Ceramide is a lipid second messenger that mediates the effects of tumor necrosis factor ␣ and other agents on cell growth and differentiation. Ceramide is believed to act via activation of protein phosphatase, prolinedirected protein kinase, or protein kinase C. Tumor necrosis factor ␣-induced common pathway of apoptosis is associated with an early impairment of mitochondria. Herein, we demonstrate that ceramide can directly inhibit mitochondrial respiratory chain function. In isolated mitochondria, a rapid decline of mitochondrial oxidative phosphorylation occurs in the presence of Nacetylsphingosine (C 2 -ceramide), a synthetic cell-permeable ceramide analog. An investigation of the site of ceramide action revealed that the activity of respiratory chain complex III is reduced by C 2 -ceramide with halfmaximum effect at 5-7 M. In contrast, N-acetylsphinganine (C 2 -dihydroceramide), which lacks a functionally critical double bond and is ineffective in cells, did not alter mitochondrial respiration or complex III activity. We suggest that these in vitro observations may set the stage for identifying a novel mechanism of regulation of mitochondrial function in vivo.The pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) 1 elicits a wide variety of cellular responses including profound alterations in transcriptional programs, perturbation of mitochondrial function, and apoptosis in a number of cell types (1, 2). Strong evidence supports a pivotal role for TNF-␣ in the genesis of septic shock (2), and it also has been implicated in ischemia reperfusion injury of heart (3). A recent report demonstrates that a physiologically relevant concentration of TNF-␣ induced apoptosis in rat cardiomyocytes as quantified by single cell microgel electrophoresis of nuclei and in situ 3Ј nick end labeling assay (4). Mitochondria are considered an early target in TNF-␣-induced cytotoxicity because they appear swollen with a reduced number of cristae, in association with profound inhibition of mitochondrial respiration (1,5,6). A growing body of evidence suggests that treatment of cells with TNF-␣ results in an electron transport inhibition at the level of complex III (1, 6) followed by an increased generation of oxygen radicals in mitochondria (7-9). However, the mechanism of TNF-␣-induced inhibition of mitochondrial respiration has not been elucidated.The sphingomyelin pathway has been implicated as a major signaling mechanism mediating the action of a number of extracellular agents (such as TNF-␣, Fas ligands, and chemotherapeutic agents) causing the activation of sphingomyelinases that cleave membrane sphingomyelin resulting in the formation of ceramide (10, 11). Synthetic cell-permeable ceramide analogs have been shown to mimic many TNF-␣-induced cell responses (10 -13). In malignant and nonmalignant cell lines, ceramides specifically induce apoptosis that involves activation of interleukin-1␤-converting enzyme-like proteases, whereas closely related dihydro-analogs are inactive (10). The intracellular targets for ceramid...

Fatty acids are the primary fuel for the heart and are ligands for peroxisome proliferator-activated receptors (PPARs), which regulate the expression of genes encoding proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the proapoptotic lipid intermediate ceramide. This study assessed cardiac function, expression of PPAR-regulated genes, and cardiomyocyte apoptosis in rats after 8 wk on either a low-fat diet [normal chow control (NC); 10% fat calories] or high-fat diets composed mainly of either saturated (Sat) or unsaturated fatty acids (Unsat) (60% fat calories) (n = 10/group). The Sat group had lower plasma insulin and leptin concentrations compared with the NC or Unsat groups. Cardiac function and mass and body mass were not different. Cardiac triglyceride content was increased in the Sat and Unsat groups compared with NC (P < 0.05); however, ceramide content was higher in the Sat group compared with the Unsat group (2.9 +/- 0.2 vs. 1.4 +/- 0.2 nmol/g; P < 0.05), whereas the NC group was intermediate (2.3 +/- 0.3 nmol/g). The number of apoptotic myocytes, assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining, was higher in the Sat group compared with the Unsat group (0.28 +/- 0.05 vs. 0.17 +/- 0.04 apoptotic cells/1,000 nuclei; P < 0.04) and was positively correlated to ceramide content (P < 0.02). Both high-fat diets increased the myocardial mRNA expression of the PPAR-regulated genes encoding uncoupling protein-3 and pyruvate dehydrogenase kinase-4, but only the Sat diet upregulated medium-chain acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and expression of PPAR-regulated genes independent of cardiac mass or function.

