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.

The effects of fasting and feeding on preproinsulin messenger RNA in rats.

The purpose of these experiments was to determine whether alterations in preproinsulin messenger (m)RNA activity could account for changes in insulin biosynthesis during fasting and refeeding. Rats were fasted 4 d and then fed for 6, 8, 24, or 48 h. With fasting, body weight decreased 25%, plasma glucose decreased from 6.1 to 2.2 mM, and pancreatic insulin content fell to 40% that of fed animals. Islet RNA decreased to 50% and protein to 55% that of control animals, while islet DNA content remained unchanged. After 6 h of refeeding, islet RNA content increased and was not significantly different from controls. Total islet and preproinsulin mRNA activity was estimated with an mRNA-dependent wheat germ cell-free protein synthesizing system. Preproinsulin and total protein synthesis was linearly dependent upon added RNA at concentrations up to 3 mug. Preproinsulin was identified by its mobility on SDS polyacrylamide gel electrophoresis and by hybrid arrested translation of preproinsulin mRNA. After an 18-h fast, islet mRNA activity decreased 33%; after 4 d mRNA activity decreased to 66% below that of control fed animals. Preproinsulin mRNA activity was decreased, but to a lesser extent, accounting for 20% of total islet protein in fed animals and 46% in the 4-d fasted animals. Total mRNA activity returned to control values after 8 h of refeeding and increased to 150% of controls at 24 and 48 h. Preproinsulin mRNA activity increased more rapidly on refeeding. By 8 h it was 160% of controls.To determine whether changes in preproinsulin mRNA activity were associated with changes in the amount of preproinsulin mRNA, nucleic acid hybridization analysis was performed. Pancreatic RNA from fed and fasted animals was electrophoresed on agarose gels, transferred to diazophenylthio paper, and hybridized to (32)P-labeled preproinsulin complementary (c)-DNA. This analysis demonstrated that changes in mRNA activity were associated with changes in the amount of hybridizable mRNA present. These studies are the first to demonstrate alterations of preproinsulin mRNA under any conditions, and the changes correlate with alterations in rates of insulin biosynthesis.

An in vivo analysis of pancreatic protein and insulin biosynthesis in a rat model for non-insulin-dependent diabetes.

The purpose of these experiments was to estimate insulin biosynthesis in vivo in a rat model for non-insulin-dependent diabetes. Insulin biosynthesis rates were determined in 4-wk-old animals that had been injected with 90 mg/kg of streptozotocin 2 d postpartum. Control and diabetic animals did not differ in body weight or fasting plasma glucose. Fed plasma glucose was significantly elevated (186 +/- 13 micrograms/dl vs. 139 +/- 7 mg/dl, P less than 0.05) and pancreatic insulin content was reduced (41 +/- 2 micrograms/g vs. 63 +/- 8 micrograms/g, P less than 0.05) in the diabetic rats. Insulin biosynthesis was estimated in vivo by measuring and comparing [3H]leucine incorporation into proinsulin with that into total pancreatic protein 45 min after injection. Insulin biosynthesis was 0.391 +/- 0.07% of pancreas protein synthesized in control rats and 0.188 +/- 0.015% (P less than 0.05) in diabetic rats. In animals of the same age, the fractional and absolute rate of pancreatic protein synthesis were determined. Total pancreatic protein synthesis was not reduced in streptozotocin treated animals (185.5 +/- 14.1%/d vs. 158.6 +/- 14.9%/d, NS) but was markedly reduced in control rats after a 48-h fast (to 70.8 +/- 5.5%/d, P less than 0.01). Because total pancreatic protein synthesis was not decreased in the diabetic rats, the decrease in the fraction of radiolabel incorporated into insulin seems to represent an absolute decrease in the rate of insulin biosynthesis in this animal model for diabetes. Through RNA blot hybridization with 32P-labeled cloned rat insulin complementary DNA, proinsulin messenger RNA (mRNA) was estimated as the rate of insulin biosynthesis in control and diabetic animals. There was a 61% reduction in proinsulin mRNA at 4 wk and an 85% reduction at 7 wk (P less than 0.001) in the diabetic animals. After streptozotocin injection in neonatal rats, there is marked beta-cell damage and hyperglycemia. Beta-cell regeneration occurs with return to normoglycemia, but with age hyperglycemia develops. The reduction in insulin synthesis and proinsulin mRNA seemed disproportionate with the more modest reduction in beta-cell number. The importance of these observations is that, in this animal model, diabetes is associated with a limited ability to regenerate beta-cell mass and to synthesize insulin. The relationship between the defect in glucose-stimulated insulin release and impaired insulin biosynthesis has yet to be determined.

Effects of glucose on proinsulin messenger RNA in rats in vivo.

