Hexose metabolism in pancreatic islets. Effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets.

Anaplerotic reactions leading to the de novo synthesis of fatty acids, were recently proposed to participate in the coupling of metabolic to secretory events in the process of glucose-stimulated insulin release. In an attempt to validate such a proposal, the effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release was investigated in glucose-stimulated rat pancreatic islets. The inhibitor of ATP citrate-lyase, when tested in the 1.0-2.0 mM range, failed to affect glucose-stimulated insulin release, but also failed to inhibit the incorporation of 14C-labelled acetyl residues derived from L-[U-14C]leucine into islet lipids. A partial inhibition of fatty acid labelling by 3H2O was only observed in islets incubated for 120 min in the presence of 5.0 mM (-)-hydroxycitrate and absence of CaCl2. These findings suggest that (-)-hydroxycitrate is not, under the present experimental conditions, a useful tool to abolish fatty acid synthesis in intact pancreatic islets.     

Glucose metabolism in insulin-producing tumoral cells

Homogenates of insulin-producing tumoral cells catalyzed the phosphorylation of glucose, mannose, and fructose. The kinetics of phosphorylation at increasing glucose concentrations, the inhibitory effect of glucose 6-phosphate, and the comparison of results obtained with distinct hexoses indicated the presence of both low-Km hexokinase-like and high-Km enzymatic activities, the results being grossly comparable to those collected in normal pancreatic islets. Relative to protein content, the glucose-phosphorylating enzymatic activity was higher in tumoral than normal islet cells. The activity of other enzymes was either lower (glutamate dehydrogenase), moderately higher (phosphoglucomutase, lactate dehydrogenase) or considerably greater (ornithine decarboxylase) in tumoral than in normal islet cells. In intact tumoral cells, incubated under increasing glucose concentrations, the oxidation of D-[U-14C]glucose and the output of lactic and pyruvic acids reached a close-to-maximal value at 2.8 mM glucose. The ratios for glucose oxidation/utilization and lactate/pyruvate output were much lower in tumoral than in normal islet cells. Although glucose caused a modest increase in insulin output from the tumoral cells, this effect was saturated at a low glucose concentration (2.8 mM) and less marked than that of other secretagogues (e.g., L-leucine, L-ornithine, or forskolin). Thus, despite a close-to-normal enzymatic equipment for glucose phosphorylation, the tumoral cells displayed severe abnormalities in the metabolism and secretory response to this hexose. These findings point to regulatory mechanisms distal to glucose phosphorylation in the control of glucose metabolism in insulin-producing cells.     

Arginine metabolism in rat enterocytes

Rat enterocytes exposed to L-arginine in the absence of any other exogenous substrate were found to actively metabolize this cationic amino acid. L-Arginine was converted to L-citrulline either directly in a NADPH-sensitive manner thought to be coupled with the generation of NO, or indirectly through the sequence of reactions catalyzed by arginase and ornithine transcarbamylase. A large fraction of L-citrulline and L-ornithine generated from exogenous L-arginine was released in the incubation medium. The production of CO2 and (poly)amines from L-arginine occurred at rates 2 to 3 orders of magnitude lower than that characterizing the net uptake of the cationic amino acid, and this despite the fact that enterocytes were equipped to allow the interconversion of L-ornithine and L-glutamate. It is concluded that the oxidative catabolism of L-arginine in enterocytes is quantitatively negligible relative to its conversion to L-citrulline and L-ornithine.     

Hexose metabolism in pancreatic islets: Effect of D-glucose on the mitochondrial redox state

The mitochondrial NADH/NAD⁺ ratio for free nucleotides in rat pancreatic islets was judged from the cell content in L-glutamate and L-alanine, 2-ketoglutarate and pyruvate, and NH ₄ ⁺ . At a physiological concentration of D-glucose, such a ratio averaged 9.6±1.1%. A rise in hexose concentrations, above a threshold value in excess of 5.6 mM, caused a rapid, sustained and rapidly reversible decrease in the mitochondrial NADH/NAD⁺ ratio. It is speculated that in the process of glucose-stimulated insulin release, the latter change participates in the coupling between metabolic and secretory events by favouring both the activity of key mitochondrial dehydrogenases and the translocation of Ca²⁺ from the mitochondria into the cytosol.     

Glucose-induced changes in cytosolic ATP content in pancreatic islets

The cytosolic and mitochondrial contents in ATP, ADP and AMP were measured in islets incubated for 45 min at increasing concentrations of D-glucose and then exposed for 20 s to digitonin. The latter treatment failed to affect the total islet ATP/ADP ratio and adenylate charge. D-Glucose caused a much greater increase in cytosolic than mitochondrial ATP/ADP ratio. In the cytosol, a sigmoidal pattern characterized the changes in ATP/ADP ratio at increasing concentrations of D-glucose. These findings are compatible with the view that cytosolic ATP participates in the coupling of metabolic to ionic events in the process of nutrient-induced insulin release.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction of L-asparagine and L-leucine in pancreatic islets

Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. L-Asparagine augmented the oxidation of L-leucine, an effect possibly attributable to activation of 2-ketoisocaproate dehydrogenase. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

The possible significance of intracellular pH in insulin release

Glucose and other nutrients which stimulate insulin release increase the rate of H⁺ generation and inhibit Na⁺Ca²⁺ counter-transport in the pancreatic B-cell. It is proposed that the latter phenomena are linked together by interference with an ionophoretic system. Indeed, in an artificial model, it is possible to mimick the process of Ca²⁺ countertransport and its inhibition in response to intracellular acidosis, by using native ionophores extracted from isolated pancreatic islets.     

