Search

We have previously shown that the activity of several mitochondrial oxidative enzymes increased greatly in the medullary thick ascending limb of Henle's loop (MTAL) of developing rat kidney between 16 days after birth and the adult stage. These changes were triggered by the postnatal rise in circulating glucocorticoids. To determine whether these increases are related to a mitochondrial biogenesis we have studied the changes in mitochondrial density of MTAL cells and mitochondrial DNA content in the inner stripe of the outer medulla during the postnatal period. The activities of respiratory chain (RC) complexes II and IV, located in the inner mitochondrial membrane (IMM), were also assayed, and developmental changes in the ultrastructure of the IMM were quantified. Quantitative electron-microscopic data showed a doubling in mitochondrial density from day 16 to the adult, accompanied by twofold increase in mitochondrial DNA, as determined by slot-blot experiments. The surface density of IMM increased by 77%, and there was a concomitant large rise in the activity of both RC enzymes. A possible role of glucocorticoids on the regulation of mitochondrial biogenesis was examined in similar experiments performed in adrenalectomized rat pups. The data demonstrated that glucocorticoids are essential for the rise in RC enzymes and the development of IMM but do not regulate postnatal changes in mitochondrial density and mitochondrial DNA content. Finally, ontogenic changes in oxidative capacities of MTAL cells could be a critical factor in the development of kidney urine concentrating mechanisms in weanling rats.

This study examines the ability of dietary lipids to regulate gene expression of mitochondrial and peroxisomal fatty acid β-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and evaluates the role of glucagon or of the α-isoform of peroxisome proliferator-activated receptor (PPARα) in mediating β-oxidation enzyme gene regulation in the immature kidney. The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels were found coordinately upregulated in renal cortex, but not in medulla, of pups weaned on a high-fat diet from day 16to21. Further results establish that switching pups from a low- to a high-fat diet for only 1 day was sufficient to induce large increases in cortical LCAD, MCAD, and ACO mRNA levels, and gavage experiments show that this upregulation of β-oxidation gene expression is initiated within 6 h following lipid ingestion. Treatment of pups with clofibrate, a PPARα agonist, demonstrated that PPARα can mediate regulation of cortical β-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically regulate gene expression of mitochondrial and peroxisomal β-oxidation enzymes in the renal cortex of suckling pups, and this might involve PPARα-mediated mechanisms.

We report on the loss of mitochondrial nicotinamide adenine dinucleotides in human cultured cells along with cell culture and acidification of the culture medium. This was established both by the direct measurement of the decrease in the mitochondrial NAD content and by the alteration of the oxidative properties of the mitochondria. In situ, this loss could be reversed in less than 2 h by changing the culture medium or by readjusting the pH of the medium at physiological pH values. By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool. This allowed the restoration of an active NAD+-dependent substrate oxidation. Depletion of mitochondrial NAD in cells grown under quiescent conditions was further confirmed by fluorimetric measurement of mitochondrial NAD, as was the influx of NAD+ into the mitochondrial matrix. These data constitute the first evidence of rapid fluxes of NAD through mitochondrial membranes in animal cells. They also point to the possible confusion between a loss of mitochondrial NAD and a defect of respiratory chain complex I in the context of screening procedures for respiratory chain disorder in human.

The activities of citrate synthase, 3-oxoacid CoA-transferase, and Na/K-ATPase were determined in the proximal convoluted tubules (PCT) of midcortical nephrons from 16-, 21- and 30-day-old and adult rats. Enzyme microassays based on NAD amplification were run on tubule segments microdissected from lyophilized tissue sections, and the activities were expressed per unit of tissue dry weight. The activities of 3-oxoacid CoA-transferase (+ 155%) and citrate synthase (+ 44%) increased between 16 and 30 days, while no significant change in Na/K-ATPase activity occurred during this period. The results obtained in PCT from subcapsular nephrons were similar. It is concluded that active transport of Na+ coupled to mitochondrial ATP production might be mature in the PCT by the time of weaning, consistent with data on the development of Na+ reabsorption. Since adrenalectomy on day 16 induced no changes in the activities of oxidative enzymes or Na/K-ATPase on day 21 in midcortical or subcapsular PCT, the physiological rise in circulating glucocorticoids, characteristic of the weaning period, does not trigger the development of oxidative enzymes and Na/K-ATPase in the PCT of the developing rat kidney.

The oxygen-consumption rates and the activities of fumarase and beta-hydroxyacyl-CoA dehydrogenase were compared in mitochondria isolated from fetal- and neonatal-rat kidney. Whole-organ ATP, phosphocreatine and creatine contents were determined in parallel. Kidney mitochondrial respiratory rates in the presence of succinate, glutamate/malate and palmitoyl-L-carnitine increased between 21 days post coitum and 1 day post partum, together with activities of oxidative enzymes. However, this postnatal maturation of oxidative metabolism was not yet initiated in mitochondria isolated from kidney 1 h post partum. An increase in ATP and phosphocreatine was observed immediately after delivery; newborn-rat kidney ATP content then remained high, whereas phosphocreatine reserves decreased considerably between 6 h and 1 day post partum. It is concluded that the increase in high-energy phosphate compounds observed at birth is not initially related to an activation of oxidative phosphorylation, and probably involves a transient stimulation of anaerobic glycolysis, while a progressive mitochondrial maturation takes place in the rat kidney during the first day of newborn life.

To explore the gene regulatory mechanisms involved in the metabolic control of cardiac fatty acid oxidative flux, the expression of muscle-type carnitine palmitoyltransferase I (M-CPT I) was characterized in primary cardiac myocytes in culture following exposure to the long-chain mono-unsaturated fatty acid, oleate. Oleate induced steady-state levels of M-CPT I mRNA 4.5-fold. The transcription of a plasmid construct containing the human M-CPT I gene promoter region fused to a luciferase gene reporter transfected into cardiac myocytes, was induced over 20-fold by long-chain fatty acid in a concentration-dependent and fatty acyl-chain length-specific manner. The M-CPT I gene promoter fatty acid response element (FARE-1) was localized to a hexameric repeat sequence located between 775 and 763 base pairs upstream of the initiator codon. Cotransfection experiments with expression vectors for the peroxisome proliferator-activated receptor alpha (PPARalpha) demonstrated that FARE-1 is a PPARalpha response element capable of conferring oleate-mediated transcriptional activation to homologous or heterologous promoters. Electrophoretic mobility shift assays demonstrated that PPARalpha bound FARE-1 with the retinoid X receptor alpha. The expression of M-CPT I in hearts of mice null for PPARalpha was approximately 50% lower than levels in wild-type controls. Moreover, a PPARalpha activator did not induce cardiac expression of the M-CPT I gene in the PPARalpha null mice. These results demonstrate that long-chain fatty acids regulate the transcription of a gene encoding a pivotal enzyme in the mitochondrial fatty acid uptake pathway in cardiac myocytes and define a role for PPARalpha in the control of myocardial lipid metabolism.