Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing ␤ cells and glucagon-positive ␣ cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcriptioncoupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of ␦ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.Insulin is synthesized, stored, and secreted by the pancreatic islet ␤ cells in a highly regulated manner and plays a vital role in glucose homeostasis. Insulin action also results in several other pleiotropic effects that are less well documented. Embryonic insulin synthesis begins early in gestation, but fetal glycemia closely follows maternal blood glucose levels. The question, therefore, arises as to what function embryonic insulin might fulfill during development. For instance, one might ask whether insulin plays an autocrine or paracrine role in pancreatic islet cell growth and differentiation, since insulin is synthesized with other hormones in developing islet cell types (1-3). Recently, this question has been addressed in a few transgenic studies. For instance, the gene encoding PDX-1 (4, 5), a homeodomain transcription factor synthesized in adult ␤ cells and capable of transactivating insulin gene expression, has been inactivated by targeted disruption (6, 7). Agenesis of pancreas resulting from PDX-1 deficiency precluded from addressing the question of the possible role of insulin in islet cell growth and differentiation. Similarly, mice lacking the LIM homeodomain transcription factor ISL1, synthesized in all classes of islet cells in the adult, were arrested in development soon after embryonic day 9.5 (8). The requirement of ISL1 in pancreatic epithelium for the differentiation of all islet cell types was, however, demonstrated by in vitro culture of explants from ISL1-deficient embryonic day 9.5 embryos that gave rise to cells that were negative for glucagon, insulin, and somatostatin. In another study, transgenic mouse embryos expressing the gene encoding the diphteria toxin A chain under control of the rat Ins2 promoter were generated (9). The resulting genetic ablation of the insulin-producing cells did not appear to alter the development of the nontarget...

Targeted disruption of the insulin receptor gene (Insr) in the mouse was achieved using the homologous recombination approach. Insr+/− mice were normal as shown by glucose tolerance tests. Normal Insr−/− pups were born at expected rates, indicating that Insr can be dispensable for intrauterine development, growth and metabolism. However, they rapidly developed diabetic ketoacidosis accompanied by a marked post‐natal growth retardation (up to 30–40% of littermate size), skeletal muscle hypotrophy and fatty infiltration of the liver and they died within 7 days after birth. Total absence of the insulin receptor (IR), demonstrated in the homozygous mutant mice, also resulted in other metabolic disorders: plasma triglyceride level could increase 6‐fold and hepatic glycogen content could be five times less as compared with normal littermates. The very pronounced hyperglycemia in Insr−/− mice could result in an increased plasma insulin level of up to approximately 300 microU/ml, as compared with approximately 25 microU/ml for normal littermates. However, this plasma level was still unexpectedly low when compared with human infants with leprechaunism, who lack IR but who could have extremely high insulinemia (up to > 4000 microU/ml). The pathogenesis resulting from a null mutation in Insr is discussed.

In the mouse, insulin is produced from two similar but nonallelic genes that encode proinsulins I and II. We have investigated expression of these two genes during mouse embryonic development, using a PCR to detect the two gene transcripts and immunocytochemistry to visualize the two corresponding proteins. At appearance of the dorsal pancreatic anlage at day 9.5 of gestation, both mRNAs could be detected in the embryos, and both proteins were present together in the same cells of the developing pancreas. At days 9.5 and 10.5, when the ventral anlage appears, there were fewer proinsulin H mRNAs than proinsulin I mRNAs. At day 12.5 this ratio was reversed. Proinsulin II mRNA, but not proinsulin I mRNA, could be detected at day 8.5 in the prepancreatic embryo. Proinsulin II mRNA, but not proinsulin I mRNA, was also found in the heads of embryos at day 9.5 and at all later stages studied. These results indicate that the two proinsulin genes are regulated independently, at least in part. They also suggest that insulin might play a role as a growth factor in the developing mouse brain.Insulin, a key hormone in metabolic homeostasis, is synthesized, stored, and secreted by beta cells of the pancreatic islets. The protein is synthetized in the form of a precursor, preproinsulin, which is highly conserved among animal species. Unlike most mammals, mice and rats express two nonallelic genes that encode proinsulins I and II. In the mouse these two proteins differ by two amino acids in the B chain and three amino acids in the C peptide. Mouse C peptide I also lacks the Gly-Ala residues present in positions 17 and 18 of C peptide 11(1, 2). The corresponding genes in mouse and rat are highly homologous, and their organization is similar, except that the preproinsulin I gene possesses only the first of two introns present in the preproinsulin II gene (1, 3).On previous studies concerning the regulation of these two nonallelic genes, mRNAs were reported to be present in nearly equal quantities in adult pancreas of mouse (1, 4), as well as of rat (5) Here we have investigated regulation of the expression of the two proinsulin genes during mouse development. In the mouse, the pancreas is derived from the duodenum as two evaginations evolving at days 9.5 (dorsal) and 10.5 (ventral) of the gestation. The evaginations coalesce at day 11, and insulin has been first detected in previous studies at day 11.5 (9, 10). We have used a reverse-transcriptase-PCR (RT-PCR) assay, which allows identification and estimation of the relative amounts of mRNA transcribed from each of the two proinsulin genes. This RT-PCR, using a single pair ofprimers and a single probe for the two transcripts, allowed comparison of the relative amounts of proinsulin I and II mRNAs in the samples (11). Immunocytochemistry with antibodies specific for each of the two proinsulins allowed us to distinguish the products of the two genes in embryo sections. We have, thus, been able to detect insulin mRNAs and proteins much earlier, to distinguish the two forms, ...