GLUT2 mRNAs Research Articles

1365-P: Intermittent Fasting Modifies Pancreatic Glucosensor Mechanism In Diabetic Mice

Background: A specific feature in type-2 diabetes is beta-cell dysfunction, associated to reduction in glucose-stimulated insulin secretion. This function is high-regulated by the glucose sensor mechanism, conformed by GLUT2 and glucokinase, and their expresión is decreased in type-2 diabetes. Intermittent fasting is a recent option in diet control, with alternate cycles of fasting and feeding. In this study we evaluated intermittent fasting in diabetic mice, and its long-term effect in pancreatic GLUT2 and glucokinase expression. Methods: C57BL6 mice were fed with high fat diet for eight weeks, and then intermittent fasting cycles among four weeks. Weight, total caloric intake, fasting and glucose tolerance were determinated. Changes in insulin and glucokinase expression were evatualed by immunohistochemistry assays. Glucose-stimulated insulin secretion, and PDX1, insulin, GLUT2 and glucokinase mRNAs were analyzed in isolated islets. Results: Intermittent fasting caused weight loss, reduction in total caloric intake decrease and enhanced glucose tolerance. In diabetic mice, glucose-stimulated insulin secretion was restored after intermittent fasting, without significant changes in hormone content. Treated groups with fasting increased gene expression of PDX1, insulin and GLUT2, but decreased glucokinase mRNA and localization in beta-cells, but enhanced alpha-cell possitive presence. Conclusion: Intermittent fasting modifies glucosensor mechanism in pancreatic islets from diabetic mice, through GLUT2, PDX1 and insulin expression, but glucokinase. On future studies we will determine changes glucokinase enzymatic activity, and another transcription factors involved in these effects. Disclosure A.Vilches-flores: None. Y.Padilla-romero: None. Funding UNAM PAPIIT IN225819

The Postnatal Offspring of Finasteride-Treated Male Rats Shows Hyperglycaemia, Elevated Hepatic Glycogen Storage and Altered GLUT2, IR, and AR Expression in the Liver

Background: A growing body of data indicates that the physiology of the liver is sex-hormone dependent, with some types of liver failure occurring more frequently in males, and some in females. In males, in physiological conditions, testosterone acts via androgen receptors (AR) to increase insulin receptor (IR) expression and glycogen synthesis, and to decrease glucose uptake controlled by liver-specific glucose transporter 2 (GLUT-2). Our previous study indicated that this mechanism may be impaired by finasteride, a popular drug used in urology and dermatology, inhibiting 5α-reductase 2, which converts testosterone (T) into dihydrotestosterone (DHT). Our research has also shown that the offspring of rats exposed to finasteride have an altered T–DHT ratio and show changes in their testes and epididymides. Therefore, the goal of this study was to assess whether the administration of finasteride had an trans-generational effect on (i) GLUT-2 dependent accumulation of glycogen in the liver, (ii) IR and AR expression in the hepatocytes of male rat offspring, (iii) a relation between serum T and DHT levels and the expression of GLUT2, IR, and AR mRNAs, (iv) a serum glucose level and it correlation with GLUT-2 mRNA. Methods: The study was conducted on the liver (an androgen-dependent organ) from 7, 14, 21, 28, and 90-day old Wistar male rats (F1:Fin) born by females fertilized by finasteride-treated rats. The control group was the offspring (F1:Control) of untreated Wistar parents. In the histological sections of liver the Periodic Acid Schiff (PAS) staining (to visualize glycogen) and IHC (to detect GLUT-2, IR, and AR) were performed. The liver homogenates were used in qRT-PCR to assess GLUT2, IR, and AR mRNA expression. The percentage of PAS-positive glycogen areas were correlated with the immunoexpression of GLUT-2, serum levels of T and DHT were correlated with GLUT-2, IR, and AR transcript levels, and serum glucose concentration was correlated with the age of animals and with the GLUT-2 mRNA by Spearman’s rank correlation coefficients. Results: In each age group of F1:Fin rats, the accumulation of glycogen was elevated but did not correlate with changes in GLUT-2 expression. The levels of GLUT-2, IR, and AR transcripts and their immunoreactivity statistically significantly decreased in F1:Fin animals. In F1:Fin rats the serum levels of T and DHT negatively correlated with androgen receptor mRNA. The animals from F1:Fin group have statistically elevated level of glucose. Additionally, in adult F1:Fin rats, steatosis was observed in the liver (see Appendix A). Conclusions: It seems that treating male adult rats with finasteride causes changes in the carbohydrate metabolism in the liver of their offspring. This can lead to improper hepatic energy homeostasis or even hyperglycaemia, insulin resistance, as well as some symptoms of metabolic syndrome and liver steatosis.

