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.

Time course for effects of hypoglycemia on insulin gene transcription in vivo.

The purpose of these studies was to determine the time course for onset of effects of hypoglycemia on insulin gene transcription in vivo. Using insulin infusions, we found that insulin-induced hypoglycemia decreased levels of precursors for insulin mRNA, reflecting changes in new mRNA synthesis, to new steady-state values within 100 min. These changes were followed by declines in processed insulin mRNA. An alternate infusion technique was developed to lower plasma glucose levels from a constant level of 120-130 to 50-60 mg/dl in < 10 min without changing insulin levels from those maintained during a preceding 1-h control period. Using this protocol, we found that levels of precursors for insulin mRNA remained constant for the first 20 min of hypoglycemia, then decreased rapidly at 40 and 60 min. The initial delay followed by rapid decline suggests that the decrease of insulin gene transcription in response to hypoglycemia is an active process requiring one or more inductive events before implementation.

Allele specific inactivation of insulin 1 and 2, in the mouse yolk sac, indicates imprinting.

Genomic imprinting, gene inactivation during gametogenesis, causes maternal and paternal alleles of some genes to function unequally. We examined the possibility of imprinting in insulin genes because the human insulin gene (ins) and its mouse homologue (ins2) are adjacent to the known imprinted genes, igf2 and H19, and because imprinting has been implicated in the transmission of an ins linked risk for Type I diabetes. We show, by single strand conformational polymorphism (SSCP) analysis of cDNAs from parents and progeny of interspecies mouse crosses, that insulin genes are imprinted. While both alleles of the two mouse insulin genes were active in embryonic pancreas, only paternal alleles for both genes were active in the yolk sac.

Insulin gene expression and insulin synthesis in mammalian neuronal cells.

To demonstrate the presence of de novo synthesis in central mammalian neurons, we cloned and sequenced a rabbit insulin cDNA from pancreas and used it to define sequences encoding insulin mRNA from postnatal rabbit brain. We observed transcription/elongation of nascent insulin transcripts, characterized the size of these transcripts, and localized them to specific neurons in certain catecholaminergic-rich areas of the central nervous system. RNase protection assays using a rabbit probe spanning a region from 14 bases 5' to the translation start site through all but 18 bases of the sequence encoding the A-chain of insulin showed two bands in rabbit brain RNA and only one band in pancreas. The larger band in brain was the same size as that in pancreatic RNA; the other was approximately 10 bases shorter. Because the sequence of a reverse transcription-polymerase chain reaction product from brain RNA was identical to pancreatic RNA sequence in the region corresponding to the 3' region of the probe, the smaller band in brain is most consistent with a sequence mismatch in some brain mRNA in the region corresponding to the 5'-end of the probe. In situ hybridization localized insulin mRNA to anatomical regions involved with olfaction and higher association of the limbic system. High performance liquid chromatography, radioimmunoassay, and [35S]cysteine metabolic labeling of cultured neuronal and glial cells indicated extracellular secretion of immunoprecipitable insulin by neurons only. Presence of insulin transcripts within specific neurons with extracellular secretion of the peptide suggests a specialized biological role.

Insulin II gene expression in rat central nervous system.

Controversy persists concerning the origin of insulin in the central nervous system. While there has been convincing evidence in vitro to demonstrate the presence of neuronal insulin mRNA, conventional assays have failed to detect the same in whole brain preparations. Here we employed RNAse-protection and sensitive reverse transcription-polymerase chain reaction (RT-PCR) assays in attempts to detect insulin I and II mRNAs in rat brains obtained from different developmental stages. The RNAse-protection assay did not detect insulin I or insulin II transcripts in fetal (13 to 21 day gestation) or adult brains. RT-PCR, while detecting low amounts of insulin I transcripts in other extrapancreatic tissues such as the rat yolk sac and fetal liver previously shown to express insulin II, failed to detect insulin I in brain at any age examined. Insulin II mRNA was detected by RT-PCR in fetal, neonatal and adult rat brains, just as in yolk sac, fetal and adult livers. We conclude that while the duplicated insulin I gene is not expressed, the ancestral insulin II gene is expressed in fetal, neonatal and adult rat brains. Our observations support the concept of de novo brain insulin II synthesis beyond the pre-pancreatic stage of embryonic development.

