The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.

Chromatographically determined haemoglobin A1c concentration was measured during short-term (1-24h) changes in glucose concentration in vitro and in vivo. In vitro at 37 degrees C the HbA1c concentration increased with glucose concentration and time both in normal and diabetic erythrocytes. In normal erythrocytes incubated in 20--100 mmol/l glucose, the increases in the HbA1c concentration were maximal after 4--6 h and then stable for the next 18--20 h. During the first hour, increases in the HbA1c concentration were linear with time and on average 0.034% HbA1c x h-1 x mmol/l glucose-1. In erythrocytes, after a rapidly produced increase (2 h), HbA1c decreased to preincubation concentrations during a further incubation of the erythrocytes in a glucose-free medium at 37 degrees C for 4--6 h. The mean rate of linear decrease was 0.017% x h-1 x mmol/l glucose-1. After incubation of erythrocytes in 100 mmol/l glucose for 24 h, 1.3% HbA1c remained stable for 6 h in saline. The rapid increase in HbA1c concentration, as determined by chromatography, was not due to stable HbA1c (ketoamine linked glucose) as no increase was found in the HbA1c concentrations determined by the thiobarbiturate method. In juvenile diabetics controlled by an artificial beta-cell, rapid changes of blood glucose concentration (up to 20 mmol/l) resulted in increases in HbA1c concentration of as much as 1.9% within 12 h (mean 1.1%). Rapid in vivo increases in HbA1c concentration were reversible by normalization of the blood glucose concentration. That rapid changes in HbA1c may occur in daily diabetic life was evidenced by differences in HbA1c concentration between blood samples from out-patient diabetics incubated in saline for 16 hours at 4 degrees C and 37 degrees C (range of differences 0.2--1.4% HbA1c). The differences correlated to the blood glucose concentration at the time of sampling blood for HbA1c determination. Thus, incubation of blood at a low glucose concentration prior to determination of the glycosylated haemoglobin concentration may overcome interference from rapidly produced HbA1c.

We previously established pluripotent transformed rat islet cell lines, MSL-cells, of which certain clones have been used to study processes of islet beta-cell maturation, including the transcriptional activation of the insulin gene induced by in vivo passage. Thus, successive sc transplantation in NEDH rats resulted in stable hypoglycemic insulinoma tumor lines, such as MSL-G2-IN. Occasionally, hypoglycemia as well as severe weight loss were observed in the early tumor passages of MSL-G and the subclone, NHI-5B, which carry the transfected neomycin and human insulin genes as unique clonal markers. By selective transplantation, it was possible to segregate stable anorectic normoglycemic tumor lines, MSL-G-AN and NHI-5B-AN, from both clones. These tumors cause an abrupt onset of anorexia when they reach a size of 400-500 mg (< 0.3% of total body weight), and the observed weight loss parallels that of starved rats until death results from cachexia. After tumor resection, animals immediately resume normal feeding behavior. Comparative studies of hormone release and mRNA content in anorectic lines, MSL-G-AN and NHI-5B-AN, vs. those in the insulinoma line, MSL-G2-IN, revealed selective glucagon gene expression in both of the anorectic tumors, whereas insulin and islet amyloid polypeptide gene expression were confined to the insulinoma. Both tumor phenotypes produced cholecystokinin and gastrin in variable small amounts, making it unlikely that these hormones contribute to the anorectic phenotype. Tumor necrosis factor (cachectin) was not produced by any of the tumors. Proglucagon was processed as in the fetal islet to products representative of both pancreatic alpha-cell and intestinal L-cell phenotypes, with glucagon and Glp-1 (7-36)amide as the major extractable products. In contrast to the administration of cholecystokinin, neither glucagon, Glp-1 (7-36)amide, nor their combination, affected feeding behavior in fasted mice, suggesting the presence of a hitherto unidentified anorectic substance released from the glucagonoma. We conclude 1) that glucagonomas and insulinomas can be derived from a common clonal origin of pluripotent MSL cells, thus supporting the existence of a cell lineage relationship between islet alpha- and beta-cell during ontogeny; and 2) that our glucagonomas release an anorexigenic substance(s) of unknown nature that causes a severe weight loss comparable to that reported in animals carrying tumor necrosis factor-producing experimental tumors.