About a decade ago calcitroic acid was isolated as a major side chain cleaved water-soluble metabolite of 1,25-dihydroxyvitamin D3 [Esvelt, R. P., Schnoes, H. K., & Decula, H. F. (1979) Biochemistry 18, 3977]. Presently, calcitroic acid is being considered as the major excretory form of 1,25-dihydroxyvitamin D3. However, the exact site or sites of calcitroic acid production and the possible side chain modified intermediary metabolites that may be formed during the conversion of 1,25-dihydroxyvitamin D3 into calcitroic acid are not fully understood. In the mean time there have been many advances in our understanding of the side-chain metabolism of 1,25-dihydroxyvitamin D3. It is now well established that both the kidney and the intestine metabolize 1,25-dihydroxyvitamin D3 through the C-24 oxidation pathway according to the following steps: 1,25-dihydroxyvitamin D3----1,24,25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3-----1,23,25-trihydroxy-24-oxovitamin D3. Recently, we identified 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-23 alcohol) as a major side chain cleaved lipid-soluble metabolite of 1,25-dihydroxyvitamin D3 and further extended the aforementioned C-24 oxidation pathway in the kidney by demonstrating 1,23,25-trihydroxy-24-oxovitamin D3 as the precursor of C-23 alcohol [Reddy, G. S., Tserng, K. Y., Thomas, B. R., Dayal, R., & Norman, A. W. (1987) Biochemistry 26, 324]. In this present study, we investigated the metabolic fate of 1,25-dihydroxyvitamin D3 (3 X 10(-10) M) in the perfused rat kidney and identified calcitroic acid as the major water-soluble metabolite of 1,25-dihydroxyvitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

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Intracardiac accumulation of lipid and related intermediates (e.g., ceramide) is associated with cardiac dysfunction and may contribute to the progression of heart failure (HF). Overexpression of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) increases intramyocellular ceramide and left ventricular (LV) dysfunction. We tested the hypothesis that activation of fatty acid metabolism with fat feeding or a PPARalpha agonist increases myocardial triglyceride and/or ceramide and exacerbates LV dysfunction in HF. Rats with infarct-induced HF (n = 38) or sham-operated rats (n = 10) were either untreated (INF, n = 10), fed a high-fat diet (45% kcal fat, INF + Fat, n = 15), or fed the PPARalpha agonist fenofibrate (150 mg.kg(-1).day(-1), INF + Feno, n = 13) for 12 wk. LV ejection fraction was significantly reduced with HF (49 +/- 6%) compared with sham operated (86 +/- 2%) with no significant differences in ejection fraction (or other functional or hemodynamic measures) among the three infarcted groups. Treatment with the PPARalpha agonist resulted in LV hypertrophy (24% increase in LV/body mass ratio) and induced mRNAs encoding for PPARalpha-regulated genes, as well as protein expression and activity of medium chain acyl-CoA dehydrogenase (compared with INF and INF + Fat groups). Myocardial ceramide content was elevated in the INF group compared with sham-operated rats, with no further change in the INF + Fat or INF + Feno groups. Myocardial triglyceride was unaffected by infarction but increased in the INF + Fat group. In conclusion, LV dysfunction and dilation are not worsened despite upregulation of the fatty acid metabolic pathway and LV hypertrophy or accumulation of myocardial triglyceride in the rat infarct model of HF.