Hypoglycemia but not hyperglycemia induces rapid changes in pancreatic beta-cell gene transcription.

The purpose of these studies was to quantify several mRNAs expressed specifically in pancreatic islet cells and known or postulated to be important for insulin release after acute well defined alterations in levels of plasma glucose. Glucose levels were maintained at 50, 120, or 180 mg/dl (2.8, 6.7, or 10 mM) for 3 h in conscious unrestrained rats. Hypoglycemia (for 3 h) caused significant decreases in pancreatic content of mRNAs for insulin 2 and GLUT-2 to 55 and 34% of control values, respectively. There were no significant changes in insulin 1, amylin, glucokinase, or glucagon mRNAs. Unprocessed insulin 1 and 2 mRNA precursors were decreased to 17 and 10% of levels in controls, consistent with effects of short-term hypoglycemia on new mRNA synthesis. Hyperglycemia (for 3 h) caused no increase in pancreatic content of any mRNA measured. To discriminate between effects of hypoglycemia and hyperinsulinemia in the hypoglycemic animals, rats were made hypoglycemic by infusion with etomoxir, a carnitine palmitoyltransferase I inhibitor that lowers glucose in the fasted (glycogen-depleted) state by inhibiting hepatic gluconeogenesis. A single dose of this agent caused a decrease in glucose from 120 mg/dl (6.7 mM) to 80 mg/dl (4.4 mM) and significantly decreased insulin mRNA and pre-mRNA. These results are consistent with the hypothesis that glucose modulates islet cell gene transcription directly. They indicate that the range of glucose concentrations that modulate gene transcription differs from the levels of glucose that alter both insulin biosynthetic and secretion rates.

Insulin receptor gene expression during development: developmental regulation of insulin receptor mRNA abundance in embryonic rat liver and yolk sac, developmental regulation of insulin receptor gene splicing, and comparison to abundance of insulin-like growth factor 1 receptor mRNA.

Insulin gene expression has been demonstrated in nonpancreatic tissues early in development, suggesting that this hormone might have actions significant for the differentiating embryo. Because such actions imply ligand-receptor binding, we quantified mRNAs encoding the two known forms of insulin receptor in rat liver and yolk sac, two endodermally derived tissues shown to express insulin genes, between gestation days (E) 13 and E21 (mid-organogenesis to parturition). Because of its presumed importance for fetal growth, we estimated the abundance of mRNA encoding insulin-like growth factor 1 (IGF 1) receptor in the same samples for comparison. The abundance of insulin receptor mRNA exceeded that for IGF 1 receptor mRNA in liver and yolk sac at all times studied. This difference was greater in liver, where insulin receptor mRNAs were three to more than 50 times more abundant than IGF 1 receptor mRNA on gestation days E13-E16, times which antedate the development of significant hepatic metabolic actions of insulin. The marked abundance of mRNAs encoding insulin receptors is consistent with the hypothesis that insulin has significant actions in specific tissues during the organogenic period.

Differential regulation of rat insulin I and II messenger RNA synthesis: effects of fasting and cyproheptadine.

