Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells.

Expression of peroxisome proliferator-activated receptor alpha (PPARalpha) and enzymes of fatty acid (FA) oxidation is markedly reduced in the fat-laden, dysfunctional islets of obese, prediabetic Zucker diabetic fatty (fa/fa) rats with mutated leptin receptors (OB-R). Leptin, PPARalpha/retinoid x receptor ligands, and FA all up-regulate PPARalpha and enzymes of FA oxidation and stimulate [3H]-palmitate oxidation in normal islets but not in islets from fa/fa rats. Overexpression of normal OB-R in islets of fa/fa rats corrects all of the foregoing abnormalities and reverses the diabetic phenotype. PPARalpha is a OB-R-dependent factor required for normal fat homeostasis in islet cells.

Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.

To elucidate the mechanism of the basal hyperinsulinemia of obesity, we perfused pancreata from obese Zucker and lean Wistar rats with substimulatory concentrations of glucose. Insulin secretion at 4.2 and 5.6 mM glucose was approximately 10 times that of controls, whereas beta-cell volume fraction was increased only 4-fold and DNA per islet 3.5-fold. We therefore compared glucose usage at 1.4, 2.8, and 5.6 mM. Usage was 8-11.4 times greater in Zucker islets at 1.4 and 2.8 mM and 4 times greater at 5.6 mM; glucose oxidation at 2.8 and 5.6 mM glucose was > 12 times lean controls. To determine if the high free fatty acid (FFA) levels of obesity induce these abnormalities, normal Wistar islets were cultured with 0, 1, or 2 mM long chain FFA for 7 days. Compared to islets cultured without FFA insulin secretion by FFA-cultured islets (2 mM) perifused with 1.4, 3, or 5.6 mM glucose was increased more than 2-fold, bromodeoxyuridine incorporation was increased 3-fold, and glucose usage at 2.8 and 5.6 mM glucose was increased approximately 2-fold (1 mM FFA) and 3-fold (2 mM FFA). We conclude that hypersecretion of insulin by islets of obese Zucker fatty rats is associated with, and probably caused by, enhanced low Km glucose metabolism and beta-cell hyperplasia, abnormalities that can be induced in normal islets by increased FFA.

Expression of normal and novel glucokinase mRNAs in anterior pituitary and islet cells.

The glucose-phosphorylating enzyme glucokinase likely plays an important role in regulating glucose-stimulated insulin secretion from the islets of Langerhans and has previously been thought to be expressed only in that tissue and in liver. In this study, we demonstrate high levels of glucokinase mRNA in the anterior pituitary cell line AtT20ins, which has been engineered to secrete correctly processed insulin, as well as in primary anterior pituitary tissue. Unlike islet or liver cells, expression of glucokinase mRNA in anterior pituitary cells was not accompanied by expression of the high Km glucose transporter (GLUT-2) mRNA. The glucokinase transcript in anterior pituitary cells was similar in size to islet glucokinase mRNA, which has a unique, elongated 5'-end relative to the liver glucokinase message. Amplification and sequence analysis of the glucokinase mRNA expressed in islets, RIN1046-38 cells, and anterior pituitary cells confirmed that the glucokinase transcripts in these cell types contain the same 5'-sequence. In addition, a novel alternative transcript was identified that contains a 52-nucleotide deletion and that predicts a 58-amino acid peptide as a result of a frame shift. Both the deleted and undeleted transcripts were found in islets, RIN cells, and AtT20ins cells, whereas only the deleted product was identified in primary anterior pituitary tissue. An antibody prepared against a peptide found at the N terminus of the islet isoform of glucokinase easily detected a protein with a size predicted by the undeleted transcript in extracts prepared from islets, RIN1046-38 cells, and AtT20ins cells. Since both the glucokinase protein and mRNA are naturally expressed in AtT20ins and RIN1046-38 cells, we compared the effect of varying concentrations of glucose on insulin secretion from the two lines. Insulin secretion from RIN1046-38 cells was stimulated by glucose in a dose-dependent manner over the range 0-2.5 mM, where it reached a maximum. AtT20ins cells, in contrast, exhibited no response to glucose at any concentration tested, despite the fact that insulin secretion from both cell lines was stimulated by incubation with dibutyryl cAMP. We conclude that glucokinase expression in AtT20ins cells may be necessary, but is not sufficient to confer glucose-stimulated insulin secretion.

