Hans Kofod

Tissue and Plasma Concentrations of Amidated and Glycine-Extended Glucagon-Like Peptide I in Humans

Using specific radioimmunoassays, we studied the occurrence of amidated and glycine-extended glucagon-like peptide I (GLP-I) molecules in the human small intestine and pancreas and in the circulation system in response to a breakfast meal. Through gel permeation chromatography of extracts of the human pancreas (n = 5), we found that 71% of the GLP-I immunoreactivity eluted as a large molecule corresponding to the major proglucagon fragment, 24% corresponded to GLP-I 1–36 amide, and 5% to GLP-I 1–37. By gel permeation chromatography of extracts of human small intestine (n = 6), we found that all immunoreactivity eluted in one peak at the common elution position of the two insulin-releasing peptides, GLP-I 7–36 amide and GLP-I 7–37. Of the GLP-I immunoreactivity, 80% corresponded to GLP-I 7–36 amide and 20% to GLP-I 7–37. The mean concentrations of amidated GLP-I and glycine-extended GLP-I in fasting plasma were 7 ± 1 and 6 ± 1 pM, respectively (n = 6). In response to a breakfast meal, the concentration of amidated GLP-I rose significantly amounting to 41 ± 5 pM 90 min after the meal ingestion, whereas the concentration of glycine-extended GLP-I only rose slightly to a maximum of 10 ± 1 pM. Thus, both amidated and glycine-extended GLP-I molecules are produced in the small intestine and in the pancreas in humans. Both amidated and glycine-extended GLP-I are measurable in fasting plasma. The higher meal response of amidated GLP-I compared with glycine-extended GLP-I probably reflects the larger amount of amidated GLP-I produced in the tissues compared with glycine-extended GLP-I.

Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells.

SummaryThe present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse ΒTC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open probability of single ATP-sensitive K+-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (<5 min). The influence of repaglinide and glibenclamide on the ATP-sensitive K+ current was studied using the whole-cell patch clamp configuration. A half-maximal steady-state inhibition of the ATP-sensitive K+ currents is observed at 89 pmol/l repaglinide and at 47 pmol/l glibenclamide in whole-cell experiments of longer duration (30 min). Applying digital Ca2+ imaging on single ΒTC3 cells we found that repaglinide and glibenclamide induced a concentration-dependent increase in intracellular free Ca2+ concentration ([Ca2+]i) with a half-maximal effect at 0.5 nmol/l for both drugs in long-term experiments (30 min). The rise in [Ca2+]i results from Ca2+ entry through voltage-dependent L-type Ca2+-channels since it is inhibited by verapamil (10 Μmol/l). The effect of repaglinide and glibenclamide is partly reversible (≈80%).

Differential Expression of Glutamic Acid Decarboxylase in Rat and Human Islets

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were β-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to β-cells, also surprisingly localized to some α-cells, δ-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained β-cell specific as observed in vivo, whereas GAD67 was localized not only to the β-cells but also in the α-cells and δ-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.

Immunocytochemical evidence for a paracrine interaction between GIP and GLP-1-producing cells in canine small intestine.

Abstract. Glucagon-like-peptide 1 (GLP-1) released from the intestine is considered to be an important incretin. We have recently demonstrated that glucose-dependent insulinotropic peptide (GIP) stimulated GLP-1 secretion from canine ileal L cells in culture. To investigate further the interplay between GLP-1- and GIP-secreting cells, we set out to determine the exact location and abundance of both cell types throughout the canine intestine. Canine small intestine was subdivided into 15–20 segments and investigated by immunocytochemistry with computer-assisted imaging. The abundance of GIP-, GLP-1- and somatostatin-immunoreactive cells was determined. GIP-secreting K cells were equally distributed in duodenum and jejunum, with the GLP-1-secreting L cells concentrated in the jejunum (5% duodenum, 73% jejunum and 22% ileum). These results indicated that the middle section of the small intestine containing 69% of the K cells also contained 51% of the L cells. Double immunostaining confirmed this overlap and furthermore over 30% of the L cells in this region were found adjacent to K cells. These results suggest the existence of a paracrine interaction between the K and L cells and indicate the importance of the jejunum in the regulation of insulin release by enteric-derived incretins.

On the effects of human galanin in man

SummaryHuman galanin was recently isolated and sequenced and was found to differ from porcine galanin, hitherto used for studies in humans, in several important respects. We therefore synthesized and purified human galanin and infused it i.v. at a rate of 74 pmol · kg−1· min−1 into six healthy volunteers for 60 min during a hyperglycaemic clamp. The clamp was achieved by i. v. infusion of glucose at a rate which in a control experiment had been demonstrated to maintain the plasma glucose level at 12–13 mmol/l for 90 min. Galanin concentrations reached a plateau of approximately 1500 pmol/l throughout the infusion as opposed to pre-infusion and control levels of 20–30 pmol/l. The glucose levels obtained in the two experiments were indistinguishable. Plasma levels of C-peptide and insulin increased significantly in both experiments and the dynamic concentration curves were almost identical. Glucagon concentrations in plasma decreased significantly and similarly. Growth hormone levels, however, increased eight-fold during galanin infusions. Galanin was eliminated from plasma with a half-life of 3.7±0.4 min, similar to that of porcine galanin. It is concluded that human galanin powerfully stimulates growth hormone secretion in man, but has no effect on pancreatic endocrine secretion or glucose metabolism in the concentrations obtained in this study.

Impact of fetal and neonatal environment on beta cell function and development of diabetes

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

Pancreatic islet insulin secretion and metabolism in adult rats malnourished during neonatal life.

