Uptake of D-mannoheptulose by normal and tumoral pancreatic islet cells.

D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.

D-glucose metabolism in normal dispersed islet cells and tumoral INS-1 cells.

The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of 3HOH from D-[5-(3)H]glucose and 14C-labelled CO2, acidic metabolites and amino acids from D-[U-14C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-14C]glucose oxidation and D-[5-(3)H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types.

Antitumoral action of 2-deoxy-D-glucose tetraacetate in human melanoma cells.

The ester 2-deoxy-D-glucose tetraacetate (2-DOGTA) was recently shown to display cytostatic and cytotoxic activity in various lines of tumoral cells. In the present work, it was found to inhibit cell growth and confer chemosensitivity to cisplatin in two lines of human melanoma cells, poorly responsive to cisplatin. The inhibition of cell growth by 2-DOGTA was apparently not attributable to alteration of either D-glucose utilization or oxidation in these melanoma cell lines. In freshly isolated human melanoma cells, 2-DOGTA also inhibited cell growth, even in cells resistant to standard chemotherapeutic agents, such as temozolomide, cisplatin and/or vindesine. It is proposed, therefore, that 2-DOGTA should be further investigated for the treatment of melanoma patients, whether alone or in combination with known chemotherapeutic agents.

Altered metabolic priming by D-glucose in pancreatic islets from Goto-Kakizaki rats.

The possible priming by D-glucose of metabolic events in islets from control rats and Goto-Kakizaki rats (GK rats) was investigated by first incubating the islets for 120 min either in the absence of any exogenous nutrient or presence of 16.7 mM D-glucose. The islets were then incubated for a second period of 120 min either at 2.8 mM or 16. 7 mM D-glucose, the hexose being now mixed with tracer amounts of D-[U-14C]glucose and D-[5-3H]glucose. In islets from control rats first incubated in the absence of exogenous nutrient the hierarchy in the 16.7 mM/2.8 mM ratio for metabolic variables was as follows: D-[U-14C]glucose oxidation > D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to amino acids > D-[U-14C]glucose conversion to acidic metabolites. When the islets from control rats were first incubated in the presence of 16.7 mM D-glucose, the preferential stimulation of mitochondrial oxidative events at high hexose concentration, as documented by the increase in the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, was further enhanced. The 16.7 mM/2.8 mM ratio for the conversion of D-[U-14C]glucose to amino acids, relative to that for D-[U-14C]glucose conversion to acidic metabolites, was much lower, however, after a first incubation in the presence of D-glucose, rather than in its absence, probably as a result of the progressive exhaustion of endogenous amino acids considered as transamination partners. The major differences between these results and those obtained in islets from GK rats consisted, in the latter animals, in i) higher absolute values for all metabolic fluxes, ii) lower 16.7 mM/ 2.8 mM ratios, iii) lower paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, and iv) absence of a priming effect of D-glucose (16.7 mM) upon such a paired ratio in the islets incubated at 16.7 mM D-glucose during the second incubation. Taken as a whole, these observations confirm that the preferential stimulation of mitochondrial oxidative events, in response to a rise in D-glucose concentration, is impaired in islets from GK rats and extend this knowledge to the priming action of D-glucose, in high concentration, on the catabolism of the hexose during a subsequent incubation.

Isomeric and anomeric specificity of the metabolic and secretory response of rat pancreatic islets to glucose pentaacetate.

In the presence of 2.8 mM D-glucose, beta-D-glucose pentaacetate (1. 7 mM) augmented insulin release from isolated rat pancreatic islets more than alpha-D-glucose pentaacetate. Likewise, the further increment in insulin output evoked by nateglinide (0.01 mM) was higher in islets exposed to beta- rather than alpha-D-glucose pentaacetate. Inversely, in the presence of 2.8 mM unesterified D-glucose, alpha-L-glucose pentaacetate, but not beta-L-glucose pentaacetate, significantly augmented insulin output. The higher insulinotropic potency of the beta-anomer of D-glucose pentaacetate coincided with the fact that it significantly increased the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, whereas alpha-D-glucose pentaacetate failed to do so. These findings are interpreted to support the concept that the stimulation of insulin release by these esters is largely attributable to their direct interaction with a stereospecific receptor, with preference for the configuration of the C1 common to beta-D-glucose pentaacetate and alpha-L-glucose pentaacetate.

Protective effects of succinic acid dimethyl ester infusion in experimental endotoxemia.

