Evidence for biosynthesis of preproinsulin in gut of rat.

Glucagon and other pancreatic peptides are made in the gut, but there is little evidence for the formation of insulin. The demonstration of insulin receptors on the mucosa of gut epithelium suggests that there may be an autocrine or paracrine role for insulin made in the gut. Such insulin may control cell division, the secretion of other peptides from the same or neighboring cells, or motility and absorption. To search for the ability of the gut to make insulin, sections of freshly excised segments of rat gut were treated with an antiserum against porcine insulin. Intracellular immunoreactivity appeared in glandular cells in the stomach and colon but not in the small intestine. Preproinsulin mRNA was detected in similar cells in the stomach and colon by in situ hybridization, using specific oligonucleotide probes. Rat preproinsulin 1 and 2 mRNAs were transcribed by reverse transcriptase to the corresponding cDNAs, which were then amplified by polymerase chain reaction, utilizing specific oligonucleotide primers. Restriction analysis confirmed the identity of rat preproinsulin 1 and 2 mRNA in the colon and rat preproinsulin 1 mRNA in the stomach. Neither was found in the small intestine. Base sequences of the cDNAs were identical to the coding regions of pancreatic rat preproinsulin 1 and 2 messages. These observations are strong evidence for the synthesis of preproinsulin in the gut of the rat.

The drug line: the clinical expression of the pigmentary Voigt-Futcher line in turn derived from the embryonic ventral axial line.

BACKGROUND: Observation of patients with morbilliform eruptions reveals a distinctive demarcation line on the upper arms. OBJECTIVE: We studied the origin of this sharp "drug line." METHODS: We performed a literature search of all known variables. RESULTS: The drug line represents clinical expression of the pigmentary Voigt-Futcher line. CONCLUSION: The drug line reveals the otherwise inapparent embryologic ventral axial line, which marks the precise border between the sensory innervation of the lateral and medial upper arm. The significance of the cutaneous sensory nerves is thus apparent. The drug line is further evidence of the segmental nature of human skin, as evidenced in evolutionary and embryologic studies.

The transverse nasal line: an embryonic fault line.

The transverse nasal line has long been neglected and frequently overlooked. This narrow pink or hyperpigmented line or groove extends transversely between the upper two-thirds and the lower third of the nose. It is often hereditary and may be the locus of comedones and milia. Eighteen examples are reported by us, along with a possible embryological interpretation.

Preproinsulin messenger ribonucleic acid in the rat adrenal gland.

Many studies support the concept of insulin synthesis in tissues other than the pancreas. Our previous investigations have demonstrated the presence of insulin immunoreactivity in the adrenal medulla of the rat. This immunoreactivity was found to be associated with the chromaffin granule. This study is directed at isolating the messenger ribonucleic acid that encodes for preproinsulin. Reverse transcription coupled with polymerase chain reaction was used. Complementary deoxyribonucleic acid (cDNA) was amplified, extracted and reamplified. It was then subjected to digestion with four different restriction endonuclease enzymes. Its resemblance to the corresponding cDNA that encodes for preproinsulin I in the rat was established. Our results suggest that insulin is synthesized in the rat adrenal gland for autocrine, paracrine, neuromodulation or local physiologic function.

Preproinsulin mRNA in the rat eye.

PURPOSE: The goal of this study was to extend the results of previous immunoassay, immunocytochemistry, and in situ hybridization studies showing the presence of insulin-related peptide in the rat retina by confirming the expression of insulin genes in the rat eye. METHODS: Total and poly(A)+ RNA were isolated from whole rat eyes, and separately from the retina, choroid, iris, lens, and vitreous. The poly(A)+ RNA was used for preparation of insulin-specific cDNA according to a coupled reverse transcription polymerase chain reaction (RT-PCR) protocol under high stringency conditions. Southern transfers, restriction fragment analyses, and nucleotide sequencing were used to characterize and identify the amplified cDNA products. RESULTS: Amplified cDNA fragments of 329 +/- 6 base pairs (bp) were derived from whole rat eye and rat retina poly(A)+ RNA, but not from other regions of the eye. Southern blots probed with preproinsulin-specific primer demonstrated homology with similar-sized cDNA from rat pancreas. Restriction digests with 10 restriction enzymes and direct nucleotide sequencing confirmed that the 329-bp cDNA was identical to the previously known coding sequence for rat pancreatic preproinsulin1 DNA. CONCLUSIONS: The identification of retinal preproinsulin1 mRNA was confirmed. This correlates with previous studies showing insulin immunoreactivity in rat eyes and in cultured retina, and verifies in situ hybridization evidence for the presence of insulin-related mRNA in retinal glial cells.

Human retinoblastoma Y79 cells contain both insulin-specific mRNA and insulin-binding sites.