November 17, 2006; doi:10.1152/ajpheart.01021.2006.-Lipid accumulation in nonadipose tissue due to enhanced circulating fatty acids may play a role in the pathophysiology of heart failure, obesity, and diabetes. Accumulation of myocardial lipids and related intermediates, e.g., ceramide, is associated with decreased contractile function, mitochondrial oxidative phosphorylation, and electron transport chain (ETC) complex activities. We tested the hypothesis that the progression of heart failure would be exacerbated by elevated myocardial lipids and an associated ceramide-induced inhibition of mitochondrial oxidative phosphorylation and ETC complex activities. Heart failure (HF) was induced by coronary artery ligation. Rats were then randomly assigned to either a normal (10% kcal from fat; HF, n ϭ 8) or high saturated fat diet (60% kcal from saturated fat; HF ϩ Sat, n ϭ 7). Sham-operated animals (sham; n ϭ 8) were fed a normal diet. Eight weeks postligation, left ventricular (LV) function was assessed by echocardiography and catheterization. Subsarcolemmal and interfibrillar mitochondria were isolated from the LV. Heart failure resulted in impaired LV contractile function [decreased percent fractional shortening and peak rate of LV pressure rise and fall (ϮdP/dt)] and remodeling (increased end-diastolic and end-systolic dimensions) in HF compared with sham. No further progression of LV dysfunction was evident in HF ϩ Sat. Mitochondrial state 3 respiration was increased in HF ϩ Sat compared with HF despite elevated myocardial ceramide. Activities of ETC complexes II and IV were elevated in HF ϩ Sat compared with HF and sham. High saturated fat feeding following coronary artery ligation was associated with increased oxidative phosphorylation and ETC complex activities and did not adversely affect LV contractile function or remodeling, despite elevations in myocardial ceramide. oxidative phosphorylation; electron transport chain; ceramide; lipotoxicity FATTY ACIDS (FA) are the dominant energy source for the adult mammalian heart and also are utilized for membrane biosynthesis, generation of lipid signaling molecules, posttranslational protein modification, and transcriptional regulation (43). Chronic exposure to FA can result in an imbalance between FA uptake and utilization that potentially can trigger cytotoxic mechanisms, leading to cell dysfunction or death, a phenomenon known as lipotoxicity. Extensive clinical and animal studies have shown that excess lipid accumulation in nonadipose tissue due to enhanced circulating FA may play an important role in pathophysiological conditions such as heart failure, obesity, insulin resistance, and diabetes (15,43,58).A loss of synchronization between FA availability and utilization in cardiomyocytes, despite otherwise normal or upregulated ␤-oxidation capacity, can lead to an increase in the accumulation of tissue ceramide (24). Ceramide, a lipid signaling molecule, has been implicated in the formation of reactive oxygen species and peroxidation of membrane lipids (11), a...

To estimate "true" rate of glucose production, as well as glucose carbon recycling in humans, a mass spectrometric method with [U-13C]glucose as tracer was developed. Because the opportunity for the uniformly labeled glucose, [13C6]glucose, to recycle back as [13C6]glucose is negligible, the true glucose production rate was obtained by measuring the plasma isotope enrichment of [13C6]glucose with chemical ionization mass spectrometry. In contrast, when the isotopic enrichment of C-1 of glucose was measured by enzymatic decarboxylation and ratio mass spectrometry, the "apparent" glucose production including carbon recycling was obtained. The difference between the two rates was the extent of glucose carbon recycling. [U-13C]glucose was administered as primed constant-rate infusion to six normal healthy adult subjects. Rates of glucose turnover were calculated from tracer dilution by applying steady-state kinetics. The true rate of glucose production estimated by the present method was 2.02 +/- 0.19 mg X kg-1 X min-1 (mean +/- SD). After an overnight fast, glucose carbon recycling contributed from 3 to 26% of the total glucose production rate. Use of stable isotopes and mass spectrometric technique permits estimation of important parameters of glucose metabolism without resorting to multiple tracers and at the same time preventing the risk of radiation.

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