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Effect of stimulation of glucokinase (GK) export from the nucleus by small amounts of sorbitol on hepatic glucose flux in response to elevated plasma glucose was examined in 6-h fasted Zucker diabetic fatty rats at 10 wk of age. Under basal conditions, plasma glucose, insulin, and glucagon were approximately 8 mM, 2,000 pmol/l, and 60 ng/l, respectively. Endogenous glucose production (EGP) was 44 +/- 4 micromol x kg(-1) x min(-1). When plasma glucose was raised to approximately 17 mM, GK was still predominantly localized with its inhibitory protein in the nucleus. EGP was not suppressed. When sorbitol was infused at 5.6 and 16.7 micromol x kg(-1) x min(-1), along with the increase in plasma glucose, GK was exported to the cytoplasm. EGP (23 +/- 19 and 12 +/- 5 micromol x kg(-1) x min(-1)) was suppressed without a decrease in glucose 6-phosphatase flux (145 +/- 23 and 126 +/- 16 vs. 122 +/- 10 micromol x kg(-1) x min(-1) without sorbitol) but increased in glucose phosphorylation as indicated by increases in glucose recycling (122 +/- 17 and 114 +/- 19 vs. 71 +/- 11 microl x kg(-1) x min(-1)), glucose-6-phosphate content (254 +/- 32 and 260 +/- 35 vs. 188 +/- 20 nmol/g liver), fractional contribution of plasma glucose to uridine 5'-diphosphate-glucose flux (43 +/- 8 and 42 +/- 8 vs. 27 +/- 6%), and glycogen synthesis from plasma glucose (20 +/- 4 and 22 +/- 5 vs. 9 +/- 4 mumol glucose/g liver). The decreased glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake may result from failure of the sugar to activate GK by stimulating the translocation of the enzyme.
The mechanism by which COX2 inhibition decreases renal cell survival is poorly understood. In the present study we examined the effect of COX2 activity on organic osmolyte accumulation in renal medulla and in cultured mouse renal medullary interstitial cells (MMICs) and its role in facilitating cell survival. Hypertonicity increased accumulation of the organic osmolytes inositol, sorbitol, and betaine in cultured mouse medullary interstitial cells. Pretreatment of MMICs with a COX2-specific inhibitor (SC58236, 10 micromol/liter) dramatically reduced osmolyte accumulation (by 79 +/- 9, 57 +/- 12, and 96 +/- 10% for inositol, sorbitol, and betaine respectively, p < 0.05). Similarly, 24 h of dehydration increased inner medullary inositol, sorbitol, and betaine concentrations in vivo by 85 +/- 10, 197 +/- 28, and 190 +/- 24 pmol/microg of protein, respectively, but this increase was also blunted (by 100 +/- 5, 66 +/- 15, and 81 +/- 9% for inositol, sorbitol, and betaine, respectively, p < 0.05) by pretreatment with an oral COX2 inhibitor. Dehydrated COX2-/- mice also exhibited an impressive defect in sorbitol accumulation (88 +/- 9% less than wild type, p < 0.05) after dehydration. COX2 inhibition (COX2 inhibitor-treated or COX2-/- MMICs) dramatically reduced the expression of organic osmolyte uptake mechanisms including betaine (BGT1) and sodium-myo-inositol transporter and aldose reductase mRNA expression under hypertonic conditions. Importantly, preincubation of COX2 inhibitor-treated MMICs with organic osmolytes restored their ability to survive hypertonic stress. In conclusion, osmolyte accumulation in the kidney inner medulla is dependent on COX2 activity, and providing exogenous osmolytes reverses COX2-induced cell death. These findings may have implications for the pathogenesis of analgesic nephropathy.
Ascorbic acid, or vitamin C, has been reported to lower erythrocyte sorbitol concentrations, and present studies were performed to determine the mechanism of this effect. Incubation of erythrocytes with increasing concentrations of glucose (5-40 mM) progressively increased erythrocyte sorbitol contents, reflecting increased flux through aldose reductase. At extracellular concentrations of 90 microM, both ascorbic acid and its oxidized form, dehydroascorbate, decreased intracellular sorbitol by 25 and 45%, respectively. This inhibition was not dependent on the extracellular glucose concentration, or on erythrocyte contents of free NADPH or GSH. To test for a direct effect of ascorbate on aldose reductase, erythrocyte hemolysates were prepared and supplemented with 100 microM NADPH. Hemolysates reduced glucose to sorbitol in a dose-dependent manner that was inhibited with a Ki of 120 microM by the aldose reductase inhibitor tetramethylene glutaric acid. Above 100 microM, ascorbic acid also lowered hemolysate sorbitol generation by about 30%. Studies with ascorbic acid derivatives showed that the reducing capacity of ascorbic acid was not required for inhibition of sorbitol production from glucose in erythrocyte hemolysates. These results show that high, but physiologic, concentrations of ascorbic acid can directly inhibit erythrocyte aldose reductase, and provide a rationale for the use of oral vitamin C supplements in diabetes.
