Flora de Pablo

Insulin antibodies retard and insulin accelerates growth and differentiation in early embryos.

The physiologic function of insulin in early embryonic life is unknown. We have shown that insulin is present in unfertilized eggs and in chick embryos at 2–3 days of development, even before the emergence of the endocrine pancreas. To define insulins role, we exposed 2-day-old chick embryos to anti-insulin antibodies and followed their development up to day 5. Antibodytreated embryos had a higher rate of growth retardation and death by days 3–5 of embryogenesis, compared with controls. Among the survivors, biochemical maturation was delayed at days 4 and 5; weight, protein, total creatine kinase activity, and creatine kinase-MB were decreased in antibody-treated embryos. By contrast, insulin (50 ng/embryo) administered to 2-day-old embryos yielded nearly symmetrical stimulatory results. These findings suggest that endogenous insulin plays a probable physiologic role regulating growth and differentiation in early embryos. In addition, the findings provide some clues to a possible function for insulin produced outside the organisms own beta cells.

Genes for the insulin receptor and the insulin-like growth factor I receptor are expressed in the chicken embryo blastoderm and throughout organogenesis

The early expression of insulin and insulin-like growth factor I (IGF-I) in the chicken embryo suggests that these peptides play an important role in early development. The receptors for insulin and IGF-I, however, had not been studied at the molecular level in this model. We report two chicken sequences that, by comparison with known tyrosine kinases, appear to correspond to the tyrosine kinase domain of the insulin receptor homologue (CTK-1) and the IGF-I receptor homologue (CTK-2). Using reverse-transcription of RNA, amplification with the polymerase chain reaction (RT-PCR), and gene-specific hybridization, we demonstrate that the two genes, CTK-1 and CTK-2, are expressed in embryos at least as early as the blastoderm (Day 0), during neurulation (Day 1), and in early (Days 2-3) and late (Day 9) organogenesis.

Insulin-like growth factor I activity is stored in the yolk of the avian egg

Growth factors of maternal origin may be incorporated into the vertebrate egg and play a role in early phases of embryo growth and differentiation. An avian insulin-like growth factor I (IGF-I) activity from unfertilized chicken egg-yolk has been partially purified by HPLC. The material is slightly more hydrophobic than recombinant human IGF-I. It reacts in a human IGF-I radioimmunoassay and is specifically depleted by anti-human IGF-I antibodies. Like authentic IGF-I, the extracts enriched in IGF-I activity stimulated the accumulation of delta-crystallin mRNA in epithelial cells from chick embryo lens with a potency approximately equivalent to its IGF-I immunoactivity.

Insulin action in early embryonic life: Anti-insulin receptor antibodies retard chicken embryo growth but not muscle differentiation in vivo

Insulin receptors are present in chicken embryos at day 2 of development and insulin stimulates embryonic growth and differentiation. Most important, anti-insulin antibodies cause either death or developmental retardation in chicken embryos of that age. To determine if the embryos endogenous insulin acts through its own receptor, we compared the effects of anti-insulin antibodies to the effects of anti-insulin receptor antibodies on growth and differentiation indexes in the chicken embryo. While the anti-insulin antibody caused a dose-dependent decrease in growth parameters like weight, total protein, DNA, RNA, total creatine kinase activity and a marker of differentiation, the creatine kinase-MB, the anti-insulin receptor antibody decreased all parameters except the creatine kinase-MB. Many, but not all, of the effects of insulin in early embryos, thus, are mediated through the insulin receptor.

Programmed cell death in the neurulating embryo is prevented by the chaperone Hsc70

Neuronal cell death is a genuine developmental process, with precise regulation and defined roles. In striking contrast, characterization of cell death that occurs at early stages of neural development is very limited. We previously showed that embryonic proinsulin increases the level of the chaperone heat shock cognate 70 (Hsc70) and reduces the incidence of apoptosis in the neurulating chick embryo [de la Rosa, et al. (1998), Proc. Natl. Acad. Sci. USA, 95, 9950]. We now demonstrate that Hsc70 is directly involved in cell survival during neurulation, as specific downregulation of endogenous Hsc70 by antisense oligodeoxynucleotide interference provoked an increase in apoptosis both in vitro and in ovo. In parallel, activation of caspase‐3 was increased after hsc70 antisense oligodeoxynucleotide treatment. Dead cells were located mostly in the developing nervous system, distributed in areas where the incidence of cell death was high. These areas coincided both in vivo and under different death‐inducing conditions, including antisense interference and growth factor deprivation. Hsc70 immunostaining was strong in at least some areas of high cell death. Apoptotic cells within these areas presented undetectable Hsc70 levels, however, suggesting that this protein acts as an intrinsic protector of neuroepithelial and neural precursor cells.