The purpose of these studies was to determine whether glucose, the principal regulator of insulin biosynthesis in mammals, controls synthesis through alterations in levels of proinsulin mRNA in whole animals. Rats were starved for 3 days and then either refed or injected with glucose or saline for 24 h. Glucose injection raised plasma glucose levels equivalent to levels seen with refeeding but provided less than 20% of caloric replacement. Pancreatic RNA was extracted and the relative concentration of proinsulin mRNA was determined by blot hybridization with a cloned rat proinsulin cDNA probe. In starved animals proinsulin mRNA levels were 15-20% that of fed controls. Glucose injection produced a specific three- to fourfold increase in proinsulin mRNA levels relative to total pancreatic RNA, within 24 h. The effect was measurable 2 h after glucose injection and appeared largely complete by 12 h. Actinomycin D blocked the glucose-induced increase in proinsulin mRNA. These studies demonstrate effects of changes of plasma glucose on levels of proinsulin mRNA. Their rapidity of onset and large magnitude are comparable to effects of glucose on rates of insulin biosynthesis in isolated islets and suggest that insulin biosynthesis is regulated at least in part by levels of proinsulin mRNA.

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.

Impaired insulin biosynthetic capacity in a rat model for non-insulin-dependent diabetes. Studies with dexamethasone.

These studies of a rat model for non-insulin-dependent diabetes mellitus (NIDDM) were performed to determine whether hyperglycemia occurs when capacity to synthesize insulin is exceeded. The neonatal streptozocin (STZ)-treated rat has acute hyperglycemia with marked destruction of pancreatic beta-cells, followed by gradual regeneration to 50-70% normal beta-cell number. At age 4 wk, fed serum glucose concentration is only mildly elevated relative to controls. With age, the rats become progressively hyperglycemic, and by 12 wk they have marked impairment of glucose-stimulated insulin release. In these studies, dexamethasone (0.125 mg/kg/day for 4 days) was administered to control and to STZ-treated animals to produce insulin resistance. The relationship between insulin biosynthesis and serum glucose concentrations was assessed. In control rats, response to dexamethasone was similar at both 4 and 12 wk. Serum glucose levels and pancreatic insulin concentration remained unchanged. Both insulin biosynthetic rates (as measured by 3H-leucine incorporation into proinsulin) and proinsulin mRNA levels increased twofold. STZ-treated rats at age 4 wk demonstrated mild hyperglycemia. Dexamethasone injection resulted in an increase in insulin biosynthesis and proinsulin mRNA in these animals, while serum glucose did not increase. STZ-treated rats at 12 wk showed more profound hyperglycemia (serum glucose 315 +/- 38 mg/dl versus control, 187 +/- 12 mg/dl). A marked rise in serum glucose (to 519 +/- 42 mg/dl) was observed after 4 days of dexamethasone injection. Pancreatic insulin content became severely depleted relative to saline-injected, STZ-treated animals, and there was no response of levels of proinsulin mRNA.

Analysis of insulin gene expression in human pancreas.

The purpose of these studies was to determine whether insulin gene expression at the level of proinsulin mRNA could be studied in human pancreas. RNA was isolated from autopsy specimens and analyzed by RNA-blot hybridization with various 32P-human insulin gene probes spanning either the entire gene or the second intervening sequence. A major band 0.62 kilobases (kb) in length accounted for over 95% of the mRNA, consistent in size with presumed mature proinsulin mRNA. In addition, minor bands of 1.5 and 1.3 kb were seen, consistent with an initial gene transcript containing both intervening sequences and with a processed intermediate. The 1.5- and 1.3-kb RNA were confirmed to be proinsulin mRNA precursors by hybridization specifically with the IVS II probe. Total RNA and polyadenylated RNA from five normal pancreata and two insulinomas revealed the same pattern. This method provides a means of determining whether altered insulin gene expression is one cause of diabetes.

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.

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.

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.

Proinsulin I and II gene expression in inbred mouse strains.

Mice and rats express two nonidentical insulins from a pair of unlinked genes. We have applied a nuclease protection assay, which can sensitively quantify each of the mouse insulin mRNAs, to the resolution of the following questions concerning their expression. First, it has not been established whether alterations in expression of one or both of these genes cause differing total insulin biosynthetic capacity noted between several inbred mouse strains. These studies showed that the relative abundance of mRNAs encoding mouse insulins I and II was identical in four separate mouse strains. In spontaneously obese, hyperinsulinemic (db/db)C57BL/KsJ mice, both proinsulin I and proinsulin II mRNAs were increased relative to the levels in normal (+/db) C57BL/KsJ mice, but again the ratio of the two mRNAs did not differ. The ratio was nearly identical to that for the orthologous mRNAs in rats, indicating that the mechanisms which regulate insulin mRNAs in rodents are conserved in both genes in several mouse strains and between rodent species. This finding suggests that differences between mouse strains in insulin biosynthetic capacity result from differences in the glucose sensing/signalling mechanism at a point before coordinate gene transcription. Second, low levels of insulin synthesis have been suggested as an explanation for relatively high levels of insulin in several nonpancreatic tissues. We showed that the ribonuclease protection assay, sufficiently sensitive to measure 1/2000th the amount of insulin mRNA present in pancreas, was unable to detect insulin mRNA in salivary gland. This result indicates that the high levels of radioimmunoassayable insulin detected in salivary glands are not the result of insulin synthesis in situ.