We previously reported the presence of insulin-like immunoreactivity in cells from the human retinoblastoma Y79 cell line. In the present study, in situ DNA hybridization techniques were applied, using a human insulin cDNA probe to investigate whether the insulin-like activity is due to local synthesis of insulin. Our results suggest that Y79 cells contain mRNA for the synthesis of insulin or a homologous peptide. In addition, 125I-insulin binding autoradiographic studies show that these cells also contain specific insulin-binding sites. It is suggested that insulin may play an autocrine and/or paracrine role in the maintenance and metabolism of the Y79 retinoblastoma cells.

Insulin immunoreactivity in the fetal and neonatal rat retina.

The ganglion cell layer of pre- and postnatal rat retina is positive for insulin immunoreactivity. At birth the inner nuclear layer also stains for insulin. By 5 days after birth the layers characteristic of the mature retina are demonstrable. At this time the outer nuclear layer and both limiting membranes show insulin reactivity. The lens is positive for insulin at all stages studied and the retinal pigment and choroid layers are positive after birth. These observations suggest that insulin may be important in differentiation and/or maturation of the retina.

Anatomy of the pancreas. Emphasis on blood supply and lymphatic drainage.

Early diagnosis of pancreatic disease is difficult because of the position of the pancreas deep in the abdomen, where it is covered by hollow viscera, making it inaccessible for simple examination. In addition, the functional reserve of the gland is great, and over 50% of its acinar tissue must be destroyed before there is marked evidence of an effect on digestion. The stimulus to pancreatic secretion is very complicated and difficult to control because it is both humoral and neurogenic. Since the ampulla of Vater is used for both bile and pancreatic secretion and opens into the duodenum, there is a reservoir for reflux into the biliary system and the pancreas, as well as a potential route for transfer of infection. The relationship of the pancreas to the surrounding viscera, its rich blood and lymphatic supply, and its lack of complete fascial protective covering allow for the rapid and severe dissemination of malignant processes long before obvious symptoms are apparent.

Immunocytochemical demonstration of insulin or insulin-like immunoreactivity in the rat prostate gland.

The indirect immunocytochemical technique was used in conjunction with anti-insulin antisera to localize insulin-like immunoreactivity in tissue sections and primary cell cultures from rat prostate gland. Positive immunostaining for insulin-like reactivity was demonstrated in the epithelium of the prostate gland. There appeared to be some variation in the intensity and localization of the immunoreactivity within different regions of the gland. Prostatic epithelial cells grown in culture in an insulin-free medium displayed strong cytoplasmic immunostaining when treated with anti-insulin antisera, while nuclear staining was absent. These results demonstrate that insulin-like immunoreactivity is present in the epithelium of the prostate gland and suggest that there may be some local insulin or insulin-like synthesis in this organ.

Demonstration of insulin-specific mRNA in cultured rat retinal glial cells.

A number of studies have recently demonstrated that insulin may be synthesized outside the pancreas. The present study was designed to investigate whether insulin-like activity exists in retinal glial cells and if so, whether it is due to local synthesis of insulin. Immunocytochemical techniques using insulin antisera were applied to cultured rat retinal glial cells, and insulin-like immunoreactivity was demonstrated in the cytoplasm of these cells. In situ DNA-RNA hybridization studies using 3H-labeled rat insulin cDNA indicated that the glial cells, particularly the Müller cells of the retina also contained the mRNA necessary for de novo synthesis of insulin or a closely homologous peptide. This peptide may be important in neuromodulation or regulation of metabolism of retinal cells and capillaries.

Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization.

Cultured seminal vesicle epithelial cells exhibited cytoplasmic immunoreactivity following treatment with anti-insulin antisera. In addition, these cultured epithelial cells were found, by in situ hybridization with a radiolabeled insulin complementary deoxyribonucleic acid (cDNA) probe, to contain an insulin or insulin-like messenger ribonucleic acid (mRNA). Autoradiograms of the hybridized cells exhibited heavy labeling over the cytoplasm and minimal distribution of grains over the nuclei and background areas. These observations indicate that cultured mouse seminal vesicle epithelium contains an insulin or insulin-like peptide as well as the mRNA that is required for its synthesis.

Insulin or insulin-like peptides in the human pituitary.

Human pituitary cells and pancreatic islet beta cells from surgical and autopsy material showed positive immunoreactivity to anti-porcine insulin antisera demonstrating the presence in these tissues of insulin or insulin-like immunoreactive material. Glutaraldehyde-fixed, one micron epoxy sections were used and the polymerized resin was removed prior to staining with the peroxidase anti-peroxidase technique using guinea pig antisera to porcine insulin. Pancreatic beta cells served as the positive control, and appropriate negative controls were also utilized.

Detection of oncogene mRNA sequences in cultured cells by in situ hybridization.

