Ragnar Henningsson

Morphological evidence for the existence of nitric oxide and carbon monoxide pathways in the rat islets of Langerhans: an immunocytochemical and confocal microscopical study.

Aims/hypothesis. To map the cellular location of inducible and constitutive nitric oxide synthase and haem oxygenase in rat islets to clarify the morphological background to putative nitric oxide and carbon monoxide pathways. Methods. Immunocytochemistry and confocal microscopy. Results. After treatment with endotoxin, immunoreactivity for inducible nitric oxide synthase was expressed in a large number of islet cells, most of which were insulin-immunoreactive beta cells and in single glucagon-immunoreactive and pancreatic polypeptide-immunoreactive cells. Somatostatin-immunoreactive cells lacked immunoreactivity for inducible nitric oxide synthase. In untreated rats, immunoreactivity for constitutive nitric oxide synthase occurred in the majority of insulin-immunoreactive and glucagon-immunoreactive cells, in most pancreatic polypeptide-immunoreactive and somatostatin-immunoreactive cells and in islet nerves. Similarly, immunoreactivity for constitutive haem oxygenase was detected in all four types of islet cells. Endotoxin treatment did not change the pattern of immunoreactivity for constitutive and inducible haem oxygenase. After treatment with alloxan, insulin-immunoreactivity was observed only in single islet cells, being almost devoid of immunoreactivity for constitutive nitric oxide synthase and haem oxygenase. Conclusion/interpretation. In vivo endotoxin-induced expression of inducible nitric oxide synthase in insulin-producing and in scattered glucagon-producing and pancreatic polypeptide-producing cells strengthens previous suggestions of a pathophysiological role for inducible nitric oxide synthase in the development of insulin-dependent diabetes mellitus. The presence of constitutive nitric oxide synthase and haem oxygenase in all four types of islet cells, together with recent functional data of ours support roles for nitric oxide and carbon monoxide as intracellular, paracrine or neurocrine modulators of islet hormone secretion. [Diabetologia (1999) 42: 978–986]

Arginine-induced insulin release is decreased and glucagon increased in parallel with islet NO production

Nitric oxide (NO) produced by islet constitutive NO synthase (cNOS) is a putative modulator of islet hormone secretion. We show here for the first time that the release of insulin induced by L-arginine or L-homoarginine is inhibited and that of glucagon stimulated in parallel with the rate of islet NO production. It was found that L-homoarginine was approximately 25-30% less potent than L-arginine as an insulin secretagogue but equally potent as a glucagon secretagogue. Biochemical determination of islet cNOS activity revealed that the NO production with L-homoarginine as substrate was only approximately 40% of that of L-arginine. Selective inhibition of islet cNOS potentiated insulin release during amino acid stimulation. Moreover, inhibition of cNOS suppressed glucagon release, more so with L-arginine than with L-homoarginine as secretagogue, reflecting the relative rates of their NO production. In K+-depolarized islets, inhibition of cNOS enhanced the insulin response to L-arginine by 50% and that to L-homoarginine by 23%, largely corresponding to their relative NO production. The intracellular NO donor hydroxylamine dose dependently inhibited insulin but increased glucagon secretion in K+-depolarized and amino acid-stimulated islets. We conclude that both amino acids have a dual action on insulin release, since their stimulatory effects are reduced in parallel with the rates of their NO production. Furthermore, the greater NO production induced by L-arginine relative to L-homoarginine corresponds to NO-mediated increases in glucagon release. These NO effects are mainly exerted independently of membrane depolarization events.Nitric oxide (NO) produced by islet constitutive NO synthase (cNOS) is a putative modulator of islet hormone secretion. We show here for the first time that the release of insulin induced byl-arginine orl-homoarginine is inhibited and that of glucagon stimulated in parallel with the rate of islet NO production. It was found thatl-homoarginine was ≈25-30% less potent thanl-arginine as an insulin secretagogue but equally potent as a glucagon secretagogue. Biochemical determination of islet cNOS activity revealed that the NO production with l-homoarginine as substrate was only ≈40% of that ofl-arginine. Selective inhibition of islet cNOS potentiated insulin release during amino acid stimulation. Moreover, inhibition of cNOS suppressed glucagon release, more so with l-arginine than with l-homoarginine as secretagogue, reflecting the relative rates of their NO production. In K+-depolarized islets, inhibition of cNOS enhanced the insulin response tol-arginine by 50% and that tol-homoarginine by 23%, largely corresponding to their relative NO production. The intracellular NO donor hydroxylamine dose dependently inhibited insulin but increased glucagon secretion in K+-depolarized and amino acid-stimulated islets. We conclude that both amino acids have a dual action on insulin release, since their stimulatory effects are reduced in parallel with the rates of their NO production. Furthermore, the greater NO production induced byl-arginine relative tol-homoarginine corresponds to NO-mediated increases in glucagon release. These NO effects are mainly exerted independently of membrane depolarization events.

