Rajendra S. Rana

Effect of esters of succinic acid and other citric acid cycle intermediates on insulin release and inositol phosphate formation by pancreatic islets

Esters of carboxylic acids are permeable to cells and once inside the cell are hydrolyzed to carboxylic acids. Methyl and ethyl esters of succinate and other citric acid cycle intermediates were tested to find out whether they are insulin secretagogues. Monomethyl succinate stimulated insulin release from pancreatic islets in a concentration-dependent manner with maximal release attained at a concentration of 10 mM. Dimethyl succinate (10 mM) was as effective as monomethyl succinate, but pyruvate methyl ester, monoethyl succinate, and dimethyl fumarate were ineffective as primary secretagogues. However, dimethyl fumarate potentiated both leucine- and leucine-plus-glutamine-induced insulin release. Glucose, leucine, leucine plus glutamine, and monomethyl succinate increased inositol tris-, bis- and monophosphate formation in pancreatic islets and antimycin A inhibited this formation. Since mitochondrial metabolism is probably essential for glucose-induced insulin release and the metabolism of succinate and leucine (without or with glutamine) involves mitochondrial respiration exclusively, these results might indicate that mitochondrial metabolism generates conditions or factors that are transmitted to the cytosol to increase inositol trisphosphate formation and thus calcium mobilization and insulin release. Since succinate is believed to enter metabolism at site II of the mitochondrial respiratory chain, it is interesting that rotenone, an inhibitor of NADH dehydrogenase and site I of the respiratory chain, was a potent inhibitor of monomethyl succinate-induced insulin released. Rotenone also inhibited leucine (plus or minus glutamine)-induced insulin release. These results indicate that beta cell metabolism of monomethyl succinate and leucine, like glucose, influences dehydrogenases that produce NADH.

Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, glutathione peroxidase, cytochrome c peroxidase, and ascorbate free radical reductase activities in Neurospora crassa

Aging of post-mitotic cells, the conidia, of Neurospora crassa is defined as the time-dependent loss of viability under a constant laboratory environment which probably resembles the organisms tropical habitat; namely, at 30 degrees C, 85-100% relative humidity under white light. Median lifespan is defined as the age at which survival of a conidial population has declined to 50% of that of a fully viable population at birth. A collection of short (age-) and long-lived (age+) mutants were previously selected from the wild-type whose median lifespan is 22 days. Thus, five groups of strains with distinct lifespans of 7, 22, 36, 50 and 60 days were defined. The purposes of the present investigation were to determine if the activities of anti-oxygenic enzymes are correlated with lifespan and to elucidate the function of the cellular longevity determinant genes. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were highly-correlated with lifespan; whereas glutathione reductase and non-specific peroxidase activities were not correlated. The short-lived mutants were also deficient in cytochrome c peroxidase (CPX) and ascorbate free radical reductase (AFR), but not deficient in dehydroascorbate reductase. (These latter three enzymes were not examined in age+ mutants.) By isoelectric focusing analysis, the deficiencies of SOD, CAT, and GPX activities of age- mutants were defined in terms of specific isozymes. The mutants were specifically deficient in a cyanide-resistant mitochondrial isozyme of SOD. Sixteen age- genes, called the age-1 complex, were previously mapped on one arm of the seven chromosomes. On the basis of mapping and complementation data, it was inferred that the genes are spatially and functionally redundant. The hypothesis of functional redundancy is also supported by the enzyme data. Of seven mutants examined, representing seven of the age- genes, all were deficient in SOD, CAT and CPX, and six were deficient in AFR. Of four mutants examined, representing four of the genes, all were deficient in GPX. The results indicate a molecular basis for the previously observed photosensitivity of the mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Antioxidants prolong life span and inhibit the senescence- dependent accumulation of fluorescent pigment (lipofuscin) in clones of Podospora anserina☆

Culture of clones of Podospora anserina s+ with either nordihydroguaiaretic acid or reduced glutathione (GSH) at concentrations that were not inhibitory to growth significantly prolonged the average time to onset of senscence. GSH also prolonged the average time to onset of clonal death. The specific concentration of chloroform-methanol soluble fluorescent pigment was larger in senescent than in pre-senescent cells. The pigment exhibited fluorescence excitation and emission spectra and fluorescence polarization numbers characteristic of lipofuscin, an end-product of lipid peroxidation. Analyses of the lipofuscin concentration in either sub-clonal fractions of different times of origin from senescent clones, or in sub-clonal fractions of identical age in time of origin from parent clones of different age, revealed a similar concentrations distribution. Although pre-senscent cells contained rather large concentrations, a massive increase occurred during senescence prior to the time of onset of clonal death. Culture with GSH not only prolonged clonal life span but aslo inhibited the formation of lipofuscin by an average factor of 30. Furthermore, unlike untreated clones, the sub-clonal distribution of the pigment was not only low but was also independent of their age.

