Richard B. Tobin

Thyroxine-induced changes in rat liver mitochondrial cytochromes☆

The effects of 10 days of thyroxine injection (15 micrograms/100 g body weight) on rat liver mitochondrial cytochrome concentration and on the percent reduction of the individual cytochromes during succinate-driven state III and IV respiration was spectrophotometrically determined at cytochrome-specific wave-length pairs. The concentrations of cytochromes b, c, total c (c + c1) and a a3 increased in hyperthyroid rats. The concentration of cytochrome c1 remained constant in euthyroid and hyperthyroid rats. Changes in the concentration of the membrane-bound cytochromes were also determined by difference spectra in cytochrome c-depleted mitochondrial membranes. Cytochromes b and a a3 showed increased concentrations in hyperthyroid rats while the concentration of cytochrome c1 remained unchanged. Hyperthyroid mitochondria showed increased reduction of cytochromes b, c1, c and total c during state III respiration and cytochromes c1, c, and total c during state IV respiration. The percent reduction of cytochrome b decreased during state IV respiration in the hyperthyroid mitochondria. These results suggest that the increase in respiration observed in the hyperthyroid state may be related to changes both in the mitochondrial cytochrome concentration and in the cytochrome reduction level.

Free radical theory of aging: Effects of antioxidants on mitochondrial function

Mitochondria would be expected to be particularly subject to free redical-induced changes since most of the O2 utilized by mammals takes place in them. Attempts to decrease the rate of production of mitochondrial damage may be thwarted because of the highly selective permeability of the inner membrane and/or adverse effects of anti-oxidants on mitochondrial function. The purpose of this brief study was to examine the effects of six antioxldants — butylated hydroxytoluene (BHT), ethoxyquin (EQ) (santoquin), 2-mercapto-ethylemine (2-MEA), cystamine — the disulfide of 2-MEA, cyeteine and sodium ascorbate — on the function of isolated liver mitochondria.The two lipid soluble antloxldente — BHT and EQ, in contrast to the water-soluble ones — had marked deleterious effects on mitochondrial function, including State III, State IV, and uncoupled resplretlon. All the free radical inhibitors either prevented the determination of the P:O ratio or decreased the ratio by about 15 percent.Taken as a whole, the data suggest that the failure of antioxidants to increase the maximum life span of mice may be largely due to failure of the compounds to decrease the mitochondrial aging rate at concentrations below those which interfere with mitochondrial function.

Oxidative phosphorylation and respiration by rat liver mitochondria from aspirin-treated rats

Respiration and oxidative phosphorylation by liver mitochondria isolated from rats fed 0.2% dietary aspirin for 31 days were studied polarographically with pyruvate, α-ketoglutarate and succinate as substrates. There was an increase in the state 3 respiration with pyruvate (50 per cent), α-ketoglutarate (38 per cent) and succinate (20 per cent). A significant (P < 0.05) increase in state 4 respiration with all three substrates was found. The ADP/O ratios were significantly decreased with pyruvate (P < 0.025), α-ketoglutarate (P < 0.005) and succinate (P < 0.01). There were no changes in the mitochondrial ATPase activity. Addition of aspirin in vitro to isolated mitochondria decreased state 3 respiration with pyruvate (P < 0.05), α-ketoglutarate (P < 0.05) and succinate (P < 0.01). The state 4 respiration was significantly (P < 0.025 to P < 0.001) increased with all three substrates. The oxidative phosphorylation was decreased with all substrates. It is suggested that, in vivo, the uncoupling by aspirin was not due to an increase in the ATPase activity.

Oxidation and Phosphorylation in Liver Mitochondria from Alloxan and Streptozotocin Diabetic Rats

Summary Respiratory activities were determined for liver mitochondria from rats with chronic alloxan and streptozotocin diabetes. Only those rats with blood glucose levels above 300 mg %, 30 days after injection of the diabetogenic substances were used. Both alloxan and streptozotocin diabetic mitochondria showed increased rates of respiration with a-oxoglutarate and L-malate + pyruvate as substrates but the P:O ratios were not altered. Administration of alloxan (200 mg/kg) to chronically diabetic rats 60 hr prior to killing did not cause further alterations in mitochondrial function which would have been indicative of direct mitochondrial poisoning by alloxan. It can be concluded that altered efficiency of oxidative phosphorylation in the liver mitochondria is not a primary lesion in the biochemistry of chronic diabetes. Poisoning of liver mitochondria by the direct action of alloxan is not an acceptable explanation for the altered mitochondrial functions previously seen with diabetic liver mitochondria when the alloxan was administered subcutaneously more than 60 hr before rat sacrifice.

The effects of a polychlorinated biphenyl mixture (aroclor 1254) on liver gluconeogenic enzymes of normal and alloxan-diabetic rats

Normal and alloxan-diabetic rats were fed ground Purina Laboratory Chow with or without 500 ppm of Aroclor 1254 (AR) ad lib for 2 weeks. In both normal and diabetic rats, AR administration decreased food consumption, weight gain and blood glucose concentration, and increased liver weight, liver:body weight ratio, total liver lipid, liver protein and malic enzyme (ME) activity. In the normal rat, AR increased the concentrations of acetoacetate and beta-hydroxybutyrate in blood, but in the diabetic rat the concentrations were markedly reduced. AR administration decreased the activity of phosphoenolpyruvate carboxykinase (PEPck) in normal liver and the activities of pyruvate carboxylase (PC), PEPck and glucose-6-phosphatase (G6Pase) in diabetic liver.

Carnitine-14C metabolism in choline-deficient, alloxan-diabetic choline-deficient and insulin-treated rats.

