Modified oscillation behavior and decreased D-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria

One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d-3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d-3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa₃ content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b, c₁, and c content, and the P:O ratio for mitochondria oxidizing d-3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d-3-hydroxybutyrate oxidation by intact mitochondria to their normal values.     

Early events in the transport of proteins into mitochondria. Import competition by a mitochondrial presequence.

Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G. (1991) J. Biol. Chem. 266, 21693-21699). Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20. A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2. Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import. Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein. Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition. F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25%. In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25%. Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi. Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions. F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step. Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide. We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.     

Phospholipid and triacylglycerol fatty acid content and pattern in the cardiac endothelium of rats depleted in long-chain polyunsaturated omega 3 fatty acids

Rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3-depleted rats) display several features of the metabolic syndrome including hypertension and cardiac hypertrophy. This coincides with alteration of the cardiac muscle phospholipid and triacylglycerol fatty acid content and/or pattern. In the present study, the latter variables were measured in the cardiac endothelium of normal and omega3-depleted rats. Samples derived from four rats each were obtained from 16 female normal fed rats and three groups of 36-40 female fed omega3-depleted rats each aged 8-9, 15-16 and 22-23 weeks. At comparable mean age, the ratio between the square root of the total fatty acid content of phospholipids and cubic root of the total fatty acid content of triacylglycerols was lower in omega3-depleted rats than in control animals. The total fatty acid content of triacylglycerols was inversely related to their relative content in C20:4omega6. Other differences between omega3-depleted rats and control animals consisted in a lower content of long-chain polyunsaturated omega3 fatty acids in both phospholipids and triacylglycerols, higher content of long-chain polyunsaturated omega6 fatty acids in phospholipids, higher activity of delta9-desaturase (C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios) and elongase [(C16:0 + C16:1omega7)/(C18:0 + C18:1omega9) and C20:4omega6/C22:4omega6 ratios], but impaired generation of C22:6omega3 from C22:5omega3 in the former rats. These findings support the view that cardiovascular perturbations previously documented in the omega3-depleted rats may involve impaired heart endothelial function.     

Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats.

Heart mitochondria from chronically diabetic rats ('diabetic mitochondria'), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats ('normal mitochondria'). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.     

Increased peroxisomal fatty acid β-oxidation and enhanced expression of peroxisome proliferator-activated receptor-α in diabetic rat liver

To determine whether the increased fatty acid -oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR- and its target genes responsible for the metabolism of fatty acids.     

Phospholipid fatty acid pattern and D-glucose metabolism in muscles from omega3 fatty acid-depleted rats

A depletion in long-chain polyunsaturated omega3 fatty acids may affect fuel homeostasis. In such a perspective, the present study deals mainly with the in vitro fate of D-[U-(14)C]glucose in hemidiaphragms, stretched soleus and plantaris muscle pieces obtained from normal and omega3-depleted rats (second generation) and incubated in the absence or presence of insulin. When so required, the omega3-depleted rats were injected 120 min before sacrifice with either a omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (FO) or a control medium-chain triglyceride:olive oil emulsion (OO). The content of the soleus muscle in long-chain polyunsaturated omega3 fatty acids was severely decreased in the omega3-depleted rats, and modestly albeit significantly increased after injection of FO to these animals. In stretched soleus muscles from OO-injected omega3-depleted rats, the absolute values for glycogen synthesis measured in the absence or presence of insulin were about twice higher than in normal animals. In the OO-injected omega3-depleted rats, insulin augmented the output of (14)C-labelled amino acids, whilst such was not the case in normal animals. These and other findings suggest a lower catabolism of D-glucose relative to the anabolic process of glycogen synthesis and a lower availability of endogenous amino acids in the muscles of omega3-depleted rats, as compared to those of control animals. The prior injection of FO to the omega3-depleted rats restored a normal value for the paired ratio between the output of (14)C-labelled amino acids and acidic metabolites, but further increased glycogen net synthesis. It is proposed, therefore, that the perturbation of d-glucose metabolism in muscles from omega3-depleted rats involves a multifactorial determinism, only some of the concerned factors being susceptible to rapid correction after enrichment of cell phospholipids in long-chain polyunsaturated omega3 fatty acids.     

Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes.

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.     

Energy-minimized conformation of gramicidin-like channels. I. Infinitely long poly-(L,D)-alanine beta 6.3-helix.

The energy-minimized conformation of an infinitely long poly-(L,D)-alanine in single-stranded beta 6.3-helix was calculated by the molecular mechanics method. When energy minimization was started from a wide range of initial geometries, six optimized conformations were obtained and identified as the right- and left-handed counterparts of the beta 4.5-, beta 6.3-, and beta 8.2-helices. It was found that their conformation energies increase in this order, the beta 4.5-helix having the lowest energy. The backbone dihedral angles of the energy-minimized beta 6.3-helix were: phi L = -116 degrees (or -131 degrees), psi L = 122 degrees (or 111 degrees), phi D = 131 degrees (or 116 degrees), psi D = -111 degrees (or -122 degrees), omega L = 173 degrees (or 173 degrees), and omega D = -173 degrees (or -173 degrees) for the right-handed (or left-handed) helix. This helix was composed of 6.30 residues/turn with a pitch of 4.97 A. All the alpha-carbons of L- and D-configurations appeared on one common circular helix. Interestingly, small deviations (approximately 7 degrees) of the peptide bonds from the planar structure caused a considerable lowering of the conformation energy, and, at the same time, they produced more favorable fitting of the hydrogen bonds; the carbonyl oxygens and the nearest-neighbor alpha-hydrogens also took more favorable relative positions.     

Regulation of fatty acid biosynthesis as a possible mechanism for the mitoinhibitory effect of fumonisin B1 in primary rat hepatocytes

The mitoinhibitory effect of fumonisin B1 (FB1) on the mitogenic response of epidermal growth factor (EGF) was investigated in primary hepatocyte cultures with respect to the alterations in the omega6 fatty acid metabolic pathway. Fatty acid analyses of hepatocytes showed that EGF treatment resulted in a significant decrease in the relative levels of 20:4omega6 (arachidonic acid) and an increase in 18:2omega6 (linoleic acid). Supplementation of the hepatocyte cultures with 20:4omega6 in the absence of EGF resulted in an increase in the total omega6 and omega6/omega3 fatty acid ratio. Addition of 20:5omega3 (eicosapentaenoic acid) resulted in an increase of the relative levels of the long chain omega3 fatty acids at the expense of the omega6 fatty acids. When 20:4omega6 and 20:5omega3 was added in the presence of EGF, the mitogenic response of EGF was increased and decreased respectively. When compared to the fatty acid profiles in the absence of EGF, the decreased mitogenic response coincided with a decrease of total omega6 fatty acids and total polyunsaturated fatty acids (PUFA). In addition, the saturated and mono-unsaturated fatty acids increased and the polyunsaturated/saturated (P/S) fatty acid ratio decreased which implied a more rigid membrane structure. Addition of prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) stimulated and inhibited the mitogenic response respectively. Ibuprofen, a known cyclooxygenase inhibitor, and FB1 inhibited the EGF-induced mitogenic response in a dose-dependent manner. The mitoinhibitory effect of FB1 on the EGF response was counteracted by the addition of PGE2. FB1 also disrupts the omega6 fatty acid metabolic pathway in primary hepatocytes, resulting in the accumulation of C18:2omega6 in phospatidylcholine and triacylglicerol. The disruption of the omega6 fatty acid metabolic pathway and/or prostaglandin synthesis is likely to be an important event in the mitoinhibitory effect of FB1 on growth factor responses.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology

Chemical shifts of nuclei in or attached to a protein backbone are exquisitely sensitive to their local environment. A computer program, SPARTA, is described that uses this correlation with local structure to predict protein backbone chemical shifts, given an input three-dimensional structure, by searching a newly generated database for triplets of adjacent residues that provide the best match in phi/psi/chi(1 )torsion angles and sequence similarity to the query triplet of interest. The database contains (15)N, (1)H(N), (1)H(alpha), (13)C(alpha), (13)C(beta) and (13)C' chemical shifts for 200 proteins for which a high resolution X-ray (< or =2.4 A) structure is available. The relative importance of the weighting factors for the phi/psi/chi(1) angles and sequence similarity was optimized empirically. The weighted, average secondary shifts of the central residues in the 20 best-matching triplets, after inclusion of nearest neighbor, ring current, and hydrogen bonding effects, are used to predict chemical shifts for the protein of known structure. Validation shows good agreement between the SPARTA-predicted and experimental shifts, with standard deviations of 2.52, 0.51, 0.27, 0.98, 1.07 and 1.08 ppm for (15)N, (1)H(N), (1)H(alpha), (13)C(alpha), (13)C(beta) and (13)C', respectively, including outliers.     

