Effect of the streptozotocin diabetes upon the in vivo conversion of [I-14C] gamma-linolenic and dihomo-gamma-linolenic acids into arachidonic acid in rats (author's transl)]

Streptozotocin diabetic rats were administered the same tracer dose of either [1 14C] gamma-linolenic or [1 14C] dihomo-gamma-linolenic acid by stomach tube seven days after streptozotocin injection. They were killed 48 hours later and the radioactivity in individual fatty acids of the liver determined by radio-gas chromatography. Results were compared to those obtained in non diabetic rats similarly prepared. With either radioactive precursor, 14C radioactivity recovered in hepatic dihomo-gamma-linolenic acid was not significantly affected by the diabetic state. On the other hand, 14C radioactivity incorporated into arachidonic acid was considerably decreased in diabetic rats, compared with normal rats, when the weights of hepatic arachidonic acid were the same in the two groups of animals. These results suggest that streptozotocin diabetes causes a partial inhibition of the 5-desaturation of dihomo-gamma-linolenic acid in rat liver in vivo.     

Role of COX-2 in the enhanced vasoconstrictor effect of arachidonic acid in the diabetic rat kidney

In the rat isolated perfused kidney, arachidonic acid elicits cyclooxygenase-dependent vasoconstriction through activation of PGH2/TxA2 receptors; responses are enhanced in kidneys from diabetic rats. This study examined the roles of cyclooxygenase-1/cyclooxygenase-2 in the enhanced renal vasoconstrictor effect of arachidonic acid in streptozotocin-diabetic rats. Release of 20-HETE was also determined, as this eicosanoid has been reported to elicit cyclooxygenase-dependent vasoconstriction. We confirmed that vasoconstrictor responses to arachidonic acid were enhanced in the diabetic rat kidney associated with a 2-fold-greater increase in the release of 6-ketoPGF1alpha, which was used as an index of cyclooxygenase activity. One and three micrograms of arachidonic acid increased perfusion pressure by 85+/-37 and 186+/-6 mm Hg, respectively, in diabetic rat kidneys compared with 3+/-1 and 17+/-8 mm Hg, respectively, in control rat kidneys. Inhibition of both cyclooxygenase isoforms with indomethacin (10 micromol/L) abolished the vasoconstrictor response to arachidonic acid in both diabetic and control rat kidneys, whereas inhibition of cyclooxygenase-2 with nimesulide (5 micromol/L) reduced perfusion pressure responses to 1 and 3 microg arachidonic acid only in the diabetic rat kidney to 15+/-8 and 108+/-26 mm Hg, respectively, consistent with a 3-fold increase in the renal cortical expression of cyclooxygenase-2. 20-HETE release from the diabetic rat kidney was reduced almost 6-fold and was not increased in response to arachidonic acid. These results demonstrate that the renal vasoconstrictor effect of arachidonic acid is solely dependent on cyclooxygenase activity, with no evidence for a contribution from 20-HETE; in the diabetic rat, cyclooxygenase-2 activity contributes to the renal vasoconstrictor effect of arachidonic acid.     

Vitamin E intake affects serum thromboxane and tissue essential fatty acid composition in the rat

The influence of dietary vitamin E on the composition of essential fatty acids in rat tissue and plasma lipids as well as serum thromboxane B2 was studied. Diets containing deficient (0 mg/kg diet), adequate (100 mg/kg) or supplemental (1,000 mg/kg) vitamin E were fed to young male rats for 10 weeks. The ratio of dihomo-gamma-linolenic acid to arachidonic acid in phospholipids of plasma, liver, and testes was increased in vitamin-E-supplemented rats. Serum thromboxane B2 was increased in vitamin-E-deficient rats. The data support a role for vitamin E in affecting both metabolism of long chain fatty acids, i.e. dihomo-gamma-linolenic acid, and conversion of arachidonic acid to thromboxane A2.     