Abstract In the present investigation we have examined the body pool, turnover time, half-life, and tissue levels of carnitine as well as the metabolism of radioactive carnitine in choline-deficient, alloxan-diabetic choline deficient and alloxan-diabetic choline deficient insulin-treated rats. The carnitine body pool in normal rats was 25.6; choline-deficient, 12.6; alloxan-diabetic choline-deficient, 7.7; and alloxan-diabetic choline-deficient insulin-treated rats, 32.7 mg. per 100 Gm. body weight. The gastrocnemius muscle carnitine content was greatly decreased in choline-deficient and alloxan-diabetic choline-deficient rat, whereas the muscle carnitine content in alloxan-diabetic choline-deficient insulin-treated rats approached a value close to that of normal rats. The turnover time was decreased in choline-deficient (5.9 days), alloxan-diabetic (5.1 days), and alloxan-diabetic insulin-treated (9.8 days) from that of normal rats (14.8 days). The results indicate that the decreased body pool of carnitine in choline-deficient and alloxan-diabetic choline-deficient rats is primarily due to a decrease in the turn-over time and a greatly increased metabolism of carnitine.

Effect of polychlorinated biphenyls and thiamin deficiency on liver metabolism in growing rats.

Weanling rats were fed a thiamln‐deficlent diet containing 0, 10, or 500 ppm Aroclor 1254 (AR) ad libitum for 28 days. Control animals without and with AR at both levels received an oral thiamin supplement. Fasting blood glucose levels were determined at weekly intervals. The effects of AR, thiamin deficiency, and their interaction on liver cytosol and mitochondrial redox states and on cytoplasmic phosphorylation state as calculated from metabolite concentrations in freeze‐clamped livers were determined. Liver lipid and various parameters of lipid synthesis, transport, and storage were also studied. A R treatment and thiamin deficiency resulted in enlarged livers, increased liver lipid, increased lipid synthesis, decreased plasma free fatty acids, and decreased carcass lipids. AR treatment and thiamin deficiency increased the oxidized state of the mitrochondrial NAD∗/NADH couples and similarly Increased the cytoplasmic NADP∗/ NADPH couple associated with isocitrate dehydrogenase activity. These effects we...

The effect of polychlorinated biphenyls on liver gluconeogenic enzyme activities in rats during exposure to cold

Abstract Rats were fed diets containing either 0, 75, or 400 ppm Aroclor 1254. after 2 weeks on the diets, 4 animals per group were placed in the cold at 4° for an additional 2 weeks. Rats maintained at 4° gained less weight and had decreased adipose tissue weights as compared with animals at 25°. Ingestion of Aroclor 1254, a polychlorinated biphenyl (PCB), increased liver weights in the cold and at ambient temperature. Blood glucose was also significantly decreased in the cold-exposed rats fed 400 ppm PCB. At both temperatures, PCB decreased the specific activity (μmoles/g liver/min) of phosphoenol-pyruvate carboxykinase (PEPck) and fructose-1,6-diphosphatase (FDPase). Cold exposure significantly incresed the activity of PEPck and glucose-6-phosphatase (G-6-Pase) in rats fed PCB. When the activities were expressed as total μmoles/100 g body weight/min to correct for PCB-induced hepatomegaly, no significant differences were observed in the key gluconeogenic enzymes for PCB-fed rats at 25°. At 4°, FDPase and G-6-Pase activities were significantly increased with PCB feeding. Malic enzyme was significantly increased with PCB at both temperatures, regardless of the method of calculation. PCB ingestion had no effect on mitochondrial pyruvate metabolism. These studies indicate that ingestion of PCB (as Aroclor 1254) does not alter the response of the gluconeogenic enzymes to cold exposure.

Studies on the metabolism of glycerol by hyperlipemic and normolipemic rats.

Summary The metabolism of glycerol has been studied in BHE and Wistar rats using measures of transhepatic glycerol disposal, assays of glycerol and glycerol metabolites in frozen clamped hepatic tissue, and through the study of rnitochon-drial α-glycerophosphate shuttle activity and α-glycerophosphate dehydrogenase activity. Strain differences in glycerol metabolism were observed, with the BHE rats having higher serum glycerol levels, Iower presumed glycerolkinase activity, lower α-glycerophosphate shuttle activity, and lower mitochondrial α-glycerophosphate dehydrogenase activity. These findings were presumed to indicate a lack of coordination between the cellular compartments, explaining the previously reported enhanced hepatic lipogenesis seen in BHE rats.

Mode of action of 5,5'-diphenylthiohydantoin-sites of action of 5,5'-diphenylthiohydantoin in mitochondria in relationship to thyroxine-stimulated responses.

Abstract The effects of 5,5-diphenylthiohy dantoin (DPTH) on mitochondria were examined in vitro and in vivo on well-delineated sites known to be profoundly affected by thyroid hormones, in order to test the hypothesis that thyroid hormones regulate metabolic responses through mitochondrial and cytosolic processes. The effect of methimidazole, another antithyroid drug, was studied in vitro in parallel systems. Addition of DPTH greatly decreased the capacity of mitochondria to synthesize precursors for gluconeogenesis as a result of the inhibition of pyruvate carboxylation. DPTH was found to inhibit α-glycerophosphate dehydrogenase, the enzyme involved in the regulation of hydrogen transfer to cytochromes through the cytosolic and mitochondrial α-glycerophosphate cycle. DPTH also inhibited β-hydroxybutyrate dehydrogenase. The state 3 respiration with pyruvate, α-ketoglutarate, succinate and α-glycerophosphate was inhibited by DPTH. Methimidazole had no effect in blocking the peripheral action of thyroxine and was non-inhibitory in the above systems studied. In vivo , in experiments where DPTH was added to the diets, a large inhibition of liver gluconeogenic enzyme activities, α-glycerophosphate dehydrogenase and oxygen consumption by rat liver tissue slices was observed.