2066-P: Dyrk1a Antisense Oligonucleotides Conjugated to GLP-1R Agonist Promote Pancreatic Beta-Cell Proliferation

Loss of functional beta cell mass is a major contributor to the pathogenesis of both type 1 and type 2 diabetes. Enhancing proliferation of existing beta cells in the pancreatic islet is one of the promising approaches for restoring functional beta cell mass. Increasing evidence indicates inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a) stimulates beta cell proliferation. However, to date DYRK1a small molecule inhibitors have not been therapeutically viable due to their ability to increase proliferation in peripheral tissues. Previously we demonstrated that generation 2.5 antisense oligonucleotides (ASO) conjugated to a glucagon like peptide 1 receptor (GLP-1R) agonist can specifically target the pancreatic beta cell. When compared to control ASO treated isolated islets, islets administered a GLP-1/Dyrk1a ASO conjugate reduces DYRK1a mRNA and protein expression and significantly increases Ki67 mRNA, a cell proliferation marker, in a dose-response manner in vitro. Furthermore, combination treatment of a GLP-1/Dyrk1a ASO conjugate and the small molecule TGF-β inhibitor Ly364947, can synergistically increase Ki67 mRNA expression, cell cycle progression markers cyclin D1, cyclin E2, Cdk1, Cdk4 mRNAs and beta cell markers Pdx1 and Glut2 mRNAs when compared to a DYRK1a conjugated ASO or Ly364947 alone. Administration of a Dyrk1a conjugated ASO to wild type and db/db mice reduced Dyrk1a mRNA and increased Ki67 mRNA expression in islets when compared to a control ASO. Cell cycle markers cyclin D2, cyclin E2 and Cdk1 mRNAs are also significantly upregulated. In conclusion, a GLP-1/Dyrk1a ASO conjugate can specifically inhibit beta cell Dyrk1a mRNA expression and promote beta cell proliferation, suggesting that this may be an important approach for the treatment of type 1 and type 2 diabetes. Disclosure W. Fu: Employee; Self; Ionis Pharmaceuticals, Inc. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E.C. Pirie: Employee; Self; Ionis Pharmaceuticals, Inc. T. Prakash: None. S. Murray: Employee; Self; Ionis Pharmaceuticals. A. Powers: Employee; Self; Ionis Pharmaceuticals, Inc. Stock/Shareholder; Self; Ionis Pharmaceuticals, Inc. A.E. Mullick: None. R.M. Crooke: Employee; Self; Ionis Pharmaceuticals, Inc. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.

Lactobacillus plantarum improves lipogenesis and IRS-1/AKT/eNOS signalling pathway in the liver of high-fructose-fed rats

In the present study, we investigated the influence of Lactobacillus plantarum and Lactobacillus helveticus supplementation on lipogenesis, insulin signalling and glucose transporters in liver of high-fructose-fed rats. Fructose was given to the rats as a 20% solution in drinking water for 15 weeks. Lactobacillus plantarum and L. helveticus supplementations were performed by gastric gavage once a day during final 6 weeks. Dietary high-fructose increased hepatic weight, lipid accumulation and FASN expression as well as caused a significant reduction in IRS-1 expression, pAKT/total AKT and peNOS/total eNOS ratios, but an elevation in GLUT2 and GLUT5 mRNAs in the liver. Lactobacillus plantarum supplementation decreased hepatic weight, triglyceride content and FASN expression as well as improved IRS-1/AKT/eNOS pathway and GLUT2 expression in the liver of high-fructose-fed rats. However, L. helveticus supplementation exerted a restoring effect on lipid accumulation by decreasing FASN expression, and regulating effect on IRS-1 and GLUT2 expressions.