The relationship between cholesterol absorption and intestinal cholesterol synthesis in the diabetic rat model

The chylomicron remnant particle is thought to be particularly atherogenic and we have previously shown alterations in post-prandial lipoproteins which could contribute to their atherogenicity. Cholesterol metabolism is disturbed in diabetes, yet the effect of diabetes on intestinal cholesterol synthesis and absorption has rarely been investigated. The aim of this study was to examine cholesterol absorption and intestinal synthesis of cholesterol in the streptozotocin diabetic rat. Twelve diabetic rats were paired with 12 control rats. [14C]-Cholesterol emulsion was administered and the lymph duct was canulated. Lymph was collected for 4h. At sacrifice blood was taken for plasma lipoprotein measurements. Chylomicrons were prepared from the lymph by ultracentrifugation and [14C]-cholesterol content was determined by liquid scintillation counting. Lymph apolipoprotein B48 was isolated by gradient gel electrophoresis, and quantified by densitometric scanning. Serum triglyceride and cholesterol were greatly elevated in diabetic compared to control animals (260+/-90 and 9.8+/-8.0 mg/ml vs. 1.0+/-0.4 and 0.6+/-0.3 mg/ml, p < 0.0001 respectively). Lymph chylomicron apo B48 was similar in the two groups. Cholesterol absorption was not significantly different in diabetic compared to control rats but cholesterol synthesis was significantly higher in the diabetic animals (550+/-352 vs. 322+/-113 microg/h p < 0.03). There was a positive correlation between apo B48 and cholesterol absorption (r = 0.70, p < 0.01) in the diabetic rats and control rats (r = 0.71, p < 0.01) but no correlation between apo B48 and cholesterol synthesis in either group. This study demonstrates that cholesterol synthesis was increased in diabetes whereas cholesterol absorption was unaffected suggesting that intestinal cholesterol synthesis made an important contribution to the hypercholesterolaemia seen in the diabetic animals.     

Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period.

At 3-4 degrees C, the transport of 3-O-methyl-D-glucose (30 mM) was severely impaired in islets prepared from adult rats injected with streptozotocin during the neonatal period. However, at 37 degrees C, the first and second phase of glucose-stimulated insulin release were decreased to the same relative extent in perifused islets of diabetic, as compared to control, animals. Moreover, the time-related increase in the oxidative response of the islets to 16.7 mM D-glucose was less pronounced in diabetic than control rats. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in islet homogenates of diabetic rats only represented one-fifth of that found in control rats, whereas the activity of the cytosolic NAD-glycerophosphate dehydrogenase was comparable in both types of rats. This coincided with the fact that a rise in D-glucose concentration from 2.8 to 16.7 mM failed to increase significantly L-[2-3H]glycerol conversion to 3HOH in islets from diabetic rats, in contrast to the situation found in control animals. The activity of 2-ketoglutarate dehydrogenase in islet homogenates when expressed per microgram protein was not different in control and diabetic rats. Likewise, the ratio between D-[6-14C]glucose oxidation and D-[3,4-14C]glucose oxidation and the capacity of either a non-metabolized analog of L-leucine or 3-phenylpyruvate to preferentially stimulated D-[6-14C]glucose oxidation relative to D-[5-3H]glucose utilization were both unaffected in islets from diabetic rats. These findings argue against the existence of a primary defect in the Krebs cycle of diabetic rats. It is proposed that, despite an obvious alteration of the hexose transport system in the islet cells of diabetic rats, the preferential impairment of the B-cell secretory response to D-glucose, as distinct from other secretagogues, in this model of non-insulin-dependent diabetes is mainly attributable to the low activity of FAD-linked glycerophosphate dehydrogenase, resulting in a decreased metabolic flow through the glycerol phosphate shuttle and a reduced rate of aerobic glycolysis.     