Secretagogue-responsive and -unresponsive pools of phosphatidylinositol in pancreatic islets

The effect of glucose on phosphatidylinositol turnover was studied. Phosphatidylinositol of rat pancreatic islets was labeled with myo[2-3H]inositol in the presence of various secretagogues (16.7 mM D-glucose, 22 mM D-mannose, 20 mM D-glyceraldehyde) and nonsecretagogues (3.3 mM D-glucose, 20 mM pyruvate, 16.7 mM D-galactose, 16.7 mM L-glucose). Upon subsequent stimulation with 16.7 mM D-glucose, only the islets that were labeled in the presence of secretagogues showed a loss of radioactivity from phosphatidylinositol. No loss of radioactivity from phosphatidylinositol occurred in the presence of 3.3 mM D-glucose even after labeling in the presence of secretagogues. A comparison of the subcellular distribution of labeled phosphatidylinositol in islets before and after stimulation with insulinotropic glucose revealed a loss of radioactivity from the plasma membrane fraction as judged by subcellular fractionation with a sucrose gradient. These results support a hypothesis advanced previously that pancreatic islets contain a unique pool of phosphatidylinositol that undergoes rapid turnover only in the presence of insulinotropic concentrations of D-glucose or other secretagogues [R. S. Rana, R. J. Mertz, A. Kowlura, J. F. Dixon, L. E. Hokin, and M. J. MacDonald (1985) J. Biol. Chem. 260, 7861-7867]. On the basis of the subcellular fractionation studies reported here, the secretagogue-responsive phosphatidylinositol pool appears to be located primarily in the plasma membrane of pancreatic islets.

Ageing of Neurospora crassa. V. Lipid peroxidation and decay of respiratory enzymes in an inositol auxotroph.

Abstract An inositol auxotroph of Neurospora crassa was grown in liquid culture with various inositol concentrations. Although the initial exponential growth rate was independent of the initial inositol concentration and equivalent to that of wild type, growth ceased prematurely with limiting concentrations. The period from the time of premature decline of growth rate until the time when cells begin to die at an exponential rate is defined as the senescence period. Comparisons of age-dependent changes in clones cultured with optimal and limiting inositol revealed that the latter accumulate more fluorescent pigment (lipofuscin) and that their mitochondria contained excessive concentrations of malondialdehyde, exhibited excessive rates of non-enzymatic lipid peroxidation in vitro and an age-dependent decline of specific activities of respiratory enzymes of the inner membrane. Cellular respiratory capacity was abnormal at an early age and deteriorated with increasing age. The in vitro rate of non-enzymatic lipid peroxidation was inversely correlated with the activities of the enzymes at various culture ages. Most of these abnormalities were evident in 9 h-old clones prior to the onset of senescence, becoming more severe during senescence. Culture with either nordihydroguaiaretic acid or hydrocortisone completely or partly prevented the occurrence of the biochemical abnormalities. Since these drugs also alleviate deterioration of clonal growth rates and cellular death, the observation that they protect mitochondria from the development of symptoms related to abnormal membranes and lipid peroxidation offers additional support to the hypothesis that their action is that of antioxidant and membrane stabilizer, respectively, and therefore, provides a molecular basis for their therapeutic role in vivo .

Glyceraldehyde phosphate: an insulin secretagogue with possible effects on inositol phosphate formation in pancreatic islets.

The insulinotropic action of glucose, the most potent physiologic insulin secretagogue, involves its metabolism. However, no glucose metabolite has ever been identified as a key intermediate. We tested the abilities of a number of glucose metabolites to stimulate insulin release from pancreatic islets. Of all of these metabolites, glyceraldehyde 3-phosphate was the most potent insulin secretagogue. In numerous experiments over 3 years, insulin release by 4 mM glyceraldehyde phosphate ranged from 50 to 200% of that initiated by 16.7 mM glucose--a near-maximal insulin stimulus. At concentrations of 1 and 4 mM, glyceraldehyde phosphate was even more potent than the known secretagogues glucose and glyceraldehyde. Glucose metabolites were also tested for their ability to stimulate inositol tris-, bis-, and monophosphate formation by permeabilized islets. Only glyceraldehyde phosphate stimulated inositol phosphate formation and this stimulation occurred at concentrations of glyceraldehyde phosphate which could be present in the beta cell under physiologic conditions (K0.5 = 25 microM). The current results are consistent with the idea that glyceraldehyde phosphate is a key insulinotropic glucose metabolite that might act directly (or rather directly via a receptor) on the phospholipase C that forms inositol trisphosphate in the plasma membrane.