The role of apolipoprotein A-IV in regulating glucagon-like peptide-1 secretion.

Both glucagon-like peptide-1 (GLP-1) and apolipoprotein A-IV (apoA-IV) are produced from the gut and enhance postprandial insulin secretion. This study investigated whether apoA-IV regulates nutrient-induced GLP-1 secretion and whether apoA-IV knockout causes compensatory GLP-1 release. Using lymph-fistula-mice, we first determined lymphatic GLP-1 secretion by administering apoA-IV before an intraduodenal Ensure infusion. apoA-IV changed neither basal nor Ensure-induced GLP-1 secretion relative to saline administration. We then assessed GLP-1 in apoA-IV-/- and wild-type (WT) mice administered intraduodenal Ensure. apoA-IV-/- mice had comparable lymph flow, lymphatic triglyceride, glucose, and protein outputs as WT mice. Intriguingly, apoA-IV-/- mice had higher lymphatic GLP-1 concentration and output than WT mice 30 min after Ensure administration. Increased GLP-1 was also observed in plasma of apoA-IV-/- mice at 30 min. apoA-IV-/- mice had comparable total gut GLP-1 content relative to WT mice under fasting, but a lower GLP-1 content 30 min after Ensure administration, suggesting that more GLP-1 was secreted. Moreover, an injection of apoA-IV protein did not reverse the increased GLP-1 secretion in apoA-IV-/- mice. Finally, we assessed gene expression of GLUT-2 and the lipid receptors, including G protein-coupled receptor (GPR) 40, GPR119, and GPR120 in intestinal segments. GLUT-2, GPR40 and GPR120 mRNAs were unaltered by apoA-IV knockout. However, ileal GPR119 mRNA was significantly increased in apoA-IV-/- mice. GPR119 colocalizes with GLP-1 in ileum and stimulates GLP-1 secretion by sensing OEA, lysophosphatidylcholine, and 2-monoacylglycerols. We suggest that increased ileal GPR119 is a potential mechanism by which GLP-1 secretion is enhanced in apoA-IV-/- mice.

Feeding of aged garlic extract improves glucose intolerance and fatty liver in spontaneous diabetic mice

Garlic, a common herb, is widely used as medicinal plant, but its usefulness in the treatment of diabetes has not been established. Aged garlic extract (AGE) that contains unique compounds such as S‐allyl cysteine were produced as a result of aging processes. We studied the effect of AGE using two models of spontaneous diabetic animals, TSOD and ddY‐H, developed by Dr. W. Suzuki (Tsumura & Co., Japan) and Dr. T. Maeda (Univ. of Shizuoka, Japan), respectively. These mice showed various phenotypes of type 2 diabetes, including high blood glucose, glucose intolerance, high circulating level of insulin and fatty liver. TSOD mice fed diet containing 4% (w/w) AGE for 6 months, had a lower level of blood glucose compared to control, although their body weight was still high and AGE treatment had no adverse effects. In addition, glucose intolerance was ameliorated in AGE‐fed TSOD mice. Furthermore, triglyceride accumulation in the liver of these mice was reduced compared to that in control. Moreover, the level of SREBP1c, PPAR‐gamma and GLUT2 mRNAs was lower in the liver of AGE‐fed TSOD mice. Similar results were obtained by experiments using another diabetic model, ddY‐H mice. In conclusion, our data indicated that AGE prevents occurrence of glucose intolerance and fatty liver in diabetic mice, suggesting that AGE is useful for the prevention and treatment of type 2 diabetes.

Altered pancreatic morphology in the offspring of pregnant rats given reduced dietary protein is time and gender specific

