Dynamic insulin secretion from perifused rat pancreatic islets

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 μM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 μM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K⁺ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED₅₀ = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED₅₀ = 89 nM) and DA (ED₅₀ = 2.2 μM). γ-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances. Received 16 January 1998; received after revision 6 May 1998; accepted 8 May 1998     

Über die Wirkung blutzuckersenkender Sulfonylharnstoffe auf das isolierte Rattenzwerchfell

Summary The action of blood sugar depressing sulfonylureas on glucose and oxygen uptake, as well as on glycogen content and formation of C¹⁴O₂ from uniformly labelled C¹⁴-glucose was investigated in rat hemidiaphragms incubated in phosphate buffer. The following results were obtained: (1) Tolbutamide and Carbutamide increased the glucose uptake. (2) Tolbutamide decreased the glycogen-content. (3) Oxygen uptake as well as formation of C¹⁴O₂ were increased by Tolbutamide. (4) The action of Tolbutamide and insuline was equal with respect to glucose uptake but different as regarding the glycogen content, oxygen uptake and CO₂-formation.It is concluded that sulfonylureas increase glucose oxidation in the rat hemidiaphragm probably without increasing insulin sensitivity. To our knowledge this mechanism of action has hitherto not been described.     

Localization of pyruvate carboxylase in the cells ofNeurospora crassa

Summary The cell wall ofNeurospora crassa was digested enzymatically and the cytosolic and the mitochondrial fractions were separated. The activity of pyruvate carboxylase (EC 6.4.1.1) was detected entirely in the cytosolic fraction. This indicates that the location of pyruvate carboxylase ofN. crassa is in the cytosol, but is not in the mitochondria; this is different from the situation in animal tissues.     

Ethanol influence on insulin secretion from isolated rat islets

This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.     

Effects of starvation and Ca++ on glucose-induced accumulation of cyclic 3′,5′-AMP in pancreatic islets

Summary Exposure to glucose in the presence of 3-isobutyl-1-methylxanthine leads to accumulation of cAMP in islets microdissected fromob/ob-mice. This process is dependent on extracellular Ca⁺⁺ but differs markedly from the glucose action on insulin release in the same in vitro system in disappearing after 18 h of starvation.Supported by the Swedish Medical Research Council (No. 12x562).     

Ethanol influence on insulin secretion from isolated rat islets

Summary This study was done to delineate the role of - and -adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an -adrenergic blocker, phentolamine, or a -adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.Supported by the U.S. Veterans Administration     

Post-transcriptional gene silencing of ribosomal protein S6 kinase 1 restores insulin action in leucine-treated skeletal muscle

Excessive nutrients, especially amino acids, impair insulin action on glucose metabolism in skeletal muscle. We tested the hypothesis that the branched-chain amino acid leucine reduces acute insulin action in primary myotubes via a negative feedback mechanism involving ribosomal protein S6 kinase 1 (S6K1). The effect of S6K1 on glucose metabolism was determined by applying RNA interference (siRNA). Leucine (5 mM) reduced glucose uptake and incorporation to glycogen by 13% and 22%, respectively, compared to the scramble siRNA-transfected control at the basal level. Leucine also reduced insulin-stimulated Akt phosphorylation, glucose uptake and glucose incorporation to glycogen (39%, 39% and 37%, respectively), and this reduction was restored after S6K1 silencing. Depletion of S6K1 enhanced basal glucose utilization and protected against the development of impaired insulin action, in response to excessive leucine. In conclusion, S6K1 plays an important role in the regulation of insulin action on glucose metabolism in skeletal muscle. Received 22 December 2008; received after revision 19 February 2009; accepted 23 February 2009     

Thiamine deficiency and protein secretion by pancreatic slices in vitro

Summary Pancreatic slices incubated in glucose medium take up oxygen and glucose and liberate pentose, pyruvate and proteins. Thiamine deficiency decreases oxygen consumption but increases liberation of pentose, pyruvate and proteins by pancreatic slices.     