Oxytocin alleviates oxidative renal injury in pyelonephritic rats via a neutrophil-dependent mechanism

BACKGROUND: Urinary tract infection (UTI) may cause inflammation of the renal parenchyma and may lead to impairment in renal function and scar formation. Oxidant injury and reactive oxygen species (ROS) have been found responsible in the pathogenesis of UTI. The neurohypophyseal hormone oxytocin (OT) facilitates wound healing and is involved in the modulation of immune and inflammatory processes. We investigated the possible therapeutic effects of OT against Escherichia coli induced pyelonephritis in rats both in the acute and chronic setting. METHODS: Twenty-four Wistar rats were injected 0.1 ml solution containing E. coli ATCC 25922 10(10) colony forming units/ml into left renal medullae. Six rats were designed as sham group and were given 0.1 ml 0.9% NaCl. Pyelonephritic rats were treated with either saline or OT immediately after surgery and at daily intervals. Half of the pyelonephritic rats were decapitated at the 24th hour of E. coli infection, and the rest were followed for 7 days. Renal function tests (urea, creatinine), systemic inflammation markers [lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha)] and renal tissue malondialdehyde (MDA) as an end product of lipid peroxidation, glutathione (GSH) as an antioxidant parameter and myeloperoxidase (MPO) as an indirect index of neutrophil infiltration were studied. RESULTS: Blood urea, creatinine, and TNF-alpha levels were increased, renal tissue MDA and MPO levels were elevated and GSH levels were decreased in both of the pyelonephritic (acute and chronic) rats. All of these parameters and elevation of LDH at the late phase were all reversed to normal levels by OT treatment. CONCLUSION: OT alleviates oxidant renal injury in pyelonephritic rats by its anti-oxidant actions and by preventing free radical damaging cascades that involves excessive infiltration of neutrophils.     

Functional role of aquaglyceroporin 7 expression in the pancreatic beta-cell line BRIN-BD11

Both mouse and rat pancreatic islet β-cells were recently found to express aquaglyceroporin 7 (AQP7). In the present study, the expression and role of AQP7 in the function of BRIN-BD11 cells were investigated. AQP7 mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. In an isoosmolar medium, the net uptake of [2-³H]glycerol displayed an exponential time course reaching an equilibrium plateau value close to its extracellular concentration. Within 2 min of incubation in a hypotonic medium (caused by a 50 mM decrease in NaCl concentration), the [2-³H]glycerol uptake averaged 143.2 ± 3.8% (n = 24; P < 0.001) of its control value in isotonic medium, declining thereafter consistently with previously demonstrated volume regulatory decrease. When isoosmolarity was restored by the addition of 100 mM urea to the hypotonic medium, [2-³H]glycerol uptake remained higher (112.1 ± 2.8%, n = 24; P < 0.001) than its matched control under isotonic conditions, indicating rapid entry of urea and water. Insulin release by BRIN-BD11 cells was 3 times higher in hypotonic than in isotonic medium. When glycerol (100 mM) or urea (100 mM) were incorporated in the hypotonic medium, the insulin release remained significantly higher than that found in the control isotonic medium, averaging respectively 120.2 ± 4.2 and 107.0 ± 3.8% of the paired value recorded in the hypotonic medium. These findings document the rapid entry of glycerol and urea in BRIN-BD11 cells, likely mediated by AQP7. J. Cell. Physiol. 221: 424–429, 2009. © 2009 Wiley-Liss, Inc.     

Acetylcholine chloride and renal hemodynamics during postnatal maturation in conscious lambs.

To test the hypothesis that acetylcholine-induced relaxation of the renal artery decreases with postnatal age, we measured parameters of renal hemodynamics before and for 35 s after aortic suprarenal injection of acetylcholine in conscious, chronically instrumented lambs aged approximately 1 wk (n = 5) and approximately 6 wk (n = 5). Acetylcholine was administered in one of five doses ranging from 0 to 10 mg/kg body wt; doses were administered randomly, in the same volume. There were significant age- and dose-dependent changes in renal vascular resistance after acetylcholine administration, such that the response was greater in 1-wk-old lambs. After the highest dose tested, renal vascular resistance decreased by 13.6 +/- 7.3 (SD) mmHg. ml(-1). min. g kidney wt in 1-wk-old lambs and by 9.1 +/- 3.2 mmHg. ml(-1). min. g kidney wt in 6-wk-old lambs at 35 s. We also observed a transient renal vasoconstriction before the renal vasodilatation in 6-wk-old lambs but not in 1-wk-old animals. These data provide the first age- and dose-dependent effects of exogenous administration of acetylcholine on renal hemodynamics during maturation in conscious animals.