Phospholipid methyltransferase activity in pancreatic islets: Activation by calcium☆

Pancreatic islet homogenates contain a Mg2+-requiring phospholipid methyltransferase activity, the activity of which was doubled by calcium (K0.5 less than 5 microM). Other divalent metal ions stimulated the activity from 11 to 35%, but zinc and strontium were inhibitory. Cyclic AMP had no effect on the enzyme activity and cyclic GMP inhibited it slightly. Calcium increased the Vmax of the enzyme without affecting its Km with respect to S-adenosylmethionine (6 microM). Chlorpromazine, trifluoperazine, and dibucaine inhibited the calcium-stimulatable activity without affecting the activity in the absence of calcium. Phosphatidylserine stimulated, and arachidonic acid and palmitic acid inhibited, the basal enzyme activity. The methylated products were found to be primarily mono- and dimethylphosphatidylethanolamine (30%) and phosphatidylcholine (43%) and an, as yet unidentified, nonpolar lipid fraction (27%), as judged by thin-layer chromatography. In the presence of calcium, incorporation of methyl groups into phosphatidylcholine, mono- and dimethylphosphatidylethanolamine, and nonpolar lipids was increased by 131, 60, and 46%, respectively. Based on the localization of the enzyme activity in the insulin secretory granule fraction, it is proposed that phospholipid methylation plays a role in coupling the stimulus to the initial events in insulin secretion, leading to the exocytosis of insulin.

Stimulation of phospholipid methylation by glucose in pancreatic islets

A two fold stimulation in the incorporation of [3H-methyl] groups from [3H-methyl] methionine into phospholipids was seen in intact pancreatic islets within six minutes of exposure to a glucose concentration that stimulates insulin release. Nonstimulatory sugars, L-glucose and D-galactose, as well as dibutyryl cAMP, did not affect phospholipid methylation in islet cells. A calcium channel blocker, verapamil, inhibited methylation. These studies suggest that the signal for glucose-induced insulin release could involve phospholipid methylation.

Ageing of Neurospora crassa. VI. Cytochemical and cytological correlates of senescence in three model systems

Abstract Senescent cells from three model systems in Neurospora crassa exhibit excessive rates of non-enzymatic mitochondrial lipid peroxidation in vitro and release excessive amounts of material into either the growth medium or hypotonic salt solutions, indicating that both mitochondrial and plasma membranes are abnormal. Since culture with the antioxidant nordihydroguaiaretic acid not only alleviates senescence of growth rate in each of the systems, but also alleviates or prevents the occurence of a number of biochemical abnormalities related to membranes and lipid peroxidation, excessive lipid peroxidation in plasma membranes during senescence appears to lead to abnormality of their osmotic and permeability properties. In support of this hypothesis, we demonstrated that: (1) culture of cells which undergo senescence with nordihydroguaiaretic acid inhibits the excessive release of cellular material, either during culture or in hypotonic saline solutions, to a level nearly that of normal cells; and (2) incubation of normal cells in conditions favoring peroxidation of their plasma membrane lipids, i.e. , with ferrous ascorbate, enhanced the release of cellular material and nordihydroguaiaretic acid inhibited this enhancement to a level equivalent to that of untreated cells. Additional studies of the natural-death mutant revealed an acceleration of rate of senescence by hypertonic medium and a premature age-dependent decline of cytochrome oxidase activity when grown in normal medium. Analyses of growth rates, osmotic fragility, and cellular respiratory competence of sub-clonal samples of this mutant indicated that senescence also occurs in post-mitotic cells; but that the youngest cells in time of origin are the most senescent in their physiology. These latter results indicate that there may be an age-dependent accumulation of molecular errors in mitotic cells, probably in replicating molecules such as nuclear and/or mitochondrial DNA. Deleterious reactions with the products of lipid peroxidation are the most probable source of such errors.