Simon L. Howell

A Genomewide Scan for Loci Predisposing to Type 2 Diabetes in a U.K. Population (The Diabetes UK Warren 2 Repository): Analysis of 573 Pedigrees Provides Independent Replication of a Susceptibility Locus on Chromosome 1q

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.

Nitric oxide is not involved in the initiation of insulin secretion from rat islets of Langerhans

SummaryThe 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 islets, 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.

Effects of Epicatechin on Rat Islets of Langerhans

Plants containing epicatechin (a flavonoid) have been used to treat diabetes mellitus in Indian medicine. The present study reports effects of this compound on isolated islets of Langerhans. The flavonoid (1 mM) was found to increase insulin secretion from isolated rat islets of Langerhans in the presence of either 2 or 20 mM glucose, in static incubations, or in perifusion. The increase in insulin secretion mediated by epicatechin was both ATP- and temperature-dependent. Ultrastructural studies showed no deleterious changes in the structure of the B-cells after 5 days of exposure to the compound. Intraperitoneal injection of 30 mg/kg body wt of epicatechin twice daily for 4 days increased the islet insulin content by 30%. Secretion of insulin from islets isolated from epicatechin-injected rats was significantly increased when exposed to 20 mM glucose in comparison with water-injected controls. Furthermore, islets of adult rats cultured with 5.5 mM glucose for 4 days showed a significant increase in DNA synthesis in the presence of 0.05 mM epicatechin. These results suggest direct effects of epicatechin on various aspects of islet function.

Regulation of insulin secretion: the role of second messengers.

SummaryThis review summarises briefly studies performed in the last 5–6 years concerning the role of second messengers in the regulation of insulin secretion, using intact and electrically permeabilized rat islets of Langerhans. It is concluded that cyclic AMP (through protein kinase A), calcium (through calcium-calmodulin dependent protein kinases) and diacylglycerol (through protein kinase C) may be important second messengers in modulating the effects of specific secretagogues on insulin release. However, recent studies strongly suggest that neither protein kinase A nor protein kinase C are directly involved in the regulation of insulin secretion by glucose. The possible involvement of other second messengers, nitric oxide and arachidonic acid, in the regulation of secretion is also briefly reviewed.

Translocation of protein kinase C in rat islets of Langerhans. Effects of a phorbol ester, carbachol and glucose.

In unstimulated rat islets (2 mM glucose), most of the ion‐exchange purified protein kinase C (PKC) activity was associated with the cytosolic fraction. Both carbachol and phorbol myristate acetate caused a significant translocation of PKC activity from cytosolic to membrane fractions, but under the same conditions, glucose (20 mM) did not cause such a redistribution of PKC activity. PMA‐induced translocation of PKC to the membrane fraction was also observed in electrically permeabilised islets, in which recovery of the enzyme activity was enhanced by buffering the intracellular Ca2+ concentration to 50 nM and supplying the permeabilised islets with protease inhibitors.

Catecholamine inhibition of Ca2+‐induced insulin secretion from electrically permeabilised islets of Langerhans

Noradrenaline (1–10 μM) inhibited Ca2+‐induced insulin secretion from electrically permeabilised islets of Langerhans with an efficacy similar to that for inhibition of glucose‐induced insulin secretion from intact islets. The inhibition of insulin secretion from permeabilised islets was blocked by the α2‐adrenoreceptor antagonist, yohimbine. Adenosine 3′,5′‐cyclic monophosphate (cAMP) did not relieve the noradrenaline inhibition of Ca2+‐induced secretion from the permeabilised islets, although noradrenaline did not affect the secretory responses to cAMP at substimulatory (50 nM) concentrations of Ca2+. These results suggest that catecholamines do not inhibit insulin secretion solely by reducing B‐cell adenylate cyclase activity, and imply that one site of action of noradrenaline is at a late stage in the secretory process.

Time-course of Ca2+-induced insulin secretion from perifused, electrically permeabilised islets of Langerhans: effects of cAMP and a phorbol ester

The pattern of insulin secretion from electrically permeabilised islets was studied in a perifusion system. Increases in intracellular Ca2+ stimulated insulin secretion in a dose-related manner, but the secretory response to Ca2+ was only transient, with permeabilised islets becoming rapidly insensitive to a stimulatory concentration of Ca2+. Cyclic AMP and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), both stimulated Ca2+-induced insulin secretion from perifused permeabilised islets by increasing the maximum secretory response to Ca2+, and by maintaining elevated rates of secretion when the permeabilised islets had become insensitive to the stimulatory effects of Ca2+. These results suggest that cAMP and PMA may act partly by modifying the magnitude of the secretory response to Ca2+, and also by Ca2+-independent effects on the secretory mechanism.

Activation of protein kinase C is essential for sustained insulin secretion in response to cholinergic stimulation

Insulin secretion from isolated rat islets of Langerhans is enhanced by cholinergic agonists, such as carbachol (CCh), in the presence of a stimulatory concentration of glucose. Depletion of islet protein kinase C activity by prolonged exposure to a tumour-promoting phorbol ester did not prevent the initial secretory response to CCh, but markedly reduced the duration of CCh-induced elevated secretory rates. These results suggest that the major action of PKC is in maintaining rather than initiating the insulin secretory response to cholinergic agonists.

The effects of polymyxin B, a protein kinase C inhibitor, on insulin secretion from intact and permeabilised islets of Langerhans

Polymyxin B (0.01-1 mM), a polyamine antibiotic, inhibited both phorbol ester- and glucose-stimulated insulin secretion from isolated rat islets of Langerhans. This inhibition was rapidly reversible. Assay of the cytosolic protein kinase C by measurement of incorporation of labelled phosphate into a histone substrate demonstrated the presence of activity in islet extracts which could be stimulated by 12-O-tetradecanoylphorbol-13-acetate and inhibited by polymyxin B. These results suggest that protein kinase C plays a role in glucose-induced insulin secretion.

Staurosporine inhibits protein kinases activated by Ca2+ and cyclic AMP in addition to inhibiting protein kinase C in rat islets of Langerhans

Staurosporine has been used in several studies to investigate the role of protein kinase C (PKC) in secretory responses of islets of Langerhans to insulin secretagogues. We have assessed the effect of staurosporine on: [i] islet PKC activity in vitro; [ii] the stimulation of insulin secretion by nutrient secretagogues and [iii] the stimulation of protein phosphorylation and insulin secretion in electrically permeabilised islets. All experiments were carried out on rat isolated islets of Langerhans, either intact or permeabilised by high voltage discharge (3.4 kV/cm). The activity of PKC partially purified from rat islets was inhibited by staurosporine (1.6-400 nM) in a concentration-dependent manner. Staurosporine also inhibited insulin secretion stimulated by both glucose and glyceraldehyde, with maximal effects at 50 nM. After prolonged exposure of islets to the tumour-promoting phorbol ester, 4 beta phorbol myristate acetate (4 beta PMA), a procedure which depletes islet PKC activity, staurosporine still inhibited both glucose- and glyceraldehyde-stimulated insulin release. In electrically permeabilised islets, staurosporine inhibited both Ca(2+)- and cyclic AMP-stimulated protein phosphorylation and insulin secretion. These results suggest that staurosporine should not be used as a selective inhibitor of PKC in rat islets.