Dysfunction of the Islet Lysosomal System Conveys Impairment of Glucose-Induced Insulin Release in the Diabetic GK Rat*

Accumulated evidence links an important signal involved in glucose-stimulated insulin release to the activation of the islet lysosomal glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. We have analyzed the function of the lysosomal system/lysosomal enzyme activities in pancreatic islets of young (6-8 weeks), spontaneously diabetic, GK (Goto-Kakizaki) rats and Wistar control rats in relation to glucose-induced insulin release. The insulin secretory response to glucose was markedly impaired in the GK rat, but was restored by the adenylate cyclase activator forskolin. Islet activities of classical lysosomal enzymes, e.g.. acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and cathepsin D, were reduced by 20-35% in the GK rat compared with those in Wistar controls. In contrast, the activities of the lysosomal alpha-glucosidehydrolases, i.e.. acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, were increased by 40-50%. Neutral alpha-glucosidase (endoplasmic reticulum) was unaffected. Comparative analysis of liver tissue showed that lysosomal enzyme activities were of the same magnitude in GK and Wistar rats. Notably, in Wistar rats, the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase were approximately 15-fold higher in islets than in liver. Other lysosomal enzymes did not display such a difference. Normalization of glycemia in GK rats by phlorizin administered for 9 days did not influence either the lysosomal alpha-glucosidehydrolase activities or other lysosomal enzyme activities in GK islets. Finally, the pseudotetrasaccharide acarbose, which accumulates in the lysosomal system, inhibited acid glucan-1,4-alpha-glucosidase activity in parallel with its inhibitory action on glucose-induced insulin release in intact Wistar islets, whereas no effect was recorded for either parameter in intact GK islets. In contrast, acarbose inhibited the enzyme activity equally in islet homogenates from both GK and Wistar rats, showing that the catalytic activity of the enzyme itself in disrupted cells was unaffected. We propose that dysfunction of the islet lysosomal/vacuolar system is an important defect impairing the transduction mechanisms for glucose-induced insulin release in the GK rat.

Total parenteral nutrition modulates hormone release by stimulating expression and activity of inducible nitric oxide synthase in rat pancreatic islets

The expression and activities of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in relation to insulin and glucagon secretory mechanisms were investigated in islets isolated from rats subjected to total parenteral nutrition (TPN) for 10 d. TPN is known to result in significantly increased levels of plasma lipids during the infusion time. In comparison with islets from freely fed control rats, islets taken from TPN rats at d 10 displayed a marked decrease in glucose-stimulated insulin release (4.65±0.45 ng/[islet·h] vs 10.25±0.65 for controls) (p<0.001) accompanied by a strong iNOS activity (18.3±1.1 pmol of NO/[min·mg of protein]) and a modestly reduced cNOS activity (11.3±3.2 pmol of NO/[min.mg of protein] vs 17.7±1.7 for controls) (p<0.01). Similarly, Western blots showed the expression of iNOS protein as well as a significant reduction in cNOS protein in islets from TPN-treated rats. The enhanced NO production, which is known to inhibit glucose-stimulated insulin release, was manifested as a strong increase in the cyclic guanosine 5′-monophosphate content in the islets of TPN-treated rats (1586±40 amol/islet vs 695±64 [p<0.001] for controls). Moreover, the content of cyclic adenosine monophosphate (cAMP) was greatly increased in the TPN islets (80.4±2.1 fmol/islet vs 42.6±2.6 [p<0.001] for controls). The decrease in glucose-stimulated insulin release was associated with an increase in the activity of the secretory pathway regulated by the cAMP system in the islets of TPN-treated rats, since the release of insulin stimulated by the phosphodiesterase inhibitor isobutylmethylxanthine was greatly increased both in vivo after iv injection and after in vitro incubation of isolated islets. By contrast, the release of glucagon was clearly reduced in islets taken from TPN-treated rats (33.5±1.5 pg/[islet·h] vs 45.5±2.2 for controls) (p<0.01) when islets were incubated at low glucose (1.0 mmol/L). The data show that long-term TPN treatment in rats brings about impairment of glucose-stimulated insulin release, that might be explained by iNOS expression and a marked iNOS-derived NO production in the β-cells. The release of glucagon, on the other hand, is probably decreased by a direct “nutrient effect” of the enhanced plasma lipids. The results also suggest that the islets of TPN-treated rats have developed compensatory insulin secretory mechanisms by increasing the activity of their β-cell cAMP system.