The capacity of the malate-aspartate shuttle was evaluated in periportal (PP-H) and perivenous subfraction of rat hepatocytes (PV-H). The rate of glutamine production from alanine was 34-fold higher in PV-H than in PP-H. Statistically significant differences between PP-H and PV-H were found for the activities of lactate dehydrogenase and pyruvate kinase but not for the activities of NAD(+)-malate dehydrogenase, aspartate aminotransferase, and mitochondrial alanine aminotransferase. The rate of glucose production from sorbitol and the rate of ethanol utilization were higher in PP-H than in PV-H. In the presence of phenazine methosulfate (PMS), the increments in these rates were significantly greater in PV-H than in PP-H. The capacity of malate-aspartate shuttle in the presence of alanine was significantly higher in PP-H than in PV-H but in the presence of asparagine was similar in PP-H and PV-H. The results suggest that the capacity of malate-aspartate shuttle distributes heterogeneously along liver lobules with the dominance in periportal zone and that the difference of the capacity may result from the difference in the transport of aspartate across the mitochondrial membrane.
The effects of norepinephrine and glucagon on gluconeogenesis were studied in hemoglobin-free perfused liver from rats kept for 1-20 days at 4 degrees C. When rats were starved for 24 h at 4 degrees C, the plasma glucose level of rats exposed to cold for 5, 10, and 20 days was significantly higher than that of rats for 1 day, but hepatic glycogen decreased to the same level in all groups. In the isolated perfused liver, basal rates of oxygen consumption and glucose production increased slightly through 5 days of cold exposure and returned to control levels after 20 days of cold exposure. The rates of glucose production from lactate, pyruvate, sorbitol, and glycerol increased by 20-30% after 5 days of cold exposure. The stimulation of gluconeogenesis from these substrates by norepinephrine and phenylephrine increased markedly at all time periods from 1 to 20 days in the cold, with a maximum at 5 days. The stimulation of glycogenolysis by norepinephrine was not affected by cold exposure. The response to catecholamines decreased markedly in liver perfused with calcium-free medium and/or with phentolamine. The stimulation of gluconeogenesis by glucagon increased only in rats exposed to cold for 20 days. The results obtained suggest that the stimulation of hepatic gluconeogenesis by cold is due to an alpha-adrenergic response, and the activation occurs beyond the interaction of norepinephrine with its receptor.
The role of Ca2+ in stimulation of the malate-aspartate shuttle by norepinephrine and vasopressin was studied in perfused rat liver. Shuttle capacity was indexed by measuring the changes in both the rate of production of glucose from sorbitol and the ratio of lactate to pyruvate during the oxidation of ethanol. (T. Sugano et al. (1986) Amer. J. Physiol. 251, E385-E392). Asparagine (0.5 mM), but not alanine (0.5 mM) decreased the ethanol-induced responses. Norepinephrine and vasopressin had no effect on the ethanol-induced responses when the liver was perfused with sorbitol or glycerol. In the presence of 0.25 mM alanine, norepinephrine, vasopressin, and A23187 decreased the ethanol-induced responses that occurred with the increase of flux of Ca2+. In liver perfused with Ca2+-free medium, asparagine also decreased the ethanol-induced responses, but norepinephrine and vasopressin had no effect. Aminooxyacetate inhibited the effects of norepinephrine, A23187, and asparagine. Regardless of the presence or absence of perfusate Ca2+, the combination of glucagon and alanine had no effect on the ethanol-induced responses. Norepinephrine caused a decrease in levels of alpha-ketoglutarate, aspartate, and glutamate in hepatocytes incubated with Ca2+. The present data suggest that the redistribution of cellular Ca2+ may activate the efflux of aspartate from mitochondria in rat liver, resulting in an increase in the capacity of the malate-aspartate shuttle.
The effects of calmodulin antagonists on the capacity of hydrogen-translocating shuttles were studied in the perfused rat liver. The capacity was estimated by measuring the changes in the rate of production of glucose from sorbitol during the oxidation of ethanol [T. Sugano, T. Ohta, A. Tarui, and Y. Miyamae. Am. J. Physiol. 251 (Endocrinol. Metab. 14): E385-E392, 1986]. Thyroxine given to intact rats increased the activity of alpha-glycerophosphate dehydrogenase (alpha-GPD). Glucocorticoid replacement in adrenalectomized rats decreased the activity of the alpha-GPD to values obtained after treatment with PTU. In either thyroxine-treated or steroid-replaced rats, the capacity of hydrogen-translocating shuttles increased markedly. However, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine inhibited the increased capacity in steroid-replaced rats and had no effect on the increased capacity in thyroxine-treated rats. W-7 inhibited the stimulatory effects of norepinephrine on the capacity of the malate-aspartate shuttle without inhibition of efflux of intracellular Ca2+. The stimulatory effects of vasopressin on the malate-aspartate shuttle were also inhibited by W-7, trifluoperazine, and chlorpromazine. The results suggest that the malate-aspartate shuttle may be regulated by Ca(2+)-calmodulin.