The present study was undertaken to determine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultured cells. Following in situ hybridization with 32P-labeled v-src and v-Ha-ras DNA probes, src and Ha-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-Ha-ras transfected cell lines, the number of silver grains over individual cells were significantly higher (p less than 0.001, t-test) than in a non-transfected, non-tumorigenic, rat esophageal epithelial cell line. There was a highly variable number of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-Ha-ras DNA or that were pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times more oncogene related mRNAs than non-transfected cells as judged by the numbers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirmed that the expression of src and Ha-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of human cells and to human tumor cells in culture.

Immunocytochemical localization of insulin-like immunoreactivity in mouse seminal vesicle.

Insulin-like immunoreactivity was localized in tissue sections and cell cultures of mouse seminal vesicle using the indirect technique of immunocytochemistry. Seminal vesicles were cut into fragments, fixed in 2.5% glutaraldehyde, embedded in epoxy resin, sectioned at 1 micron, and transferred to glass slides. Epithelial cell cultures of seminal vesicle were grown on coverslips in Dulbecco's Minimal Essential Medium for 4-6 days and fixed in 2.5% glutaraldehyde. Sections (etched with sodium ethanolate) or coverslips were incubated in guinea pig antiporcine insulin antiserum, in antiserum immunoabsorbed with porcine insulin, or in normal guinea pig serum. For indirect immunocytochemistry, incubation with primary antiserum was followed by treatment with rabbit anti-guinea pig immunoglobulin (Ig) G conjugated to peroxidase, or with protein A and then rabbit peroxidase anti-peroxidase (PAP). Finally, treated samples were incubated in phenylenediamine-pyrocatechol-H2O2 substrate mixture for 6-8 min at room temperature. Specific immunoreactivity to insulin antisera was confined to the epithelium of the seminal vesicle in tissue sections. No staining occurred in subepithelial connective tissue. Specific immunoreactivity was also observed in the cytoplasm of cultured seminal vesicle epithelial cells.

Insulin-like immunoreactivity in human retinoblastoma Y79 cell line.

Cells from the Y79 human retinoblastoma cell line were examined by immunofluorescence immunocytochemistry using an antiserum against insulin. All the cells showed intense staining, indicating the presence of insulin-like immunoreactivity in these cells. Our observations suggest that insulin may play an important role in the metabolism of retinoblastoma cells and that it may be possible to use this cell line as an in vitro model for studies on the action of insulin in the metabolism of human retinal cells.

Detection of insulin synthesis in mammalian anterior pituitary cells by immunohistochemistry and demonstration of insulin-related transcripts by in situ RNA-DNA hybridization.

Insulin or highly homologous transcripts is shown to be synthesized in cultures of mammalian anterior pituitary cells using cloned insulin-specific cDNA probes and nucleic acid cytochemistry. The insulin-hybridizing cells are less abundant than the growth hormone-producing cells, occurring in the cultures at approximately one tenth the frequency. Immunocytochemistry demonstrates that insulin or insulin-like proteins is also synthesized by the cultured pituitary cells and that the insulin immunoreactivity is contained within secretory granules. It appears that many of these secretory granules are concentrated around the periphery of the cell, unlike the insulin-containing granules in pancreatic B-cells.

Glucagon-like immunoreactivity in mouse and rat retina.

Mouse and rat retinae were examined by the peroxidase-anti-peroxidase technique of immunocytochemistry using an antiserum against glucagon. The immunoreactivity was found in the cells of the ganglion cell layer and inner nuclear layer, including Müller cells. These observations may indicate that glucagon or a similar peptide is important in neuromodulation and/or metabolism of retinal cells.

Immunocytochemistry of mouse and human retina with antisera to insulin and S-100 protein.

Immunocytochemistry using peroxidase antiperoxidase (PAP) techniques showed insulin-like immunoreactivity in the human retina, and in the mouse retina and optic nerve. The immunoreaction product was seen in the inner nuclear, ganglion cell, outer and inner plexiform layers of the retinas, and in glial cell bodies of the optic nerve. A similar staining pattern using antiserum to S-100 protein, a marker for glial elements, was also seen in these tissues. This demonstrates that insulin or insulin-like immunoreactivity appears to be limited to glial cells of the retina and optic nerve. Our study suggests that the presence of insulin or a similar peptide in retina and optic nerve may be important for their normal function and metabolism.

Cerebral localization of insulin by immunofluorescence.

An immunohistochemical procedure was used to detect cells which appear to bind insulin in the mouse brain. Strong fluorescence was observed in the cell bodies and processes of tanycytes lining the third ventricle and in the choroid plexi. These findings suggest that insulin enters the central nervous system, and indicate a route for its possible transport. This adds credence to earlier observations that the hypothalamic ependymal cells and processes form a highly organized and functional system, with different cells selectively absorbing (or sensing) particular substances from the systemic and ventricular circulations and transporting them (or information about them) to specific neuron receptors in the hypothalamus.