Blood glucose normalization upon transplantation of human embryonic pancreas into beta-cell-deficient SCID mice.

AIMS/HYPOTHESIS: Transplanting human pancreatic islet beta cells could represent a radical new treatment of Type I (insulin-dependent) diabetes mellitus. However, beta cells available for grafting are scarce and finding new sources of such cells would be crucial for any cell therapy for diabetes. Undifferentiated precursor cells present in the human embryonic pancreas could represent such a source. METHODS: We grafted human embryonic pancreases (6-9 weeks of development) that contain very few beta cells onto NOD/scid mice. RESULTS: The human pancreatic tissue grew, increasing in weight 200 times within six months and endocrine cells differentiated, the number of human beta cells being increased by a factor 5000. Finally, the developed human endocrine tissue was mature enough to control the glycaemia of mice deficient in endogenous beta cells. CONCLUSION/INTERPRETATION: Human embryonic pancreas represent a source of immature cells that can proliferate and differentiate into mass beta cells after transplantation. Transplantation of human embryonic pancreas into NOD/scid mice is a useful model for understanding the development of the human pancreas during prenatal life.

Pancreatic pattern of expression of thyrotropin-releasing hormone during rat embryonic development.

In rodents, the first insulin-producing cells appear in the pancreas at mid-gestation around embryonic day 11 (E11). However, on the basis of various features, such as morphology or hormonal coexpression, it is apparent that these initial insulin-expressing cells are different from those that develop after E15. In the present study, the pancreatic expression of both thyrotropin-releasing hormone (TRH) mRNA and insulin was studied during embryonic and fetal life. We report here that in the rat, while insulin mRNA is detected in the pancreas as early as E12, TRH mRNA cannot be detected before E16. At that stage and later on during fetal and early postnatal life, TRH mRNA is detected in insulin-producing cells, no signal being detected in other endocrine cell types or in exocrine tissue. It was also noted, by means of triple staining performed at E17, that the expression of TRH mRNA was restricted to insulin-expressing cells negative for glucagon, whereas the few insulin-expressing cells present at that stage, which coexpress insulin and glucagon, did not express TRH mRNA. Taken together, these data indicate that TRH is a marker of insulin-expressing cells, which develop after E15.

Cell-specific localization of G protein alpha-subunits in the islets of Langerhans.

G protein alpha-subunits are involved in the transduction of receptor-mediated regulation of insulin and glucagon secretions. To get further insight into the status of G proteins in alpha- and beta-cells of the Langerhans islets, we have used immunohistochemistry to study the distribution of alpha-subunits in pancreas sections from the rat. Our results show that only insulin-immunoreactive beta-cells display immunoreactivity for selective antibodies directed against the different members of the Galphas and Galpha12-families (alphas, alphaolf, and alpha12, alpha13 respectively). Immunoreactivities for antibodies directed against members of the Galphaq- and Galphai-families showed a more diverse localization: alpha11 and alphao2 were only detected in glucagon-immunoreactive alpha-cells, whereas alphai1 was detected in all beta-cells but only in a few alpha-cells. Even though beta-cells showed immunoreactivities for alphao-non-isoform-selective antibodies, we could not identify the isoform(s) present using selective alphao1 and alphao2 antibodies. Other members of the Galphai- and Galphaq-families (alphai3, alphat2, alphaz and alphaq) were detected in both alpha- and beta-cells. In conclusion, our findings demonstrate a clear difference in the localization of G protein alpha-subunits between alpha- and beta-cells, suggesting the involvement of specific receptor transduction pathways for the neuronal/hormonal regulation of alpha- and beta-cell functions.

Fibroblast growth factor 2 promotes pancreatic epithelial cell proliferation via functional fibroblast growth factor receptors during embryonic life.

Several investigators have postulated that soluble growth factors are involved in the early development of the pancreas. In many tissues in which soluble factors are implicated in development, these factors act on their target cells through tyrosine kinase receptors. Because we had some preliminary evidence that fibroblast growth factor receptors (FGFRs) were expressed in the early pancreas, we investigated the effect of fibroblast growth factors (FGFs) during embryonic pancreatic development. For that purpose, we first studied the distribution and the functionality of FGFRs during pancreatic organogenesis. FGFR1 and FGFR4 were shown to be expressed at a high level during early pancreatic development before embryonic day 16, their levels of expression decreasing thereafter. The functionality of FGFR was studied next. It was demonstrated in vitro that both FGF1 and FGF2 induce the expression of NGFI-A mRNA, a useful indicator of functional growth factor-signaling pathways. Finally, the effect of FGF2 on embryonic pancreatic epithelial cell proliferation was studied. It was shown that FGF2 induces the proliferation of pancreatic epithelial cells during embryonic life. Taken together, these data strongly suggest that FGFs are implicated in pancreatic development during embryonic life.

Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay.

Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.

Effect of clonidine on the circulation and vasoactive hormones after aortic surgery.

After completion of abdominal aortic graft, 29 patients received an i.v. infusion of placebo (n = 16) or clonidine 7 micrograms kg-1 (n = 13) over 120 min in a double-blind study. Cardiovascular variables were measured and plasma samples obtained up to 5 h after arrival in the recovery room, for assay of noradrenaline, adrenaline, vasopressin and renin concentrations. Noradrenaline, adrenaline and vasopressin concentrations decreased in the clonidine group throughout recovery (P less than 0.001, 0.05 and 0.05, respectively, vs placebo). Heart rate was less in the clonidine group (P less than 0.01). There was no significant difference in mean arterial pressure between groups. Stroke volume was larger (P less than 0.01) and there were fewer episodes of hypertension (P less than 0.05) and tachycardia in the clonidine group. In addition, a reduction in the number of circulatory interventions (P less than 0.05) and episodes of shivering was noted in the clonidine group. Mean (SD) postoperative volume requirements were larger in the clonidine group (total postoperative input: clonidine 1462 (604) ml; placebo 1064 (348) ml (P less than 0.05]. These data are consistent with the observation that clonidine modifies endocrine and circulatory status after major surgery.

Effect of growth hormone and glucose on rat islet cells replication using 5-bromo-2-deoxyuridine incorporation.

Islet cell proliferation was quantified by labelling fetal and adult rat islets in vitro with 5-bromo-2-deoxyuridine (BrdU), a thymidine analog that cannot be reutilized by the cell. After incubation with BrdU, cells were dispersed, centrifuged onto slides and DNA labeled with BrdU detected by a fluorescent anti BrdU antibody. The fluorescence was always restricted to the nucleus without any labelling in the cytoplasm. Insulin-containing cells were localized in parallel by immunocytochemistry demonstrating that all BrdU + cells were beta cells. The effects of fetal calf serum (FCS), human recombinant growth hormone (hGH) and glucose on islet cell proliferation were studied. After a 24 hour pulse with BrdU in 10% FCS, the percentage of BrdU+ cells found in adult and fetal islets was respectively 2.3% and 19.5% (p less than 0.001); only 8.8% of fetal islet cells were BrdU+ when fetal islets were incubated in 1% FCS. GH stimulated BrdU incorporation in a dose dependent manner: the threshold concentration of GH for a significant increase in BrdU+ cells was 50 ng/ml, the maximal effect was achieved with 100 ng/ml (+86% increased vs baseline p less than 0.01) and the half maximum response was seen at 50 ng/ml. Glucose stimulated the number of cells in S phase with a maximal response seen at 11 mM.

Immunocytochemical evidence for the presence of S-100 protein in insulin-containing cells of cultured fetal rat islets.

S-100 protein was long considered to be specific to glial and Schwann cells, but was subsequently proved to be present in various organs. In particular, S-100 proteinimmunoreactivity was demonstrated in the parathyroid gland, adenohypophysis and endocrine pancreas. In the present study cultured fetal rat islets were investigated in view of the possible presence of S-100 protein immunoreactivity in their cells. In the initial 5-day period, continuity between islets and ducts could be demonstrated, and the islets appeared to bud from the ducts. During this time, S-100 protein-immunoreactive cells were found in either the budding islets or ducts. The colocalization of S-100 protein and insulin was demonstrated immunocytochemically. In contrast, the newly formed islets from endocrine monolayers did not display S-100 protein immunoreactivity. After this initial period, numerous free-floating islets were observed, but only some of them contained S-100 protein immunoreactivity. S-100 protein-immunoreactive cells had the same distribution as those storing insulin, again suggesting the coexistence of the two peptides. The results suggest that S-100 protein might be involved in the regulation of islet function.

Coexistence of thyrotropin-releasing hormone and insulin in cultured fetal rat islets: a light and electron microscopic immunocytochemical study during islet neoformation.

Pancreatic thyrotropin-releasing hormone (TRH) is without doubt localized in the insulin-containing beta-cells. A previous study reported cellular continuity between beta-cells and ducts in cultured fetal rat islets, but it is not known whether these insulin-containing beta-cells form a cell type that is different from the TRH cells producing insulin. On the other hand, the subcellular coexistence of both peptides as yet remains unresolved. To overcome these problems the present study was conducted, using light microscopic immunocytochemistry, to verify the cellular distribution of TRH in cultured fetal rat islets with particular regard to the interrelationship between beta-cells and ducts, and using electron microscopic double labeling cytochemistry, to study the subcellular distribution of TRH and insulin. Our data show that both TRH and insulin are expressed in the same cells during islet cell neogenesis, and are localized in the same secretory granules. No topographic segregation of their respective immunoreactive moieties are seen within the secretory granule.

