Improved molecular understanding of the pathogenesis of type 2 diabetes is essential if current therapeutic and preventative options are to be extended. To identify diabetes-susceptibility genes, we have completed a primary (418-marker, 9-cM) autosomal-genome scan of 743 sib pairs (573 pedigrees) with type 2 diabetes who are from the Diabetes UK Warren 2 repository. Nonparametric linkage analysis of the entire data set identified seven regions showing evidence for linkage, with allele-sharing LOD scores > or =1.18 (P< or =.01). The strongest evidence was seen on chromosomes 8p21-22 (near D8S258 [LOD score 2.55]) and 10q23.3 (near D10S1765 [LOD score 1.99]), both coinciding with regions identified in previous scans in European subjects. This was also true of two lesser regions identified, on chromosomes 5q13 (D5S647 [LOD score 1.22] and 5q32 (D5S436 [LOD score 1.22]). Loci on 7p15.3 (LOD score 1.31) and 8q24.2 (LOD score 1.41) are novel. The final region showing evidence for linkage, on chromosome 1q24-25 (near D1S218 [LOD score 1.50]), colocalizes with evidence for linkage to diabetes found in Utah, French, and Pima families and in the GK rat. After dense-map genotyping (mean marker spacing 4.4 cM), evidence for linkage to this region increased to a LOD score of 1.98. Conditional analyses revealed nominally significant interactions between this locus and the regions on chromosomes 10q23.3 (P=.01) and 5q32 (P=.02). These data, derived from one of the largest genome scans undertaken in this condition, confirm that individual susceptibility-gene effects for type 2 diabetes are likely to be modest in size. Taken with genome scans in other populations, they provide both replication of previous evidence indicating the presence of a diabetes-susceptibility locus on chromosome 1q24-25 and support for the existence of additional loci on chromosomes 5, 8, and 10. These data should accelerate positional cloning efforts in these regions of interest.

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1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.

The effects of some flavonoids, a group of naturally occurring pigments one of which has been claimed to possess antidiabetic activities, on insulin release and 45Ca2+ handling have been studied in isolated rat islets of Langerhans. Insulin release was enhanced by approximately 44-70% when islets were exposed to either (-)epicatechin (0.8 mmol/l) or quercetin (0.01-0.1 mmol/l); others such as naringenin (0.1 mmol/l) and chrysin (0.08 mmol/l) inhibited hormone release by approximately 40-60%. These effects were observed only in the presence of 20 mmol glucose/l. Quercetin (0.01 mmol/l) and (-)epicatechin (0.8 mmol/l) both inhibited 45Ca2+ efflux in the presence and absence of extracellular Ca2+. In the presence of 20 mmol glucose/l both the short-term (5 min) and steady-state (30 min) uptake of 45Ca2+ were significantly increased by either quercetin or (-)epicatechin. These results suggest that the stimulatory compounds such as quercetin and (-)epicatechin may, at least in part, exert their effects on insulin release via changes in Ca2+ metabolism.

Isolated rat islets of Langerhans permeabilised by high-voltage discharge secreted insulin in response to elevations in Ca2+ over the range 100 nM to 10 ,uM Ca 2+. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no effects on insulin secretion in the absence of Ca2+. In the presence of Ca2+ concentrations of > 10 nM, TPA produced dose-related shifts in the Ca 2+-activation curve to lower Ca2+ concentrations, together with marked increases in the maximum secretory response to Caz+. These results suggest that, in islets, the activation of protein kinase C is important in modulating both the sensitivity of the exocytotic mechanism to intracellular Ca2+, and the magnitude of the insulin secretory response.

The involvement of nitric oxide as an intracellular messenger in the control of insulin secretion from pancreatic Beta cells was studied in rat islets of Langerhans by measuring: (i) nitric oxide generation in response to physiological insulin secretagogues; (ii) the effects of inhibitors of nitric oxide synthesis on insulin secretory responses to physiological secretagogues, and on insulin synthesis; (iii) changes in islet cyclic guanosine monophosphate in response to secretagogues; (iv) the effects of exogenous cyclic guanosine monophosphate and dibutyryl cyclic guanosine monophosphate on insulin secretion from electrically permeabilised islets and from intact, respectively. These studies produced no evidence that nitric oxide generation is required for the initiation of insulin secretion by common secretagogues. However, the results of our experiments suggest that the generation of nitric oxide may be involved in long-term, glucose-dependent increases in cyclic guanosine monophosphate content of islet cells, although the physiological relevance of these changes requires further investigation.

Isolated rat islets of Langerhans which had been pretreated with 200 nM-phorbol 12-myristate 13-acetate (PMA) for 20-24 h, a treatment reported in other cell types to deplete cells of protein kinase C activity, were found not to contain detectable Ca2+/phospholipid-dependent protein kinase activity. These islets did not secrete insulin in response to a subsequent exposure to PMA (0.1 or 1 microM) during a 30 min incubation, although insulin secretion could be stimulated by 20 mM-glucose, a response which was enhanced by 20 microM-forskolin. PMA-pretreated islets that had been permeabilized by high-voltage discharge showed unimpaired secretory responses to an increase in Ca2+ concentration, cyclic AMP and forskolin. These results suggest that (i) pretreatment of islets with tumour-promoting phorbol esters may be a useful means of investigating the role of protein kinase C in stimulus-secretion coupling in the pancreatic beta-cell and (ii) protein kinase C may not play an essential role in glucose-induced insulin secretion.