Placental transfer of lactate,glucose and 2-deoxyglucose in control and diabetic Wistar rats

Placental transfer of lactate, glucose and 2-deoxyglucose was examined employing the in situ perfused placenta. Control and streptozotocin induced diabetic Wistar rats were infused with [U-14C]-glucose and [3H]-2-deoxyglucose (2DG). The fetal side of the placenta was perfused with a cell free medium and glucose uptake was calculated in the adjacent fetuses. Despite the 5-fold higher maternal plasma glucose concentration in the diabetic dams the calculated fetal glucose metabolic index was not significantly different between the 2 groups. Placental blood flow was reduced in the diabetic animals compared with controls but reduction of transfer of [U-14C]-glucose and [3H]-2-deoxyglucose and endogenously derived [14C]-Lactate to the fetal compartment, could not be accounted for by reduced placental blood flow alone. There was no significant net production or uptake of lactate into the perfusion medium that had perfused the fetal side of the placenta in either group. The plasma lactate levels in the fetuses adjacent to the perfused placenta were found to be higher than in the maternal plasma and significantly higher in the fetuses of the diabetic group compared with control group. In this model the in-situ perfused placenta does not secrete significant quantities of lactate into the fetal compartment in either the control or diabetic group.     

Diabetes causes marked changes in function and metabolism of rat neutrophils

Several studies have shown impairment of neutrophil function, a disorder that contributes to the high incidence of infections in diabetes. Since glucose and glutamine play a key role in neutrophil function, we investigated their metabolism in neutrophils obtained from the peritoneal cavity of streptozotocin-induced diabetic rats. The activities of hexokinase, glucose-6-phosphate dehydrogenase (G6PDH), phosphofructokinase (PFK), citrate synthase, phosphate-dependent glutaminase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were assayed. Glucose, glutamine, lactate, glutamate and aspartate, and the decarboxylation of [U-14C], [1-14C] and [6-14C]glucose; [U-14C]palmitic acid; and [U-14C]glutamine were measured in 1-h incubated neutrophils. Phagocytosis capacity and hydrogen peroxide (H2O2) production were also determined. All measurements were carried out in neutrophils from control, diabetic and insulin-treated (2-4 IU/day) diabetic rats. Phagocytosis and phorbol myristate acetate (PMA)-stimulated H2O2 production were decreased in neutrophils from diabetic rats. The activities of G6PDH and glutaminase were decreased, whereas that of PFK was raised by the diabetic state. The activities of the remaining enzymes were not changed. Diabetes decreased the decarboxylation of [1-14C]glucose and [U-14C]glutamine; however, [6-14C]glucose and [U-14C]palmitic acid decarboxylation was increased. These observations indicate that changes in metabolism may play an important role in the impaired neutrophil function observed in diabetes. The treatment with insulin abolished the changes induced by the diabetic state even with no marked change in glycemia. Therefore, insulin may have a direct effect on neutrophil metabolism and function.     

Branched chain amino acid metabolism in the retina of diabetic rats

Summary Diabetes is known to produce increased levels of the branched chain amino acids in plasma, heart and muscle as well as increased oxidation of [¹⁴C]-leucine by nerves and muscles from rats. Plasma and retinas from streptozotocin diabetic rats had significant elevations in branched chain amino acid levels compared to control. Retinas from diabetic rats have been found to oxidize significantly more of the branched chain amino acids, leucine, isoleucine and valine than did control retinas when incubated in media containing 16.5 mmol/1 glucose. Neither the extracellular space nor the tissue pool of leucine was significantly different in the two groups. The addition of 19 amino acids, at normal plasma concentrations, to the incubation media resulted in 80 percent suppression of leucine oxidation without significant change in incorporation of [¹⁴C] into protein. These results suggest that the major role for the branched chain amino acids in the rat retina is in protein synthesis which is not affected by short-term diabetes.This investigation was supported by a grant-in-aid from Fight-For-Sight, Inc., New York City, and by USPHS grant AM02001 from the National Institute of Arthritis, Metabolism and Digestive Diseases.     

