Myocardial metabolism of pantothenic acid in chronically diabetic rats

Transport and metabolism of [3H]pantothenic acid ([3H]Pa) was investigated in hearts from control and streptozotocin-induced diabetic rats. In isolated perfused hearts from control animals, the transport of [3H]Pa was linear over 3 h of perfusion when 11 mM glucose was the only exogenous substrate. The in vitro transport of [3H]Pa by hearts from 48-h diabetic rats was reduced by 65% compared to controls and was linear over 2 h of perfusion with no further accumulation of Pa during the third hour. The defect in transport observed in vitro could be corrected by in vivo treatment with 4 U Lente insulin/day for 2 days. In vitro addition of insulin in the presence of 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate had no effect on [3H]Pa transport in hearts from 48-h diabetic rats during 3 h of perfusion. Accumulation of [3H]Pa was not inhibited by inclusion of 0.7 mM amino acids, 1 mM carnitine, 50 microM mersalic acid or 1 mM panthenol, pantoyllactone or pantoyltaurine. Uptake was inhibited by 1 mM nonanoic, octanoic or heptanoic acid, 0.1 mM biotin or 0.25 mM probenecid, suggesting a requirement for the terminal carboxyl group for transport. Transport of pantothenic acid was reduced in hearts from diabetic rats within 24 h of injection of streptozotocin. In vitro accumulation of [3H]Pa decreased to 10% of control 1 week after streptozotocin injection and then remained at 30% of the control value over 10 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of pantothenic acid in hearts of diabetic rats

The metabolism of pantothenic acid (Pa) by cardiac muscle was studied in normal and diabetic rats. Tissue levels of Coenzyme A (CoA) are elevated in the heart during early (6 to 12 h) diabetes, remains at a high level for several days, and then returns to normal or below normal levels. The increase in total tissue CoA mainly occurs in myocytes as indicated by isolation of cardiac myocytes from control and diabetic animals and measuring their content of CoA. The CoA concentration increased from 37 to 93 microM in the cytosolic compartment and from 2.0 to 2.6 mM in the mitochondrial matrix. These effects of diabetes were reversed by insulin treatment. CoA synthesis in hearts removed from control rats and perfused in vitro was stimulated by including in the perfusate Pa, cysteine and dithiothreitol, but no exogenous energy substrate. This stimulated in vitro rate of CoA synthesis was reduced in hearts removed from diabetic animals, and the reduction increased with duration of diabetes. The reduced rate in diabetic hearts resulted from both a decreased rate of Pa phosphorylation and decreased Pa transport. Transport of Pa into myocytes was decreased by as much as 80% in hearts from diabetic animals. The low transport rate was due to a decrease in Vmax with no apparent change in Km. Treatment of the isolated heart with insulin did not correct the diabetic-induced reduction in Pa transport. The transport rate in normal and diabetic hearts was not influenced by the type of energy substrate provided to the heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase.

We tested 42 tetrapeptides for their ability to bind to the rat brain p21ras protein farnesyltransferase as estimated by their ability to compete with p21Ha-ras in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-A1-A2-X, where positions A1 and A2 are occupied by aliphatic amino acids and position X is occupied by a COOH-terminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the A1 position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CAIL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein gamma subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21Ki-rasB was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltransferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.     

Acute encephalopathy with hepatic steatosis induced by pantothenic acid antagonist, calcium hopantenate, in dogs

In Japan, acute encephalopathy with hepatic steatosis resembling Reye's syndrome has been reported to occur after treatment with the pantothenic acid antagonist, calcium hopantenate. We studied the causal relationship and the pathogenesis in dogs. The agent was administered to seven dogs at increasing doses over a period of 8 weeks. Anorexia, vomiting, and diarrhea were common clinical findings. In four dogs, coma suddenly developed after the appearance of gastrointestinal signs. Three animals died during periods when they were not under direct observation. The effects of the agent appear to be related to dose. Laboratory findings representing significant changes at the time of coma included hypoglycemia, leukocytosis, hyperammonemia, hyperlactatemia, and elevated levels of serum transaminases. Microvesicular hepatic steatosis and mitochondrial abnormalities were consistent pathological findings. The hepatic mitochondria were enlarged and characterized by an increased number of cristae and the presence of crystalloid inclusions. In a second group of four dogs, pantothenic acid was given in addition to and in the same amount as calcium hopantenate at increasing doses over a period of 8 weeks. All four dogs survived the 8 weeks and only one developed mild anorexia. No significant biochemical changes were found and neither hepatic steatosis nor mitochondrial abnormalities were observed. The addition of pantothenic acid prevented the development of the disorder in the four animals. These results show that calcium hopantenate produces acute encephalopathy with hepatic steatosis in dogs, by inducing a deficiency of pantothenic acid. The hepatic mitochondrial changes of this reaction differ from those of Reye's syndrome.     