Restriction of dietary protein during gestation and lactation in the rat results in a reduction in beta cell mass, insulin content and release in the offspring, and glucose intolerance when the offspring reach adulthood. The present study was designed to identify if a particular developmental window existed during prenatal development when endocrine pancreatic development was most susceptible to nutritional insult. Pregnant rats received a low-protein (8%, LP), but isocalorific diet from conception to parturition, during the first 2 weeks of gestation (LP (1-2)), the second week only (LP (2)), or the third week (LP (3)). At other times, they received a 20% protein (C) diet, while control animals received this diet continuously. When the offspring were examined at 130 days age, animals that had received LP diet had a significantly impaired glucose tolerance compared with control-fed animals. Pancreatic morphology was examined in the offspring on postnatal days 1 and 21. The LP diet resulted in a significant decrease in the numbers of large (more than 10 000 microm(2)) and medium (between 5000 and 10 000 microm(2)) sized islets present at postnatal day 1 for all LP treatments. Consequently, mean islet area and the mean number of beta cells were reduced. The impact of LP diet was most pronounced in LP (2) for females and in LP (3) for males, and this was greater than for continuous LP exposure. Insulin and Glut-2 mRNA expression were impacted negatively by LP in early and late gestation, but increased following administration in mid-gestation. Total pancreatic insulin content was not altered by LP treatment. Pdx-1, a transcription factor associated with both beta cell development and insulin gene transcription, was decreased in female offspring following LP (1-2) and LP (3), but not in males. Pancreatic expression of nestin mRNA, and the abundance of nestin-immunoreactive cells within islets, was decreased by all LP treatments. By postnatal day 21, the mean islet area and number of beta cells had largely recovered. However, insulin and Glut-2 mRNAs were elevated in offspring exposed to LP diet, particularly in females. The studies show that LP dietary insult in early, middle, or late gestation, all result in a relative deficiency of beta cells following birth, due to a failure to develop larger islets, but that females were particularly susceptible in mid-gestation and males in late gestation.

Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat.

Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na+ are potential candidates. In this study we examined the role of glucose and Na+ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na+-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by approximately 36%, SGLT1 by approximately 20%, and GLUT2 by approximately 100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by approximately 100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by approximately 150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na+ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule.

Prolactin induction of insulin gene expression: the roles of glucose and glucose transporter-2.

Previous studies have shown that lactogenic hormones stimulate beta-cell proliferation and insulin production in pancreatic islets. However, all such studies have been conducted in cells incubated in medium containing glucose. Since glucose independently stimulates beta-cell replication and insulin production, it is unclear whether the effects of prolactin (PRL) on insulin gene expression are exerted directly or through the uptake and/or metabolism of glucose. We examined the interactions between glucose and PRL in the regulation of insulin gene transcription and the expression of glucose transporter-2 (glut-2) and glucokinase mRNAs in rat insulinoma (INS-1) cells. In the presence of 5.5 mM glucose, the levels of preproinsulin and glut-2 mRNAs in PRL-treated cells exceeded the levels in control cells (1.7-fold, P<0.05 and 2-fold, P<0.05 respectively). The maximal effects of PRL were noted at 24-48 h of incubation. PRL had no effect on the levels of glucokinase mRNA. The higher levels of glut-2 mRNA were accompanied by an increase in the number of cellular glucose transporters, as demonstrated by a 1. 4- to 2.4-fold increase in the uptake of 2-deoxy-d-[(3)H]glucose in PRL-treated INS-1 cells (P<0.001). These findings suggested that the insulinotropic effect of PRL is mediated, in part, by induction of glucose transport and/or glucose metabolism. Nevertheless, even in the absence of glucose, PRL stimulated increases in the levels of preproinsulin mRNA (3.4-fold higher than controls, P<0.0001) and glut-2 mRNA (2-fold higher than controls, P<0.01). These observations suggested that PRL exerts glucose-independent as well as glucose-dependent effects on insulin gene expression. Support for this hypothesis was provided by studies of insulin gene transcription using INS-1 cells transfected with a plasmid containing the rat insulin 1 promoter linked to a luciferase reporter gene. Glucose and PRL, alone and in combination, stimulated increases in cellular luciferase activity. The relative potencies of glucose (5.5 mM) alone, PRL alone, and glucose plus PRL in combination were 2.2 (P<0.001), 3.4 (P<0.01), and 7.9 (P<0.0001) respectively. Our findings suggest that glucose and PRL act synergistically to induce insulin gene transcription.

Glucagon-like peptide-1 (7-36) amide as a novel neuropeptide.