In rats injected with bacterial lipopolysaccharide (LPS; 5 gamma mg/g body weight [BWT]), the toxin provokes death within 24 h in 23% of the animals and, in surviving rats, causes a decrease in BWT, hyperlactacidemia, hyperlipacidemia, and hyperketonemia, as well as depletion of both liver and muscle glycogen content. In the liver, LPS severely lowers the ATP and total adenine nucleotide content, ATP/ADP ratio, and adenylate charge. In hepatocytes from LPS-injected rats, the oxidation of D-glucose is first increased 2 h after administration of the toxin, despite close-to-normal phosphorylation of the hexose. In hepatocytes prepared from rats killed 24 h after injection of LPS, the phosphorylation of D-glucose, its incorporation into glycogen, and its oxidation are all severely impaired. This sequence of changes, which coincides with a decreased ratio between pyruvate and lactate production from exogenous D-glucose, is comparable to that found with agents that uncouple oxidative phosphorylation. The injection of LPS also alters the metabolic response of hepatocytes to the dimethyl ester of succinic acid (SAD), in terms, for instance, of the sparing action of the ester upon both the production of 14CO2 by hepatocytes prelabeled with L-[U-14C] glutamine and the output of NH4+, and its inhibitory action on glycogenolysis and futile cycling in the reactions catalyzed by glucokinase and glucose-6-phosphatase. Nevertheless, the infusion of SAD protects the rats against the deleterious effect of LPS upon such variables as the plasma concentration of free fatty acids and beta-hydroxybutyrate, the liver ATP content, and the oxidation of D-glucose, as well as the pyruvate/lactate ratio, in hepatocytes prepared from the LPS-injected rats. The infusion of SAD also virtually suppresses lethality in the LPS-injected animals. It is proposed, therefore, that the infusion of succinic acid esters may represent a novel therapeutic approach in endotoxemia and multiple-organ failure.

Insulinotropic action of methyl pyruvate: enzymatic and metabolic aspects.

The metabolism of methyl pyruvate was compared to that of pyruvate in isolated rat pancreatic islets. Methyl pyruvate was found to be more efficient than pyruvate in supporting the intramitochondrial conversion of pyruvate metabolites to amino acids, inhibiting D-[5-3H]glucose utilization, maintaining a high ratio between D-[3,4-14C]glucose or D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization, inhibiting the intramitochondrial conversion of glucose-derived 2-keto acids to their corresponding amino acids, and augmenting 14CO2 output from islets prelabeled with L-[U-14C]glutamine. Methyl pyruvate also apparently caused a more marked mitochondrial alkalinization than pyruvate, as judged from comparisons of pH measurements based on the use of either a fluorescein probe or 14C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. Inversely, pyruvate was more efficient than methyl pyruvate in increasing lactate output and generating L-alanine. These converging findings indicate that, by comparison with exogenous pyruvate, its methyl ester is preferentially metabolized in the mitochondrial, rather than cytosolic, domain of islet cells. It is proposed that both the positive and the negative components of methyl pyruvate insulinotropic action are linked to changes in the net generation of reducing equivalents, ATP and H+.

Relevance of lactate dehydrogenase activity to the control of oxidative glycolysis in pancreatic islet B-cells.

The activities of hexokinase isoenzymes, lactate dehydrogenase, cytosolic NAD-linked glycerophosphate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, and glutamate dehydrogenase were measured in homogenates of rat purified pancreatic B and non-B islet cells. In B cell homogenates, the maximal activity of hexokinase and glucokinase was one to two orders of magnitude lower than that of lactate dehydrogenase. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase was also much lower than that of the cytosolic NAD-linked glycerophosphate dehydrogenase . A comparable hierarchy in the activity of these enzymes was observed in non-B islet cells. These findings reinforce the view that the preferential stimulation of oxidative glycolysis observed in insulin-producing cells, when exposed to high concentrations of D-glucose, is attributable to a Ca2+-induced activation of the mitochondrial FAD-linked glycerophosphate dehydrogenase, rather than to saturation of the catalytic activity of lactate dehydrogenase.

Insulinotropic action of (2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinylcarbonyl) propionate. II. Ionophoretic and conformational aspects.

1. The non-sulphonylurea insulinotropic agent sodium (2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinylcarbonyl) propionate (KAD-1229) was found to display calcium ionophoretic activity in an artificial membrane model. 2. Conformation analysis indicated that a complex between calcium and KAD-1229, with a 1:2 stoichiometry, indeed displays favourable attributes for ionophoretic activity across a hydrophobic environment. 3. It is speculated that the ionophoretic property of KAD-1229 might participate to the remodelling of cationic fluxes evoked by this insulinotropic agent in pancreatic islet cells.

D-glucose metabolism in lymphocytes of patients with mitochondrial point mutation of the tRNALeu(UUR) gene.

D-Glucose metabolism was examined in the lymphocytes of six subjects with the mitochondrial tRNALeu(UUR) gene mutation responsible for the maternally inherited diabetes and deafness MIDD syndrome and compared with control subjects. No significant difference in D-[1-14C]glucose, D-[2-14C]glucose, or D-[6-14C]glucose oxidation, as well as D-[5-3H] glucose utilization, was observed between the two groups of subjects. These negative findings stress the view that impaired D-glucose metabolism, such as presumably is occurring in the beta-cells of patients with the MIDD syndrome, does not represent a universal feature found in all cell types of these patients.