Persistence of peptidylglycine alpha-amidating monooxygenase activity and elevated thyrotropin-releasing hormone concentrations in fetal rat islets in culture.

Two experimental systems were used to investigate the relationship between TRH content and peptidylglycine alpha-amidating monooxygenase (PAMase) activity of the neonatal rat pancreatic islets: freshly isolated islets from rats aged 1-14 days, and fetal islets maintained in culture for 3 weeks. TRH was present in freshly isolated islets and in newly formed fetal islets in culture. However, while the TRH concentration in freshly isolated islets measured by RIA followed the same ontogenic pattern as that in the total pancreas, peaking during the first week of life (78 pg/micrograms DNA on day 4) and decreasing thereafter to reach 4 pg/micrograms DNA on day 14, the TRH content of fetal islets in culture did not decrease with time (65 pg/micrograms DNA on day 1 and 80 pg/micrograms DNA after 3 weeks in culture). Similarly, using immunocytochemistry, immunoreactive cells were only seen at day 2 in freshly isolated islets. In contrast, in fetal islets, TRH cells were present throughout the culture period. In both experimental systems, TRH was localized in beta-cells. PAMase activity paralleled TRH concentration, peaking during the first week of life in freshly isolated islets and remaining unchanged in the fetal islets in culture. PAMase activity is, therefore, present in the endocrine pancreas. The results suggest that PAMase activity is a rate-limiting step in TRH biosynthesis in this tissue.

Diabetes insipidus in children. III. Anterior pituitary dysfunction in idiopathic types.

Seventeen patients with idiopathic diabetes insipidus occurring in childhood were observed from 4 to 26 years (mean duration 15 1/2 years). The diagnosis of idiopathic diabetes insipidus was based on routine clinical examination and careful, repeated neuroradiologic investigations. Anterior pituitary dysfunction was present in some of these patients. Growth hormone deficiency was present in six children, insufficient thyroid stimulating hormone secretion after thyrotropin-releasing hormone stimulation was demonstrated in one, and abnormal response to a metyrapone test in two. Elevated prolactin and TSH values were present in three and two patients, respectively. Some of these abnormalities were transitory. The presence of anterior pituitary dysfunction in idiopathic diabetes insipidus indicates that the destructive process is not localized to vasopressin synthesizing cells but may also involve other parts of the hypothalamus.

Diabetes insipidus in children. I. Arginine-vasopressin determination in plasma during short dehydration test.

Plasma vasopressin as well as plasma and urinary osmolality are measured during an overnight dehydration test in vasopressin is undetectable. It is present in plasma from patients with partial diabetes insipidus but the level is not appropriate for plasma osmolality. In patients with polyuria of renal origin plasma vasopressin was significantly higher than in patients with neurogenic diabetes insipidus. Plasma vasopressin measurement is of diagnostic values in partial neurogenic diabetes insipidus and may be of considerable help to distinguish this group of patients from children with polyuria of renal origin.

Evaluation of single oral dose metyrapone tests in children with hypopituitarism.

Evaluation of single-dose metyrapone tests in children with hypopituitarism; comparison with the prolonged metyrapone and insulin induced hypoglycaemia tests and their relationship with the etiology of hypopituitarism. Acta Paediatr Scand, 65:177, 1976.--Pituitary-adrenal reserve was evaluated in control and hypopituitary subjects by comparing the 8 a.m. plasma 11-deoxycorticoid response (11-DOCS) to a single midnight oral dose of metyrapone (short test) with 1) the 8 a.m. 11-DOCS increase under repeated oral doses of metyrapone (prolonged test) and 2) with the plasma corticoid response during arginine-insulin test. In the short and the prolonged metyrapone tests, the same response was obtained in 25 out of 27 patients. The short test was repeated in 22 patients and the 11-DOCS response did not show a significant difference. In 34 of 40 patients, the response to the short test was comparable to the response during the arginine-insulin test; only 3 patients with a normal 11-DOCS rise to the short test had a low response to insulin and vice versa. Among the low responders to the short test, the mean 11-DOCS value was significantly lower in subjects with operated craniopharyngiomas than in idiopathic hypopituitary patients (p less than 0.001). In the short test, the 8 a.m. baseline cortisol value was positively correlated with the 8 a.m. 11-DOCS response (p less than 0.001), the cortisol level allowing to predict the 11-DOCS response in 28 out of 53 patients. Thus, the short oral metyrapone stimulation was found to be a reliable test in hypopituitary children.