Myron A. Mehlman

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.

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.

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.

Ultrastructural and Biochemical Alterations of Livers from Rats Treated With 5,5-Diphenyl-2-thiohydantoin (DPTH) and Thyroxine

Summary The ultrastructure and chemical composition of rat livers were examined after the animals were fed 5,5-diphenyl-2-thiohydantoin (DPTH) and/or injected with L-thyroxine (T4). Mitochondria from T4-treated rats were more numerous (with respect to normal livers) and were larger with extremely electron-lucent matrices. That glycogen was radically reduced in the livers of T4-treated animals was confirmed by both electron microscopic and biochemical analysis. Cisternae of the RER were usually non-parallel and somewhat dilated. No other ultrastructural changes were easily discernible. Analysis for lipid and RNA content showed small increases on a wet weight basis. DPTH caused significant ultrastructural changes which included proliferation of the SER and the appearance of electron-lucent matrices in mitochondria, absence of mitochondrial granules and, in some cells, appearance of myelin figures. In addition, DPTH was shown to block conversion of acylcarnitines to ketone bodies by mitochondria in vitro and in vivo. The T4-stimulated increase in RNA content was also inhibited by DPTH. Both electron microscopy and biochemical analysis showed that DPTH caused lipid accumulation and prevented the T4-stimulated decrease in glycogen content. The pattern of alterations of the liver as seen by electron microscopy and biochemical analysis correlated well.

Subcellular Distribution of Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Dog Liver and Kidney

Summary The distribution of PC and PEPck in dog liver and kidney cortex has been determined. Kidney PC is totally mitochondrial and liver PC is 60% mitochondrial. Kidney and liver PEPck are also predominately of mitochondrial origin (60-70%), while 15-16% is nuclear, and 25-30% is in the soluble fraction. It is concluded that since this distribution is similar to that found in guinea pig, rabbit, and human, the mitochondrial regulation of gluconeogenic precursor synthesis may also be similar.

The inhibitory effects of acetylsalicylic acid feeding on gluconeogenic enzymes in rat liver and kidney

Abstract Rats fed a diet containing 0.25% acetylsalicylic acid for a period of 5 weeks grew normally and did not exhibit symptoms characteristic of toxicity. Acetylsalicylic acid feeding decreased the concentration of glycogen in liver, the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in liver, and the activity of phosphoenolpyruvate carboxykinase in kidney. The activities of glucose-6-phosphatase and fructose-1,6-diphosphatase in liver and pyruvate carboxylase in kidney were not affected. Intraperitoneal injection of acetylsalicylate (5 mg/100 g body wt.) to control rats and in vitro addition of acetylsalicylate (5mM) to the enzyme assays did not inhibit any of the tested enzymes. The findings suggest that chronic acetylsalicylic acid ingestion may inhibit gluconeogenesis by decreasing the activities of the regulatory enzymes.

The effect of riboflavin deficiency on key gluconeogenic enzyme activities in rat liver.

The sequel of riboflavin deficiency, as it is now known, may be initiated by a selective decrease in the activity of several fiavoproteins involved in cellular oxidation (1–3). This in turn may affect protein turnover and lead to an increased pool of free amino acids (4) which alone, or in conjunction with altered endocrine activity (5), may stimulate the activity of several enzymes involved in amino acid metabolism (6–8). It has been hypothesized that the stimulation of alanine transaminase activity by this mechanism may be responsible for greater fasting liver glycogen levels (6) and in vivo 14C-alanine incorporation into liver glycogen of riboflavin deficient rats than in pair-fed controls (9). However riboflavin deficiency is also associated with a large decrease in mitochondrial respiration and energy production (1). Since these factors would be expected to inhibit several key steps in the gluconeogenic pathway, it was of interest to study some of the key gluconeogenic enzyme activities which are known to be rate limiting under a variety of conditions. These enzymes activities are increased in many situations where gluconeo-genesis is increased such as fasting, diabetes, high fat feeding, and glucocorticoid administration (10–15). Phosphoenolpyrurate carboxykinase (PEPck) activity is especially well correlated with gluconeogenesis (16). The regulation of these enzyme activities is a control system which appears to operate in intact liver under physiologic conditions (16). We sought to determine if the increased glyconeogenesis reported in riboflavin deficient rat liver was associated with an increased activity of one or more of these enzymes. Materials. Animals and Diet. Male Sprague-Dawley weanling rats were used in these studies. Animals weighed 50–60 g at the start of the experiment and these were randomly divided into three initial groups: Group A was fed a commercial riboflavin-deficient diet (Nutritional Biochemicals Co., Cleveland, OH) ad libitum; Group B was pair-fed the same diet supplemented with 30 mg of riboflavin/kg of diet; Group D was fed the control diet ad libitum.

α-Oxoglutarate Carboxylation in Liver Mitochondria from Normal, Alloxan Diabetic and Streptozotocin Diabetic Rats

Summary Rat liver mitochondria, incubated in media containing 4 mM ATP, 10 mM MgCl2, 6.7 mM Pi 13.3 mM TEA, 6.7 mM α-oxoglutarate, and 20 mM HCO3 -, oxidized α-oxoglutarate through the tricarboxylic acid cycle and utilized a portion of the reducing equivalents produced by this oxidation to drive α-oxoglutarate to citrate via reductive carboxylation. Streptozotocin and alloxan diabetes increased the rates of oxidation and citrate accumulation. L-Octanoylcarnitine inhibited both α-oxoglutarate oxidation and carboxylation. Insulin, glucagon, and dibutyryl cyclic AMP, added in vitro, did not affect α-oxoglutarate oxidation or carboxylation.

A comparison of the metabolic effects of bovine growth hormone and growth factor from Spirometra mansonoides on rat liver in vivo.

The effects of bovine growth hormone(GH) and Spirometra growth factor (SGF) on the in vivo concentrationions of glycolytic and citricacid (TCA) cycle intermediates in rat liver were investigated. The effects of these agents on the mitochondrial and cytoplasmic redox states, liver phosphorylation states, and adenylate kinase activity were also evaluated. The effects of SGF were studied in both normal and hypophysectomized rats,wheras GH effects ere examined only in hypophysectomized rats. Growth homrone(0.25mg/rat) was injected dayily for 9 days...