This study provides explanation for conflicting evidence in the literature relating to changes in mitochondrial function and metabolic parameters during chemically induced diabetes. Diabetes of 3 days' duration (early ketosis) did not alter heart, kidney, or liver mitochondrial respiratory rates with glutamate or succinate even though serum glucose and triglycerides were elevated. Diabetes of 5 weeks' duration did not alter kidney or liver mitochondrial function in thefed adult rat although weight gain was depressed. The amount of kidney mitochondrial protein isolated per gram of tissue was increased by 30% in the diabetic. This increase was reversed by insulin treatment as were the other biochemical modalities measured. Superimposition of a 24hr fast resulted in enhanced gluconeogenesis as measured by an animal weight loss of 17% within 24 hr (liver weight loss, 21%) and an elevation of serum urea nitrogen by 180% compared to fasted control. Respiratory rates of diabetic kidney mitochondria with glutamate were unaffected in the fasted animal whereas diabetic liver mitochondrial respiratory rates during succinate oxidation were reduced by 43%. Respiratory control was unchanged in the fasted diabetic rat. All the observed changes were reversed by insulin. Variation in the serum and liver metabolic indices (urea nitrogen, creatinine, glycerol, free fatty acids, free amino acids, triglycerides, and glucose) and liver mitochondrial responses to 7 weeks of chemically induced diabetes was affected by the rat strain, Sprague-Dawley versus Sherman, and rat weight, 72 g versus 222 g. Liver mitochondrial respirations in fed Sherman rats were not depressed by diabetes, Both rat strains had elevated liver free fatty acids and glutamate dehydrogenase activity in the diabetic state. Serum leucine, isoleucine, and valine were more elevated and serum lysine and arginine were more depressed in the diabetic Sprague-Dawley rat than in the Sherman rat. Conjectures on these results are presented in the text. 0

Recently, we have examined organ differences in mitochondrial capacities for conversion of reducing equivalents to phosphate bond energy potential and have related these differences to lipophilic properties of the mitochondrial inner membrane (1). The lipophilic (hydrophobic) nature of NADH dehydrogenase and succinate dehydrogenase sites differed in mitochondria from heart, spleen, liver, kidney and brain. These differences were not correlated with the embryonic origin of the tissues.In this communication, we wish to extend our work to the morphologically distinct tissues within one organ, the kidney. Variation in respiratory control as well as in NADH dehydrogenase lipophilicities were monitored for mitochondria isolated from renal cortex, red medulla and white medulla tissues. Variation in mitochondrial function might reflect the reported variations in the capacities of these tissues for protein and DNA syntheses (2, 3). We have already shown that inhibition of protein synthesis in rat liver by ethidium bromide was in part due to inhibition of phosphorylating oxidation in the mitochondria (4). Materials and methods.Renal mitochondria were prepared from male Sprague-Dawley rats as described previously (1). A Stadie-Riggs apparatus having 0.5 mm clearance was used to prepare slices which were dissected into cortex, red medulla, and white medulla (2). Respiratory rates, respiratory control ratios (RCR) and mitochondrial protein were determined as previously reported (1). Tetrabutylammonium bromide was a product a Eastman Organic Chemicals. Substrates and other biochemicals were purchased from Sigma Chemical Company.Results. The yields of mitochondrial protein obtained from each dissected renal tissue were 12.6 mg per g of decapsulated intact kidney, 12.2 mg/g of cortex, 8.5 mg/g of red medulla, and 1.3 mg/g of white medulla. Electron micrographs (Fig. 1) showed relatively few mitochondria in the isolated pellet of white medulla mitochondria (inset 6) and in the tissue slice of white medulla (inset 5 ) from the decapsulated kidney. Considerably more red medulla and cortical mitochondria were found in either the pellet preparations (insets 2 and 4, respectively) or tissue slices (insets 1 and 3, respectively). Mitochondria isolated from red medulla (inset 2) were somewhat larger and more spherical than those from cortex (inset 4).Variation in respiratory properties were recorded for the mitochondrial preparations in Table I. Cortical mitochondrial respiration was not significantly different from that of the intact kidney. This was not unexpected since the bulk of the kidney was composed of cortical tissue (84%), whereas only 13% was red medulla and 3% was white medulla (2,3). The response of mitochondria from red medulla to stimulation by ADP (state 3 respiration) was about 1.4 times that of organelles from cortex or whole kidney ( P < 0.05). This indicated that the former were more efficient and tightly coupled (higher RCR, P < 0.05).Although only one experiment was conducted with white medulla (kidneys from 28 rat...

Ethidium bromide (2,7-diamino-9-phenylphenanthridinium-1 0-ethyl bromide) inhibits mitochondria1 DNA (1), RNA (2), and protein syntheses (3, 4). Ethidium bromide can intercalate with the nucleic acids (5) and it is commonly thought that inhibition of these processes occurs by this mechanism rather than by inhibiting ATP generation. Ethidium bromide can combine with mitochondrial membranes (6) producing a color shift in the ethidium spectrum which indicates interaction with a low dielectric site on the membrane. This interaction is enhanced by the presence of ATP but not ADP nor AMP (7). Degradation of mitochondrial DNA may imply that energy processes are inhibited by ethidium bromide and ethidium bromide was considered an uncoupler of oxidative phosphorylation (8).Since an intact energy transducing membrane is required to show temperature discontinuities in Arrhenius plots for state 3 and state 4 respirations (9), we have studied the interaction of ethidium bromide on respiratory control in intact mitochondria. This communication presents evidence that ethidium bromide, at mutagenic concentrations of 1 pg/106 liver cells, is a phosphorylation inhibitor and not an uncoupler of mitochondrial respiratory control processes. Exhaustive washing of mitochondria did not remove inhibition caused by ethidium bromide nor did it effect more than 27% removal of bound dye.Materials and Methods. Hepatic mitochondria were prepared from Sprague-Dawley rats as described previously (10). Respiratory rates were determined polarographically (10) and the conventions of Chance and Williams (11) were used to identify respiratory states and in computing respiratory control ratios (RCR). High amplitude volume changes of the organelles (swelling and contraction cycles) and mito-chondrial ATPase (ATP phosphohydrolase, EC 3.6.1.4) activity were monitored and assayed as already described (1 2). Uncoupler stimulated ATPase and energized contraction of the organelles were both inhibited completely by oligomycin (3 and 6 pg/mg mitochondrial protein, respectively). Mitochondrial protein was solubilized with 1 % deoxycholate and estimated by a biuret method (13). Sucrose, EDTA, and sodium deoxycholate were from Fisher Scientific Co. ; mannitol was from Pfanstiehl Laboratories; ethidiilm bromide, ADP, ATP, substrates, and other biochemicals were from Sigma Chemical Co. Ethidium bromide solutions were protected from light at all times.Results. Ethidium bromide depressed Pi acceptor control of respiration in mitochondria oxidizing either glutamate or SUCcinate (Fig. 1). The effect was concentration dependent and resulted from selective inhibition of respiration in the active state (state 3). This inhibition was specifically responsible for complete depression of the RCR to unity; at no time were the organelles released from control by uncoupling, i.e., the resting state respiration (state 4) was unaltered by ethidium bromide throughout its effective concentration range. As in the case of depression of the RCR by a large number of lipophilic organic c...

Induction of hepatic steatosis and suppression of hepatic ATP levels, protein synthesis and gluconeogenesis subsequent to administration of ethionine may be consequences of interference by this compound with mitochondrial phosphorylation of ADP. The mitochondrial dysfunction is not a direct action of ethionine on the organelle.

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