Insulin receptors and insulin-like growth factor I receptors are functional during organogenesis of the lens

Insulin and insulin-like growth factor I (IGF-I) stimulate overall growth and development of the chick embryo in early organogenesis. Turning to individual organs, to clarify the cellular effects of these peptides and the activity of the receptors involved, we had demonstrated with developing lens that insulin and IGF-I increase the accumulation of delta-crystallin mRNA, a marker for lens differentiation, in part by stimulation of transcription. In this study we expand our previous work on lens receptors to an earlier time in organogenesis, day 4, which marks the beginning of differentiation of the lens epithelial cells into elongated fibers. Insulin receptors are demonstrable by affinity cross-linking in epithelial cells at day 6, and specific binding of [125I]insulin and [125I]IGF-I is detectable in day 4 lenses. Insulin and IGF-I stimulation of substrate phosphorylation in the presence of solubilized receptors occurs only with high concentrations (10-100 nM) of either peptide in day 4 lenses, while a clear response with low concentrations (1 nM) is elicited by day 6 of development. Low concentrations of both insulin and IGF-I (0.1-1 nM) increase the incorporation of [3H]leucine and [3H]uridine in day 6 lens cells, suggesting that each peptide acts through its own receptor. These results confirm and extend the finding of insulin and IGF-I receptors in the developing chicken lens, and demonstrate their functional activity. This embryonic model should be valuable for further analysis of the action of insulin and IGF-I in growth and differentiation processes during early development.

Insulin-related molecules and insulin effects in the sea urchin embryo

Insulin, the polypeptide hormone secreted by the differentiated pancreas, may play a role in vertebrate development at prepancreatic stages. In an invertebrate embryo, the sea urchin Strongylocentrotus purpuratus, we now find that insulin modulates the levels of developmentally regulated mRNAs of different lineages (one ectoderm-specific, one mesoderm-specific, and one found in all cell types). Using indirect immunofluorescence, we have localized a molecule which shares antigenic determinants with mammalian insulin in the unfertilized egg as well as in the gut of pluteus larva sea urchins. In addition, Southern hybridization reveals high similarity between sea urchin DNA sequences and the human insulin receptor gene. Our results suggest the presence of an insulin/insulin receptor-related system in sea urchin development.

Chapter 5 Molecules of intercellular communication in vertebrates, invertebrates and microbes: do they share common origins?

Publisher Summary This chapter reviews (1) materials in microbes that resemble vertebrate receptors for hormones and other intercellular messengers, (2) intercellular communication in microbes, and (3) materials that resemble vertebrate hormones in microbes, higher plants, amphibian skin, and very young avian embryos. The term “glandulocentric” is introduced to indicate a traditional approach whereby each hormone is thought to be a unique product of one endocrine gland. Because the typical vertebrate endocrine gland is limited to vertebrates, its hormonal product had been thought to be similarly restricted in distribution. The one-to-one relationship between hormone and gland is similarly applied conceptually to the endocrine system in insects and in mollusks. Intercellular communication is widespread in microbes. In Saccharomyces cerevisiae, the common yeast, there are two sex types—α and A—which communicate one with the other via peptide messengers, designated α factor and A factor. Materials that resemble vertebrate-type messenger peptides are present in plants. The paleocentric approach, which suggests that these messengers originated early in evolution before the divergence of life forms into separate kingdoms, easily accommodates these observations. Many of the messenger peptides of vertebrate as well as their receptors and many of their post-receptor components have their evolutionary origins among the microbes and appear to be distributed among a very wide range of forms of life.

The regulated expression of chimeric tyrosine hydroxylase–insulin transcripts during early development

Biological complexity does not appear to be simply correlated with gene number but rather other mechanisms contribute to the morphological and functional diversity across phyla. Such mechanisms regulate different transcriptional, translational and post-translational processes and include the recently identified transcription induced chimerism (TIC). We have found two novel chimeric transcripts in the chick and quail that result from the fusion of tyrosine hydroxylase (TH) and insulin into a single mature transcript. The th and insulin genes are located in tandem and they are generally transcribed independently. However, it appears that two chimeric transcripts containing exons from both the genes can also be produced in a regulated manner. The TH–INS1 and TH–INS2 chimeras differ in their insulin gene content, and they encode two novel isoforms of the TH protein with markedly reduced functionality when compared with the canonical TH. In addition, the TH–INS1 chimeric mRNA generates a small amount of insulin. We propose that TIC is an additional mechanism that can be employed to further regulate TH and insulin expression according to the specific needs of developing vertebrates.

Insulin Receptors in Disorders of Glucose Tolerance and Insulin Sensitivity

It has long been known that glucose stimulates the β-cells of the pancreas to release insulin which acts on target cells to promote glucose utilization and storage. Moreover, only glucose could be measured so that the system was oversimplified — hyperglycemia was due to insulin deficiency and hypoglycemia was due to insulin excess. With the advent of insulin radioimmunoassay it became clear that only a minority of patients conform to this scheme (Yalow and Berson, 1960). The diabetics who are truly insulin-requiring are indeed insulin-deficient, but the majority of hyperglycemic patients are not absolutely insulin-dependent and have normal or supernormal concentrations of circulating insulin. Since the plasma insulin in these subjects was shown not to be bound to any macromolecules (e.g. to insulin antibodies or to other binding proteins) and was qualitatively normal, these patients had insulin resistance at the tissue level.