Pancreatic islets were isolated from rats that had been nursed by dams fed with a control or an 8.7% protein diet during the first 12 d of the lactation period. Glucose-induced insulin secretion from islets in the 8.7% protein group was reduced 50%. The islet insulin and DNA content were similar, whereas the pancreatic insulin content was reduced by 30 % in the rats fed 8.7 % protein. In order to elucidate the mechanism responsible for the attenuation of insulin secretion, measurements were performed of the activity of several islet enzymes that had previously been supposed to be involved in the coupling of glucose stimulation to insulin secretion. Islet glucose oxidation was unaffected, but glucose-stimulated hydrolysis of phosphatidylinositol was reduced by one-third in the islets of rats fed 8.7% protein. The activity of mitochondrial glycerophosphate dehydrogenase was similar in islets of rats fed the 8.7% protein diet and those fed the control diet. The activity of Ca-independent phospholipase A2 was increased fourfold in the islets of rats fed 8.7% protein. It is concluded that impairment of glucose-induced insulin secretion in rats fed a low-protein diet may be caused by attenuation of islet phosphatidylinositol hydrolysis, and it is tentatively suggested that the increased activity of Ca-independent phospholipase A2 in islets of rats fed a low-protein diet may participate in the stimulation of apoptosis.

Effect of cholecystokinin and secretin on somatostatin release from cultured antral cells.

BACKGROUND Both secretin and cholecystokinin (CCK) inhibit gastric acid secretion. However, their mode of action has yet to be determined. A newly developed primary culture of human antral epithelial cells has been used to examine the effect of secretin and cholecystokinin on somatostatin release. METHODS Normal human antral epithelial cell cultures enriched for D cells were maintained in culture for 2 days before release studies. RESULTS Native human secretin at 10(-8) mol/L stimulated somatostatin release threefold. Porcine secretin and the secretin analogs, Tyr10 human secretin, Tyr13 porcine secretin, and Tyr10,13 porcine secretin were equipotent to native human secretin. CCK stimulated somatostatin release with the greatest response (eight times basal) at 10(-7) mol/L. The response to CCK was inhibited in a competitive manner by the addition of the benzodiazepine analog, MK-329. Addition of secretin in the presence of 10(-8) mol/L CCK resulted in a potentiation of somatostatin release, with the greatest response at 10(6) mol/L secretin, resulting in a 12-fold increase above basal. CONCLUSIONS The stimulation observed after the addition of CCK was the result of activation of the CCK-A receptor subtype. The secretin receptor resembles that of the pancreatic D cells and acts through increasing intracellular cyclic adenosine monophosphate levels. Finally, these data indicate that the inhibitory action of CCK and secretin on gastric acid secretion may result from a synergistic action on antral D cells to release somatostatin, which in turn decreases antral gastrin release.

Carboxypeptidase-B-like processing of the C-terminus of glucagon-like peptide-2 in pig and human small intestine

We developed specific, C‐terminal radioimmunoassays for three proglucagon (PG) fragments: PG 151–158, PG 151–160 and PG 126–159 (glucagon‐like peptide‐2 (GLP‐2)) in order to determine the exact C‐terminal sequence of the newly isolated GLP‐2 in man and pig. The antigens and the antisera showed no mutual cross‐reactivity. By gel filtration of extracts of pig and human small intestine, the immunoreactivity eluting at the position of GLP‐2 was identified by the radioimmunoassays for glucagon‐like peptide‐2 (PG 126–159) and for PG 151–158, whereas the assay for PG 151–160 was completely negative. We conclude that the C‐terminal amino acid residue of pig and human ileal GLP‐2 is PG 158. Thus the basic residues, PG 159 and 160 are removed during its processing in the small intestine.

The insulinotropic effect of endothelin-1 is mediated by glucagon release from the islet alpha cells.

Aims/hypothesis. The circulating concentrations of endothelin-1 (ET-1), a peptide derived from endothelium, are increased in hypertension and diabetes. Endothelin-1 has recently been shown to be an insulinotropic agent. The mechanism of action of endothelin-1 on the endocrine pancreas has not yet been clarified. Methods. We investigated the action of endothelin-1 on the insulin secretion, the binding of 125I-ET-1 to beta cells as well as its effects on purified beta and non-beta cells from normal rats. The expression of endothelin receptors in alpha- and beta-cell lines and in normal rat islets was also studied. Results. First, we studied the effects of endothelin-1 on insulin secretion from beta-cell lines (INS-1, βTC3 and MIN6). At all endothelin-1 concentrations applied (1 pmol/l to 1 μmol/l) no change in insulin secretion was found. Ligand-binding experiments on βTC3 cells showed no specific binding of 125I-ET-1. A prominent expression of ETA-receptor mRNA in an alpha-cell line (αTC1.9) and in normal rat islets was found whereas no expression was found in INS-1 cells. No influence of endothelin-1(1 μmol/l) on insulin secretion stimulated by glucose was detected from purified beta cells. Endothelin-1-(100 nmol/l) increased, however, both insulin and glucagon secretion from a mixture of purified beta and non-beta cells indicating that alpha cells seem to have a key role for the action of ET-1 on insulin secretion. Conclusion/interpretation. The insulinotropic impact of endothelin-1 is not caused by a direct action on the beta cells but seems to be mediated by a paracrine action, probably secondary to enhanced release of glucagon from the endothelin receptor positive alpha cells. [Diabetologia (1999) 42: 1302–1307]