Diabetes causes marked changes in lymphocyte metabolism

An enhanced susceptibility to infections is well known to occur in a poorly controlled diabetic state. Since glucose and glutamine are essential for lymphocyte function, we investigated whether their metabolism is changed in lymphocytes obtained from mesenteric lymph nodes of alloxan-induced diabetic rats (40 mg/kg body weight). The activities of hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase (G6PDH), citrate synthase and phosphate-dependent glutaminase were determined. Decarboxylation of metabolites [U-14C]-, [1-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvic acid, [U-14C]-palmitic acid and [U-14C]-glutamine was evaluated in incubated lymphocytes isolated from mesenteric lymph nodes. The measurements were carried out in cells following three experimental protocols: (1) lymphocytes freshly obtained from control and alloxan-induced diabetic rats, (2) lymphocytes from insulin-treated (2 U/rat per day) diabetic rats and (3) lymphocytes obtained from control and diabetic rats and cultured in the presence of insulin (1 mU/ml) for 6 h. The activities of hexokinase, G6PDH and citrate synthase were decreased by the diabetic state, whereas that of phosphofructokinase was raised. Decarboxylation of [U-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvate and [U-14C]-glutamine were also decreased in lymphocytes from diabetic rats, whereas [U-14C]-palmitic acid decarboxylation was increased. Insulin administration in vivo or added to the culture medium reversed the changes observed in freshly obtained lymphocytes. Alloxan-induced diabetes did change lymphocyte metabolism and this may be an important mechanism leading to impairment of lymphocyte function.     

Luteinizing hormone-releasing hormone stimulates arachidonic acid release in rat granulosa cells

The effect of LHRH on arachidonic acid release was studied in rat granulosa cells in primary culture. In cells prelabeled with [3H]arachidonic acid, LHRH caused an increase in the level of [3H]arachidonic acid released in the culture medium, to 125-150% of control levels at the end of a 60-min incubation period. In subsequent time-course and dose-response experiments, a significant effect on [3H]arachidonic acid release could be observed as early as 15 min after LHRH addition, and the lowest effective dose was 10(-8) M LHRH. Addition of LH, FSH, prostaglandin F2 alpha, or (Bu)2cAMP was without effect. Likewise, an agonistic LHRH analog (LHRHa, 10(-8) M) also markedly stimulated [3H]arachidonic acid from cultured granulosa cells, and the effects of both LHRH and LHRHa were blocked by concomitant presence of a potent LHRH antagonist. In addition to [3H]arachidonic acid release in the culture medium, the effect of LHRH on the level of radiolabel present in cellular phospholipids was also examined. In granulosa cells prelabeled with [3H] arachidonic acid, LHRH significantly depleted the level of radioactivity previously incorporated into cellular phosphatidylinositol, as early as 5 min after its addition, to 85% of control levels. The level of radiolabel found in other major phospholipids such as phosphatidylserine/phosphatidylcholine and phosphatidylethanolamine, as well as the intracellular level of unesterified [3H]arachidonic acid, were not significantly affected by LHRH. The effect of LHRH on [3H]arachidonic acid release from prelabeled phospholipids as well as the LHRH-induced loss of radioactivity previously incorporated into phosphatidylinositol could be reversed by verapamil, suggesting a possible calcium dependency. Taken together, these data support the notion that arachidonic acid liberation from phospholipids may be associated with the mechanism of action of LHRH on ovarian cells.     

Preferential alteration of oxidative relative to total glycolysis in pancreatic islets of two rat models of inherited or acquired Type 2 (non-insulin-dependent) diabetes mellitus

Summary In islets from both adult rats injected with streptozotocin during the neonatal period and spontaneously diabetic rats obtained by repeated selective breedings (GK rats), the ratio between d-[3, 4-¹⁴C]glucose oxidation and d-[5-³H]glucose conversion to ³HOH was 25% lower than in islets from control rats, indicating an impaired contribution of oxidative to total glycolysis. No primary defect in the Krebs cycle was found in the islets of diabetic rats, as judged from the ratio between either d-[2-¹⁴C]glucose or d-[6-¹⁴C]glucose and d-[3, 4-¹⁴C]glucose oxidation. Therefore, we propose that a preferential alteration of oxidative glycolysis in the pancreatic beta cell may contribute to the impairment of glucose-induced insulin release not only in a cytotoxic but also in a spontaneous model of non-insulin-dependent diabetes mellitus.     