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)

Rapid alteration of pancreatic proinsulin and preproinsulin messenger ribonucleic acid in rats treated with cyproheptadine.

Cyproheptadine (CPH) appears to be unique among islet beta-cell toxicants by virtue of its ability to rapidly and reversibly inhibit insulin biosynthesis in rats. These studies examined the mechanism of CPH-induced insulin depletion by determining the time course for CPH-induced changes in pancreatic preproinsulin mRNA, proinsulin, and insulin levels. A single oral dose of CPH decreased proinsulin levels to 35% of the control value by 3 h. Proinsulin stayed depressed for up to 24 h. Preproinsulin mRNA declined more slowly, reaching 35% of the control value at 6 h, and increased more rapidly, returning to the control value by 24 h. Pancreatic insulin levels did not decrease significantly until 24 h. Exposure of isolated rat islets to CPH for 30 min in vitro selectively inhibited proinsulin synthesis by 62%, without affecting preproinsulin mRNA levels. The dissociation between changes in proinsulin and preproinsulin mRNA levels suggests that the decrease in preproinsulin mRNA in vivo is associated with but does not cause CPH-induced changes in insulin biosynthesis.

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.

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.

Shortening of poly (A) tail of glucose transporter--one mRNA in experimental diabetes mellitus.

To determine the molecular mechanisms of diabetes-related changes in the expression of GLUT-1 in cerebral tissue, streptozotocin-induced diabetic rats and vehicle injected controls were studied after 4 weeks of diabetes. The GLUT-1 mass in cerebral microvessels was reduced in diabetic rats by approximately 38% (P < 0.01). The GLUT-1 concentration in insulin-treated diabetic group was not significantly different from controls. The GLUT-1 mRNA content of cerebral tissue in diabetic rats (0.064 +/- 0.007) was significantly reduced compared to control rats (0.122 +/- 0.011) or insulin-treated diabetic rats (0.122 +/- 0.015) P < 0.01. The in vitro translation of GLUT-1 mRNA of diabetic rats (0.793 +/- 0.047 arbitrary units) was also significantly lower than that in control rats (1.403 +/- 0.153) P < 0.01 or insulin-treated diabetic rats. (1.124 +/- 0.083) P < 0.01. These changes occurred in asssociation with a reduction in poly (A) tail length of GLUT-1 mRNA which decreased from a control value of 200-350 nt to only 50-100 nt in diabetic rats. Shortening of poly (A) tail of mRNAs is a novel mechanism of diabetes-related changes in the expression of specific genes which are regulated at a translational level.

CYP4A1 gene transfection studies and the peroxisome proliferator-activated receptor: development of a high-throughput assay to detect peroxisome proliferators.

An in vitro reporter gene assay has been established to examine cytochrome P4504A1 (CYP4A1) induction. A response element from the upstream region of the rat CYP4A1 gene containing a peroxisome proliferator response element (PPRE) has been linked to the chloramphenicol acetyl-transferase (CAT) gene in a reporter vector (1). This CYP4A1 reporter construct has been co-transfected into human HepG2 cells in the presence and absence of expression vectors encoding the transcription factors PPAR alpha and RXR alpha. The assay employs calcium phosphate-DNA co-precipitate mediated transfection. Reporter gene products have been quantitated using chemiluminescent based assays. We have shown that, in the presence of PPAR alpha, the above CYP4A1 construct is transcriptionally activated by a range of structurally different peroxisome proliferators including Wy-14,643, ciprofibrate, clofibric acid and nafenopin. Our future efforts will focus on the establishment of a high-throughput assay for the detection of peroxisome proliferators. Such an assay would provide an invaluable in vitro test for the screening of developmental drug candidates prior to in vivo studies.

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.

Rat insulin II gene expression by extraplacental membranes. A non-pancreatic source for fetal insulin.

Using cDNA cloning, ribonuclease protection, and Northern hybridization analysis, we showed that insulin gene expression occurs in yolk sac-derived fetal extraplacental membranes throughout the last half of rat fetal development. The mRNA product of the ancestral rat insulin II but not the duplicated rat insulin I gene was present in high copy number, and its abundance was regulated during development. Insulin mRNA was present in extraplacental membranes before pancreatic differentiation; membrane insulin mRNA content greatly exceeded that in pancreas until the last 2 days of gestation when content in each tissue became similar. Polyadenylation and intron splicing occurred at the same sites used in pancreas, but initiation of transcription occurred at multiple sites in membranes. Minces of membranes maintained in culture produced approximately 10 ng of radioimmunoassayable insulin/mg membrane protein/day. Over a 4-day period, approximately 50 times more insulin accumulated in medium than that present in membranes at the time of isolation. These studies indicate that yolk sac is a source for insulin during fetal development and that the mechanisms regulating insulin gene expression in this tissue differ from those in pancreatic beta cells.