Rats and mice retain a duplicated insulin (I) gene. Because the duplicated gene shares only incomplete homology with the ancestral insulin (II) gene it may be regulated differently. In the studies presented here we measured changes in abundance of these distinct insulin mRNAs and their precursors in response to fasting and fasting plus a single dose of cyproheptadine, two experimental manipulations that cause changes in the level of total insulin mRNA in rats. Both diminished rat insulin II mRNA to a greater extent than rat insulin I mRNA. Rat insulin II mRNA comprised 41% of the total insulin mRNA in 0 h controls and decreased to 33% of the total insulin mRNA after a 10-h fast. Insulin II mRNA decreased to 26% of the total insulin mRNA 10 h after treatment with cyproheptadine. To determine whether these manipulations had effects on insulin mRNA synthesis, precursors for each of the two mRNAs were quantified. Fasting for 24 h had only small effects on insulin I mRNA precursor, but diminished rat insulin II pre-mRNA to 32% of the 0 h control values. One and a half hours after fasting plus cyproheptadine administration, pre-mRNA for rat insulin II levels had decreased to 38%, while rat insulin I pre-mRNA remained at levels present in 0 h controls. Levels of rat insulin I and II pre-mRNAs were both maximally depressed at 10 h, but rat insulin II pre-mRNA decreased to 3%, while rat insulin I pre-mRNA diminished to only 49% of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective expression and developmental regulation of the ancestral rat insulin II gene in fetal liver.

Previous studies have indicated that high levels of insulin synthesis occur in the yolk sac of fetal rats. Because the yolk sac is an early site for synthesis of several tissue-specific proteins synthesized by liver later in development, these studies were performed to determine whether insulin gene expression also occurs in fetal liver. To this purpose, liver RNA obtained on consecutive days of rat fetal development from embryo day (E) 13 to E21 was evaluated for the presence of insulin or insulin-like mRNA species using Northern hybridization with a uniformly labelled rat insulin II genomic antisense RNA probe. Two species were detected. The larger was approximately 2.4 kilobases in length, was very low in abundance, and was present only during the earliest days studied (E13-15). The second species was approximately 720 bases in length, increased in abundance between days E13-16, and decreased between days E16-21. Maximum abundance of this mRNA was 0.3 pg/microgram total liver RNA, or 1/10th to 1/20th the abundance of total insulin mRNA in adult rat pancreas. Sequencing of multiple cloned products of E15 rat liver cDNA amplified by polymerase chain reaction using insulin I or II gene-specific primers indicated that the bands detected on Northern hybridization were (ancestral) rat insulin II gene transcripts. Analysis of products of polymerase chain reactions also indicated that the duplicated rat insulin I gene was not expressed in fetal liver. The content of insulin mRNA in fetal liver is sufficient to suggest that the liver may be a significant source for insulin at specific times during fetal development.

Selective expression of the insulin I gene in rat insulinoma-derived cell lines.

Four rat insulinoma-derived cell lines were found to express only the rat insulin I gene, although an apparently normal insulin II gene was present in each cell line. This finding was reflected by the absence of insulin II and the presence of insulin I gene transcripts in the products of run-on transcription assays, the absence of insulin II and the presence of insulin I unprocessed (pre-) mRNA, and the absence of mature insulin II and the presence of insulin I mRNA. Analysis of insulin II genes in cell lines by gene amplification indicated that the lack of insulin II gene transcription is not explained by the absence of or gross alterations in the insulin II genes per se. These studies indicate that the two nonallelic insulin genes do not function equivalently in pancreas-derived cells.

The two nonallelic rat insulin mRNAs and pre-mRNAs are regulated coordinately in vivo.

These studies compared the regulation of expression of the two nonallelic rat insulin genes in vivo. The relative abundance of mRNAs for rat insulins I and II was the same in sucrose-treated and fasted rats despite 2-fold higher total insulin mRNA levels in sucrose-treated groups. The ratio of rat insulin I to rat insulin II mRNA also remained constant when demand for insulin was increased by pregnancy, administration of dexamethasone, or excess growth hormone. The two mRNAs for insulin appeared at the same time during fetal pancreatic development, increased in constant proportion to maximum values during the neonatal period, and fell in tandem to adult values by age 6 weeks. Taken together, these data suggest that similar or identical mechanisms modulate steady-state levels of both insulin mRNAs in rats. Concentrations of precursors for each mRNA were also greater in sucrose-treated rats, indicating that changes in mRNA concentrations were mediated at least in part by changes in either gene transcription rates or in mRNA precursor stability.