The critical time window for androgen-dependent development of the Wolffian duct in the rat

Androgens are thought to separately regulate stabilization and differentiation of the Wolffian duct (WD), but the time windows for these effects are unclear. To address this, fetal rats were exposed to flutamide within either an early window (EW) [embryonic day 15.5 (E15.5) to E17.5], when the WD degenerates in the female, or a later window (LW) (E19.5-E21.5), when the WD morphologically differentiates in the male, or during the full window of WD development (FW) (E15.5-21.5). WDs were examined for abnormalities during fetal (E21.5) or postnatal life, and anogenital distance and prostate presence/absence were recorded. Exposure to FW- or EW-flutamide, but not to LW-flutamide, induced comparable abnormalities in the fetal WD at E21.5, namely reduced WD coiling, reduced cell proliferation, reduced epithelial cell height, altered epithelial vimentin expression, and reduced expression of smooth muscle actin in the WD inner stroma. Exposure to EW- or FW-flutamide, but not to LW-flutamide, resulted in incomplete/absent WDs in more than 50% of males by adulthood, although such abnormalities were infrequent in fetal life. These findings suggest that androgen action during the EW is sufficient to promote WD morphological differentiation several days later. Because the androgen receptor is expressed in the WD stroma but not in the epithelium during this EW, WD differentiation is likely to be dependent on androgen-mediated signaling from the stroma to the epithelium. In conclusion, the critical window for androgen action in regulating WD development in the rat is between E15.5 and E17.5. This window is also important for prostate formation and anogenital distance masculinization.     

Further comments on the ascorbic acid requirement.

Recommended Daily Allowances (US RDA) of the Food and Drug Administration for ascorbic acid are higher than Recommended Dietary Allowances (set by the Food and Nutrition Board) for adults. There is a 6-fold margin between the requirement to prevent scurvy and the US RDA. The high requirement reported for the rhesus monkey may be needed to compensate for oxidative catabolism of ascorbic acid in this species. The rate of production of ascorbic acid, in mammals that synthesize it has been listed as 3-19 g/70 kg per day. If this high rate of synthesis represents the requirement of such animals, mutations that caused a loss of ascorbic-acid-synthesizing ability would be eliminated by natural selection on diets that failed to supply these large quantities. The loss of ascorbic-acid-synthesizing ability by human beings could indicate a low requirement, which has enabled our species to spread to regions of the earth where dietary sources of ascorbic acid are poor.     

The site-specific delivery of ursodeoxycholic acid to the rat colon by sulfate conjugation

Because ursodeoxycholate has been shown to act as a tumor-suppressive agent in the colon, the absorption and metabolism of its sulfate conjugates were examined in rats to show that sulfation would facilitate the site-specific delivery of ursodeoxycholate to the colon. Bile acids were measured in intestinal contents, feces, urine, plasma, and liver tissue after oral administration of ursodeoxycholate and its C-3, C-7, and C-3,7 sulfate derivatives. Ursodeoxycholate was found in the jejunum after administration of all bile acids, but the mass was greatest for ursodeoxycholic acid administration. In the colon, lithocholic acid, normally found in negligible amounts, became the major bile acid after ursodeoxycholate administration. In contrast, reductions in mass and proportions of lithocholate and deoxycholate occurred after administering the C-7 sulfates. The fecal lithocholate/deoxycholate ratio, a risk marker for colon cancer, increased markedly after administration of ursodeoxycholate and its C-3 sulfate, but did not change after administering the C-7 sulfates. Unlike ursodeoxycholate or its C-3 sulfate, which increased liver concentrations of lithocholate and ursodeoxycholate, the C-7 sulfates had the opposite effect, which was consistent with poor absorption. Sulfation of ursodeoxycholate, specifically at the C-7 position, protects the molecule from bacterial degradation and inhibits its intestinal absorption, thereby facilitating delivery to the colon.     