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production.

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.

Abnormally decreased NO and augmented CO production in islets of the leptin-deficient ob/ob mouse might contribute to explain hyperinsulinemia and islet survival in leptin-resistant type 2 obese diabetes

The role of the gaseous messengers NO and CO for β-cell function and survival is controversial. We examined this issue in the hyperglycemic-hyperinsulinemic ob/ob mouse, an animal model of type 2 obese diabetes, by studying islets from obese vs lean mice regarding glucose-stimulated insulin release in relation to islet NO and CO production and the influence of modulating peptide hormones. Glucose-stimulated increase in ncNOS-activity in incubated lean islets was converted to a decrease in ob/ob islets associated with markedly increased insulin release. Both types of islets displayed iNOS activity appearing after ~60 min in high-glucose. In ob/ob islets the insulinotropic peptides glucagon, GLP-1 and GIP suppressed NOS activities and amplified glucose-stimulated insulin release. The insulinostatic peptide leptin induced the opposite effects. Suppression of islet CO production inhibited, while stimulation amplified glucose-stimulated insulin release. Nonincubated isolated islets from young and adult obese mice displayed very low ncNOS and negligible iNOS activity. In contrast, production of CO, a NOS inhibitor, was impressively raised. Glucose injections induced strong activities of islet NOS isoforms in lean but not in obese mice and confocal microscopy revealed iNOS expression only in lean islets. Islets from ob/ob mice existing in a hyperglycemic in vivo milieu maintain elevated insulin secretion and protection from glucotoxicity through a general suppression of islet NOS activities achieved by leptin deficiency, high CO production and insulinotropic cyclic-AMP-generating hormones. Such a beneficial effect on islet function and survival might have its clinical counterpart in human leptin-resistant type 2 obese diabetes with hyperinsulinemia.

Nitric oxide (NO) — Production and regulation of insulin secretion in islets of freely fed and fasted mice

Production of nitric oxide through the action of nitric oxide synthase (NOS) has been detected in the islets of Langerhans. The inducible isoform of NOS (iNOS) is induced by cytokines and might contribute to the development of type-1 diabetes, while the constitutive isoform (cNOS) is thought to be implicated in the physiological regulation of insulin secretion. In the present study we have detected and quantified islet cNOS- and iNOS-derived NO production concomitant with measuring its influence on insulin secretion in the presence of different secretagogues: glucose, L-arginine, L-leucine and α-ketoisocaproic acid (KIC) both during fasting and freely fed conditions. In intact islets from freely fed mice both cNOS- and iNOS-activity was greatly increased by glucose (20 mmol/l). Fasting induced islet iNOS activity at both physiological (7 mmol/l) and high (20 mmol/l) glucose concentrations. NOS blockade increased insulin secretion both during freely fed conditions and after fasting. L-arginine stimulated islet cNOS activity and did not affect islet iNOS activity. l-leucine or KIC, known to enter the TCA cycle without affecting glycolysis, did not affect either islet cNOS- or iNOS activity. Accordingly, insulin secretion stimulated by L-leucine or KIC was unaffected by addition of L-NAME both during feeding and fasting. We conclude that both high glucose concentrations and fasting increase islet total NO production (mostly iNOS derived) which inhibit insulin secretion. The insulin secretagogues L-leucine and KIC, which do not affect glycolysis, do not interfere with the islet NO-NOS system.