Although earlier studies indicated that GLP-1 (7-36) amide was an intestinal peptide with a potent effect on glucose-dependent insulin secretion, later on it was found that several biological effects of this peptide occur in the brain, rather than in peripheral tissues. Thus, proglucagon is expressed in pancreas, intestine, and brain, but post translational processing of the precursor yields different products in these organs, glucagon-like peptide-1 (7-36) amide being one of the forms produced in the brain. Also, GLP-1 receptor cDNA from human and rat brains has been cloned and sequenced, and the deduced amino acid sequences are the same as those found in pancreatic islets. Through these receptors, GLP-1 (7-36) amide from gut or brain sources induces its effects on the release of neurotransmitters from selective brain nuclei, the inhibition of gastric secretion and motility, the regulation of food and drink intake, thermoregulation, and arterial blood pressure. Central administration (icv) of GLP-1 (7-36) amide produces a marked reduction in food and water intake, and the colocalization of the GLP-1 receptor, GLUT-2, and glucokinase mRNAs in hypothalamic neurons involved in glucose sensing suggests that these cells may be involved in the transduction of signals needed to produce a state of fullness. In addition, GLP-1 (7-36) amide inhibits gastric acid secretion and gastric emptying, but these effects are not found in vagotomized subjects, suggesting a centrally mediated effect. Similar results have been found with the action of this peptide on arterial blood pressure and heart rate in rats. Synthesis of GLP-1 (7-36) amide and its own receptors in the brain together with its abovementioned central physiological effects imply that this peptide may be considered a neuropeptide. Also, the presence of GLP-1 (7-36) amide in the synaptosome fraction and its calcium-dependent release by potassium stimulation, suggest that the peptide may act as a neurotransmitter although further electrophysiological and ultrastructural studies are needed to confirm this possibility.

Age-related changes in pancreatic islet cell gene expression

Previous studies have indicated that insulin secretion in response to glucose diminishes with age but insulin synthesis and gene transcription do not. To determine whether expression of genes other than those that encode insulin are subject to age-related changes that could alter pancreatic islet function, mRNAs for insulins I and II, amylin, glucose transporter 2 (GluT2), glucagon, and glucokinase were quantified in 2-, 6-, 12-, and 24-month-old Fischer 344 rats using species-specific ribonuclease (RNase) protection assays. There was only a modest (1.2- to 1.3-fold) increase in insulin I and insulin II mRNAs between ages 2 and 12 months. There were no statistically significant changes in levels of glucokinase mRNA with age. In contrast, the abundances of amylin, GluT2, and glucagon mRNAs all doubled during the same period. Variance in values from 24-month-old rats was too great to allow conclusions, except that the ratio of insulin II mRNA to insulin I mRNA increased with age. This change was not related to islet mass or total insulin mRNA abundance because it persisted at age 24 months, when total mRNA abundance had decreased. These results indicate that aging is associated with significant alterations in the relative proportion of expression of pancreatic islet cell genes implicated in insulin secretion and in intraislet glucose metabolism.

Feedback inhibition of insulin gene expression by insulin.

To examine the possible involvement of insulin and glucose in regulation of pancreatic islet gene expression, hyperinsulinemic (insulin infusion 4.1 mU/kg per min) clamps were performed for 12 h in rats at two different levels of glycemia (either 3 or 8 mM). A control group received a saline infusion for 12 h. At the end of the 12-h study period, pancreatic RNA was extracted, proinsulin and amylin mRNAs were measured on total RNA, and glucokinase and glucose transporter (GLUT-2) mRNAs were measured on poly(A)+ RNA by dot blot analysis. In insulin-infused hypoglycemic rats, there was a 58% decrement in proinsulin mRNA (P less than 0.01) relative to levels in controls, with no change in amylin, glucokinase, or islet GLUT-2 mRNAs. In insulin-infused hyperglycemic rats, there was a comparable decrement (44%, P less than 0.01) in proinsulin mRNA and a smaller decrement in GLUT-2 mRNA (32%, P less than 0.05), with no change in amylin or glucokinase mRNAs relative to levels in control animals. These studies suggest that insulin has a negative feedback inhibitory effect on its own synthesis. The mechanism of inhibition is unknown. It could be a direct effect of insulin on its own transcription, or alternatively an indirect effect mediated by humoral or neural factors. Sustained hyperinsulinemia may lead to suppression of normal islet beta cells and may contribute to the ultimate hypoinsulinemia of noninsulin-dependent diabetes mellitus.