The endomembrane requirement for cell surface repair

The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca(2+)-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.     

Thyroid hormone transport by the rat fatty acid translocase

We examined the hypothesis that rat fatty acid translocase (rFAT) mediates the cellular uptake of T(3) and other iodothyronines. Uninjected Xenopus laevis oocytes and oocytes injected 4 d previously with rFAT cRNA were incubated for 60 min at 25 C in medium containing 0.01-10 micro M [(125)I]T(3) and 0.1% BSA, or 1-100 micro M [(3)H]oleic acid and 0.5% BSA. Injection of rFAT cRNA resulted in a 1.9-fold increase in uptake of T(3) (10 nM) and a 1.4-fold increase in uptake of oleic acid (100 micro M). Total T(3) uptake was lower in the presence than in the absence of BSA, but relative to the free T(3) concentration, uptake was increased by BSA. The fold induction of T(3) uptake by rFAT was not influenced by BSA. By analyzing uptake as a function of the ligand concentration, we estimated a K(m) value of 3.6 micro M for (total) T(3) and 56 micro M for (total) oleic acid. In addition to T(3), rFAT mediates the uptake of T(4), rT(3), 3,3'-diiodothyronine, and T(3) sulfate. The injection of human type III deiodinase cRNA with or without rFAT cRNA resulted in the complete deiodination of T(3) taken up by the oocytes, indicating that T(3) is indeed transported to the cytoplasm. In conclusion, our results demonstrate transport of T(3) and other iodothyronines by rFAT.     

The metabolism of oleic acid by the perfused rat liver in experimental diabetes induced by antiinsulin serum

The metabolism of varying quantities of oleic acid was examined in isolated perfused livers from normal fed rats and from animals made diabetic by pretreatment with guinea pig antiinsulin serum (AIS). The data presented reemphasize the fact that the quantity of free fatty acid (FFA) coming to the liver is a necessary, but not the most important, factor affecting the subsequent metabolism of the FFA. Rates of ketogenesis and output of triglyceride and the terminal concentration of hepatic triglyceride were proportional to uptake of FFA in certain concentration ranges. For equal rates of uptake of FFA, ketogenesis was greater, and the quantity of triglyceride secreted or accumulated within the liver was less, with livers from diabetic animals than with livers from normal animals. In confirmation of previous data, the liver was observed to have a maximal capacity to secrete triglyceride. Triglyceride accumulated in livers from normal-fed and diabetic animals only when uptake of FFA was more than sufficient to saturate the secretory process. Since proportionately more FFA was catabolized by livers from AIS treated animals, greater uptake of FFA was required to produce maximal rates of output of triglyceride and accumulation in livers from diabetic than from normal animals. Rates of ketogenesis by livers from normal fed animals increased minimally with increasing uptake of FFA (up to 1.0 mM free fatty acid). Even when uptake increased considerably with FFA concentrations of approximately 2.5 mM, rates of ketogenesis by livers from normal animals were less than half those of livers from diabetic rats, and maximal rates were not achieved by the normal controls. It is evident that changes in hepatic metabolism of FFA in the intact diabetic animal result from simultaneous alterations of supply of FFA and hormonally induced metabolic changes in the liver. Moreover, although hepatic secretion and accumulation of triglyceride is greater in isolated perfused livers from normal rats than from diabetic animals when the livers are exposed to equal quantities of FFA, the diabetic livers can accumulate more triglyceride, secrete more triglyceride, and oxidize more FFA to ketone bodies than can the normal under conditions in which considerably more substrate is available to the diabetic rather than to the normal livers. These differences might also be expected to occur in the acutely insulin deficient intact animal, in which changes in hormonal status and substrate (FFA) availability occur simultaneously, and might, in part, explain the ketonemia, hypertriglyceridemia, and hepatic steatosis often observed in vivo.