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass.

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.

The high Km glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence.

The liver has been shown to contain a facilitated diffusion glucose transporter with high Km for glucose that is structurally distinct from the low Km glucose transporters found in most other tissues. We find that 3-O-methyl glucose is greater than 90% equilibrated across dispersed islet cells within 60 s, consistent with a facilitated diffusion transport mechanism. L-Glucose uptake was minimal throughout the time course, indicating stereospecificity. Measurement of glucose transport over a range of 3-O-methyl glucose concentrations from 0.05 to 60 mM revealed the presence of a component of glucose transport with an apparent Km of 17 mM, a value essentially identical to that previously reported for liver. Interestingly, a second component of glucose transport was also observed with an apparent Km of 1.4 mM, as has been reported for other tissues such as erythrocytes that are known to contain the "HepG2" or "erythroid/brain" type glucose transporter. Further evidence for the existence of two transport components is provided by the observation that a low concentration of cytochalasin B (0.4 microM) completely inhibits the low Km transport activity but has no effect on the high Km transporter. The kinetic similarity of high Km glucose transport in liver and islets is readily understood in light of our structural analysis. Sequence analysis of cDNA clones indicates that the liver and islet glucose transporters have identical sequences and, thus, are the products of the same gene.

Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.

Glucokinase, a key regulatory enzyme of glucose metabolism in mammals, provides an interesting model of tissue-specific gene expression. The single-copy gene is expressed principally in liver, where it gives rise to a 2.4-kilobase mRNA. The islets of Langerhans of the pancreas also contain glucokinase. Using a cDNA complementary to rat liver glucokinase mRNA, we show that normal pancreatic islets and tumoral islet cells contain a glucokinase mRNA species approximately 400 nucleotides longer than hepatic mRNA. Hybridization with synthetic oligonucleotides and primer-extension analysis show that the liver and islet glucokinase mRNAs differ in the 5' region. Glucokinase mRNA is absent from the livers of fasted rats and is strongly induced within hours by an oral glucose load. In contrast, islet glucokinase mRNA is expressed at a constant level during the fasting-refeeding cycle. The level of glucokinase protein in islets measured by immunoblotting is unaffected by fasting and refeeding, whereas a 3-fold increase in the amount of enzyme occurs in liver during the transition from fasting to refeeding. From these data, we conclude (i) that alternative splicing and/or the use of distinct tissue-specific promoters generate structurally distinct mRNA species in liver and islets of Langerhans and (ii) that tissue-specific transcription mechanisms result in inducible expression of the glucokinase gene in liver but not in islets during the fasting-refeeding transition.

Rat amylin: cloning and tissue-specific expression in pancreatic islets.

Amyloid deposits in the islets of Langerhans of the pancreas are a common finding in non-insulin-dependent diabetes mellitus. The main protein constituent of these deposits is a 37-amino acid peptide known as amylin that resembles calcitonin gene-related peptide, a neuropeptide. We have isolated cDNA clones corresponding to the rat amylin precursor from an islet cDNA library and we show that this peptide is encoded in a 0.9-kilobase mRNA that is translated to yield a 93-amino acid precursor. The amylin peptide is bordered by dibasic residues, suggesting that it is proteolyzed like calcitonin gene-related peptide. The peptide sequences flanking the amylin sequence do not resemble the calcitonin gene-related peptide flanking sequences. RNA hybridization studies show that amylin mRNA is abundant in the islets of Langerhans but is not present in the brain or seven other tissues examined. Dietary changes, such as fasting or fasting and refeeding, have little effect on amylin mRNA expression. This tissue specificity suggests that amylin is involved in specific signaling pathways related to islet function.