Pancreatic islet β-cell deficit and glucose intolerance in rats with uninephrectomy

This study was performed to examine the effect of chronic renal impairment and renin-angiotensin system (RAS) activation induced by unilateral nephrectomy (UNX) on the development of pancreatic islet β-cell deficit and glucose intolerance. Sprague-Dawley rats were randomized into three groups: untreated UNX (n = 10), UNX treated with the angiotensin-converting enzyme inhibitor lisinopril (n = 8) and sham operation (n = 10). Blood glucose, serum insulin, renal function and histological changes of kidney and pancreas were examined 8 months postoperation. Compared with the sham rats, UNX rats developed renal impairment, insulin deficiency and glucose intolerance. Histological staining revealed an islet β-cell deficit associated with increased immunoreactivity for angiotensin and angiotensin type 1 receptor in UNX rats. Treatment with lisinopril significantly improved renal dysfunction, hyperglycemia, insulin secretion and islet RAS expression. These data suggest that chronic renal impairment and RAS activation may contribute to islet β-cell loss and glucose intolerance. RAS blockade may therefore prevent these disorders. Received 29 August 2007; received after revision 25 September 2007; accepted 27 September 2007     

Stimulation of glucagon and inhibition of insulin secretion evoked from carotid baroreceptors

Summary The influence from carotid baroreceptors on portal immuno-reactive glucagon and insulin levels and on arterial plasma glucose concentration was studied in vagotomized cats by sectioning of the sinus nerves. Such a complete elimination of the afferent baroreceptor discharge caused a prompt and pronounced increase in the glucose and glucagon levels, whereas the insulin concentration significantly decreased. The role of vascular baroreceptors in the hyperglycemic response to hemorrhage is discussed.     

Sphingosine 1-phosphate increases glucose uptake through trans-activation of insulin receptor

Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts through a family of G-protein-coupled receptors. Herein, we report evidence of a novel redox-based cross-talk between S1P and insulin signaling pathways. In skeletal muscle cells S1P, through engagement of its S1P₂ receptor, is found to produce a transient burst of reactive oxygen species through a calcium-dependent activation of the small GTPase Rac1. S1P-induced redox-signaling is sensed by protein tyrosine phosphatase-1B, the main negative regulator of insulin receptor phosphorylation, which undergoes oxidation and enzymatic inhibition. This redox-based inhibition of the phosphatase provokes a ligand-independent trans-phosphorylation of insulin receptor and a strong increase in glucose uptake. Our results propose a new role of S1P, recognizing the lipid as an insulin-mimetic cue and pointing at reactive oxygen species as critical regulators of the cross-talk between S1P and insulin pathways. Any possible implication of S1P-directed insulin signaling in diabetes and insulin resistance remains to be established.     

Stimulation of glucagon and inhibition of insulin secretion evoked from carotid baroreceptors.

The influence from carotid baroreceptors on portal immuno-reactive glucagon and insulin levels and on arterial plasma glucose concentration was studied in vagotomized cats by sectioning of the sinus nerves. Such a complete elimination of the afferent baroreceptor discharge caused a prompt and pronounced increase in the glucose and glucagon levels, whereas the insulin concentration significantly decreased. The role of vascular barorecptors in the hyperglycemic response to hemorrhage is discussed.     

Dysfunctional insulin secretion in type 2 diabetes: role of metabolic abnormalities

Insulin secretion is finely tuned to the requirements of tissues by tight coupling to prevailing blood glucose levels. The normal regulation of insulin secretion is coupled to glucose metabolism in the pancreatic B cell, a major but not exclusive signal for secretion being closure of K⁺ATP (adenosine triphosphate)-dependent channels in the cell membrane through an increase in cytosolic ATP/adenosine diphosphate. Insulin secretion in type 2 diabetes is abnormal in several respects due to genetic causes but also due to the metabolic environment of the pancreatic B cells. This environment may be particularly important for the deterioration of insulin secretion which occurs with increasing duration of diabetes. Factors in the environment with potential importance include overstimulation, a negative effect of hyperglycemia per se (‘glucotoxicity’) as well as adverse effects of elevated fatty acids (‘lipotoxicity’). Elucidating the mechanisms behind these factors as well as their clinical importance will pave the way for treatment which could preserve B-cell function in type 2 diabetic patients. Received 4 October 1999; received after revision 1 November 1999; accepted 3 December 1999     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of 45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Vaspin inhibits kallikrein 7 by serpin mechanism

The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin–hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion. By application of vaspin and generated inactive mutants, we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance.     

Insulin acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia in the rat

Microinjection of insulin (0.04–0.12 IU/l) into the anterior hypothalamus or the lateral hypothalamus, but not the vertromedial hypothalamus of the rat brain, caused a dose-dependent rise in blood glucose and in serum insulin. The majority (71.5%) of the glucose-facilitated neurons recorded in the lateral hypothalamic area were excited by intracerebral injection of insulin. The data indicate that insulni acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia. It is unknown whether insulin normally reaches the hypothalamic area, or how it might do so.This work was supported by grants from the National Science Council (Taipei, Taiwan, Republic of China).