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Altered filtration of macromolecules due to decreased electrical charge of the glomerular basement membrane might be the initial step in the development of albuminuria in patients with Type 1 (insulin-dependent) diabetes mellitus. We therefore investigated the selectivity index, i.e. renal clearance of non-glycated plasma albumin/clearance of glycated plasma albumin in 38 patients with Type 1 diabetes mellitus. The two albumin molecules differed slightly in charge, non-enzymatic glycated albumin being more anionic at physiological pH compared with unmodified plasma albumin. Glycated albumin in plasma and urine was determined by a specific, sensitive and highly reproducible chromatographic procedure. In diabetic patients with normal urinary albumin excretion, the selectivity index was increased three-fold compared with that of non-diabetic subjects (2 p less than 0.01). A significant correlation (r = 0.53, 2 p less than 0.01) between haemoglobin A1c and selectivity index was demonstrated in these patients, indicating a change in charge-dependent renal filtration could possibly be attributed to non-enzymatic glycation of components in the glomerular basement membrane and tubuli. Diabetic patients with increased albumin excretion rate had a significantly lower selectivity index compared with patients with normal albumin excretion (2 p less than 0.01). A significant negative correlation (r = 0.85, 2 p less than 0.001, exponential curve fit) was seen between urinary albumin excretion and selectivity index in the diabetic patients, indicating that the capability of differentiating between macromolecules of different charges is again lost with increasing urinary albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

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A B S T R A C T Thyrotropin-releasing hormone immunoreactivity (TRH-IR) was measured in isolated islets and in medium from rat pancreatic islets maintained in organ culture. TRH-IR in methanol extracts of both islets and culture medium was eluted in the same position as synthetic TRH by ion-exchange and gel chromatography and exhibited dilution curves parallel with synthetic TRH in radioimmunoassay.[3H]Histidine was incorporated into a component that reacted with TRH antiserum and had the same retention time as synthetic TRH on reversed-phase highperformance liquid chromatography.A continuous release of TRH-IR into the culture medium was observed from islets of both 5-d-old (newborn) and 30-d-old (adult) rats with a maximum on the second day of culture (28.7±7.0 and 13.3±3.6 fmol/islet per d, respectively). The content of TRH-IR was higher in freshly isolated islets from newborn rats (22.4±2.3 fmol/islet) than in adult rat islets, which, however, increased their content from 1.3±0.5 to 7.0±0.5 fmol/islet during the first 3 d of culture. Adult rat islets maintained in medium with 20 mM glucose released significantly more TRH-IR than islets in 3.3 mM glucose medium (13.0±0.7 vs. 4.3±0.3 fmol/islet per d). In contrast, the content of TRH-IR in the islets was reversed (1.4±0.3 vs. 4.7±1.6 fmol/ islet). By exposing islets from newborn rats to streptozotocin 0.7 mg/ml for 30 min, a 50% reduction of (3,4), and seems t6 be located mainly in the islets of Langerhans (5,6). The amount of TRH-IR in the rat pancreas and islets is highest during the first 24 h after birth, but declines rapidly with age, concomitantly with the appearance of TRH in the hypothalamus (7,8 METHODSIslet isolation and culture. Pancreatic islets were isolated from 5-d-old Wistar rats (newborn) of both sexes and from overnight-fasted 30-d-old male Wistar rats (adult) (M0lle-gaard, Lille Skensved, Denmark).The excised pancreas was digested by the collagenase method and the islets were isolated after. gradient centrifugation on PercollT (11). Isolated islets were placed in 5-cm plastic petri dishes (Nunc, Roskilde, Denmark). Each dish contained 100 islets in 5 ml medium RPMI 1640 (Flow Laboratories, Irvine, UK) containing 11 mM glucose and 10% heat-inactivated newborn calf serum (NBCS) (Gibco Laboratories, Paisly, UK). The dishes were incubated at 370C in a humidified atmosphere containing 5% carbon dioxide. Medium and islets were withdrawn at various intervals as indicated in the figures. Islets were homogenized in distilled water by sonication. Hormones were measured in both medium and islets.Glucose stimulation. Islets from adult rats were preincubated for 1 d in RPMI 1640 medium containing 10% NBCS and 11 mM glucose and then transferred to RPMI 1640 medium containing 0.5% human serum (HS) and either 3.3 or 20 mM glucose. After 1 d in 3.3 mM glucose one group of islets was transferred to 20 mM glucose, while others were maintained in either 3.3 or 20 mM glucose. The medium was changed daily and hormones were measured in medium and islets.Streptozo...