose transport in the small intestine and kidney, we provided rats with "control" diets (46% glucose as starch) and with diets enriched in fructose, glucose, or sucrose (60% each of simple carbohydrate) and measured the concentration of facilitative glucose transporter isoform (GLUT5) protein and mRNA in these tissues. The fructose-enriched diet resulted in a five- and eightfold increase in GLUT5 protein at 1 and 7 days, respectively, in the small intestine, which declined rapidly with reversion to control diet. No change in GLUT5 protein levels was seen after glucose- or sucrose-enriched diets. Glucose, and to a lesser extent fructose, feeding resulted in an increase in the basolateral GLUT2 protein. Feeding glucose to the rats caused a rise in sodium-dependent glucose transporter isoform (SGLT1) protein levels compared with the control diet. There was a transient increase in the small intestine GLUT5 mRNA 1 day after fructose feeding, which returned to normal by 7 days. In the kidney, both fructose and sucrose increased GLUT5 protein levels three- to fourfold, whereas glucose had no effect. Fructose-enriched diet did not increase the levels of GLUT5 protein or mRNA in a segment of small intestine that was isolated from the rest of the small intestine but continued to have mesenteric blood supply. The results suggest that the levels of GLUT5 protein are regulated by fructose, its in vivo substrate, in both the small intestine and kidney, and the regulation requires fructose to interact with the brush border of the small intestine, possibly stabilizing the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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