CD4 and CD8 may adopt a similar mode of binding to their MHC ligands.

A theoretical scheme is proposed by which the type-specific cell surface receptors of T-lymphocytes, CD8 and CD4, bind class I and II MHC proteins in a similar manner. The scheme has equivalent residues in the C'/C' loop-C' strand-C'/D loop region in domain 1 of CD4 and CD8 alpha binding to equivalent residues in the C and D beta-strands and C/D loops in HLA-DR beta 2 (class II) and HLA-A2 alpha 3 (class I) respectively through a series of electrostatic, hydrogen and hydrophobic bonds.     

Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin

The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M omega, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K+ concentration of the medium and magnitude of the K+ diffusion potentials was consistent with the presence of a high internal concentration of K+, (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay of membrane leakiness. Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication.     

Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary modulation of retina phospholipids and photoreceptor function with particular reference to phosphatidylinositol

The effect of dietary partially hydrogenated herring oil (HHO) on the fatty acid composition of the phospholipids of rat retina and on the amplitude of the a-wave of the electroretinogram (ERG) was studied in rats raised for several generations on an essential fatty acid poor diet. The most significant effect of the dietary treatment was a decreased content of arachidonic acid (C20:4 omega 6) and an increased concentration of docosahexaenoic acid (C22:6 omega 2) and 18:1 isomers associated with the phosphatidylinositol (PI). The a-wave of the ERG showed a decreasing amplitude during the experiment and was reduced with about 30% (P less than 0.004) of the initial value in all rats at the end of the experiment. The pronounced change in the arachidonic content in PI and the decreased amplitude of the a-wave of the ERG suggest that arachidonic acid of the PI has an important function in the visual phototransduction process.     

Glucose intolerance associated to insulin resistance and increased insulin secretion in rats depleted in long-chain omega3 fatty acids.

An intragastric D-glucose tolerance test was performed, after overnight starvation, in female rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3D rats) and control rats of same age and gender. The plasma D-glucose and insulin concentrations, insulinogenic index, and HOMA for insulin resistance were all higher, after overnight starvation, in omega3D rats than in control animals. Over the 120-minute period following the intragastric administration of D-glucose, the area under the curve for the same four variables was also higher in omega3D rats than in control animals. In addition to visceral obesity, liver steatosis, hypertension, and cardiac hypertrophy, the omega3D rats thus display further features of the metabolic syndrome, namely glucose intolerance and insulin resistance, despite hyperinsulinemia.     

Crystal structure of tert.-butyloxycarbonyl-L-prolyl-D-alanyl-D-alanyl-N-methylamide. Dimeric beta-sheet formation.

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH3, monohydrate, (C17H30N4O5.H2O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are monoclinic, space group P2(1), a = 9.2585(4), b = 9.3541(5), c = 12.4529(4)A, beta = 96.449(3) degrees, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (delta omega = 5.2 degrees and delta omega = 3.7 degrees, respectively). The backbone torsion angles are: phi 1 = -60 degrees, psi 1 = 143.3 degrees, omega 1 = -174.8 degrees, phi 2 = 148.4 degrees, psi 2 = -143.1 degrees, omega 2 = -179.7 degrees, phi 3 = 151.4 degrees, psi 3 = -151.9 degrees, omega 3 = -176.3 degrees. The pyrrolidine ring of the proline residue adopts the C2-C gamma conformation. The molecular packing gives rise to an antiparallel beta-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric beta-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient beta-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH3.     

Effect of dietary lipid and vitamin E on mitochondrial lipid peroxidation and hepatic injury in the bile duct-ligated rat.

To assess whether lipid peroxidation of hepatic mitochondria is associated with cholestatic hepatic injury we examined the effect of bile duct ligation (BDL) versus sham surgery on mitochondrial lipids of rats maintained on one of seven diets. Diets included vitamin E-deficient (E-) and vitamin E-sufficient (E+) combined with normal lipid (11.9% calories as stripped corn oil), high lipid (35% calories as stripped corn oil), or n-3 fatty acid (fish oil) supplementation. Rats were killed 17 days after surgery, mitochondria were isolated by differential centrifugation, and lipid-conjugated dienes and thiobarbituric acid-reacting substances (TBARS) were measured in mitochondrial lipids as indices of lipid peroxidation. BDL resulted in significant increases in lipid peroxidation in all dietary groups. The E- high lipid diets (with either corn oil or fish oil) were associated with higher lipid peroxide and serum bilirubin values in BDL rats compared to the normal lipid diets. Fish oil supplementation did not ameliorate cholestatic or oxidative injury. Serum alanine aminotransferase, bilirubin, alkaline phosphatase, and cholylglycine levels correlated significantly with levels of mitochondrial conjugated dienes and TBARS. These data suggest that free radical stress occurs during BDL in the rat and may result in mitochondrial lipid peroxidation, and that diets high in lipid may increase free radical damage to hepatic mitochondria. The role of free radicals in cholestatic hepatic injury requires further investigation.     

Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria.

1. Evidence is given for three sites of phosphorylation in the alpha-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. 2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1, 2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. 3. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations ranging from 0.15 to 0.4 mM. The results showed that the concentration of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 greater than site 2 greater than site 1. 4. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporation of 32Pi into site 3, but it had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. It is concluded that site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. 5. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.     

Rapid effects of the intravenous injection of a medium-chain triglyceride: fish oil emulsion on the triglyceride fatty acid pattern of soleus muscle from omega3 fatty acid-depleted rats.

Second generation rats depleted in long-chain polyunsaturated omega3 fatty acids display several features of the metabolic syndrome, including visceral obesity, liver steatosis, insulin resistance, hypertension, and cardiac hypertrophy. In the framework of an extensive study on such metabolic, hormonal and functional perturbations, the phospholipid fatty acid pattern and ex vivo metabolism of D-glucose were recently investigated in the soleus muscle of these omega3-depleted rats. The present study deals with the triglyceride fatty acid content and pattern of the soleus muscle in control animals and omega3-depleted rats. Some of the latter rats were injected intravenously 60-120 minutes before sacrifice with either an omega3 fatty acid-rich medium-chain triglyceride/fish oil emulsion (omega3-FO rats) or a control medium-chain triglyceride/olive oil emulsion (omega3-OO rats). The total fatty acid content of triglycerides was comparable in control and omega3-depleted rats and, in both cases, inversely related to their C20:4omega6 relative content. At variance with the situation found in control rats, no long-chain polyunsaturated omega3 fatty acid (C18:3omega3, C20:5omega3, C22:5omega3, C22:6omega3) was detected in the omega3-depleted rats. Unexpectedly, the triglyceride content in most long-chain polyunsaturated omega6 fatty acids (C18:2omega6, C20:3omega6, C20:4omega6 and C22:4omega6) had also decreased in the latter rats. Moreover, the activity of Delta9-desaturase was apparently increased in the omega3-depleted rats, as judged from the C16:1omega7/C16:0 and C18:1omega9/C18:0 ratios. The omega3-FO rats differed from omega3-OO rats by a lower contribution of C18:2omega6 metabolites (C18:3omega6, C20:3omega6, C20:4omega6 and C22:4omega6). These findings indicate that the prior injection of the medium-chain triglyceride/fish oil emulsion, known to increase the muscle phospholipid content in long-chain polyunsaturated omega3 fatty acids, may nevertheless accentuate the depletion in long-chain polyunsaturated omega6 fatty acids otherwise found in the triglycerides of omega3-depleted rats. Such a dual effect is reminiscent of that observed, under the same experimental conditions, for selected variables in D-glucose metabolism in the soleus muscle.