The effect of diabetes, insulin, and the redox potential on leucine metabolism by isolated rat hemidiaphragm.

The oxidation of leucine by hemidiaphragms of control and diabetic rats was studied in vitro. Rats were rendered diabetic with streptozotocin. Hemidiaphragms of diabetic rats produced approximately 50% more 14CO2 during incubation with 0.1 mM [1-14C]leucine than did control muscles. This was observed during incubation with or without glucose and in the presence or absence of a full complement of plasma amino acids. The concentration of leucine in the tissue water of hemidiaphragms from diabetic rats was greater than that in the control muscles before incubation. The specific activity of leucine at the end of 60 min incubation was not significantly different in diabetic and control muscles, indicating that the increased 14CO2 production represented stimulation of leucine oxidation. Hemidiaphragms of diabetic rats released more leucine into the medium during incubation than did control muscles. The stimulating effect of diabetes on leucine oxidation in vitro was reversible by insulin therapy prior to sacrifice. The addition of 5 mM pyruvate to a medium containing glucose inhibited 14CO2 production from [14C]leucine in control muscles, but stimulated leucine oxidation by hemidiaphragms of diabetic rats. Leucine oxidation by hemidiaphragms of diabetic rats was markedly stimulated by the addition of an electron acceptor, 0.02 mM methylene blue, suggesting that the NADH/NAD ratio may be rate-limiting for branched chain amino acid oxidation in muscles of diabetic rats, but not in muscles of controls. We suggest that the accelerated oxidation of branched chain amino acids by muscles may play a role in the acceleration of the muscle protein catabolism and gluconeogenesis which develop during insulin deficiency. The restraining effect of the cellular redox potential on branched chain amino acid oxidation may play a role in the eventual deceleration of protein catabolism during a prolonged fast.     

Natural occurrence of poly(ADP-ribosyl) histones in rat liver.

Poly(ADP-ribose) bound to histones has been isolated from rat liver. When [14C]ribose was administered intraperitoneally to rats at a dosage of 300-750 mug (100-250 muCi)/10o g, approximately 1% of the radioactivity was recovered in the acid (5% CLCCCOOH)-INSOLUBLE MATERIAL OF THE LIVER NUCLEI 2 HR AFTER INJECTION. Of the acid-insoluble radioactivity, 4.5-9% was extractable with 0.25 N HCL. Carboxymethyl-cellulose column chromatography of the HCl-extracted material revealed that the radioactivity cochromatographed with histone subfractions f1 and, to a lesser extent, f2 and f3. Part of the protein-bound radioactivity was rendered acid-soluble by treatment with either snake venom phosphodiesterase or neutral NH2OH. From the enzyme digest, 5'-AMP and psiADP-ribose [2'-(5"-phosphoribosyl)-5'-AMP] were recovered, while the NH2OH treatment yielded ADP-ribose monomer and, presumably, oligomer. These observations indicate that ADP-ribose is attached to histones in vivo and is present both as a monomer and a polymer.     

Effect of dietary cholesterol on the release of prostacyclin from the mesenteric vascular bed in diabetic rat

Streptozotocin-induced diabetic female rats and age-matched control rats were fed a regular chow with or without the addition of 1% cholesterol in the diet. The release of 6-keto-PGF1 alpha, a prostacyclin metabolite, from the mesenteric vascular bed was significantly increased in diabetic rats. The production of PGI2 in diabetic rats was significantly reduced whereas that in the control was not affected by cholesterol feeding. The examination of the fatty acid composition of phospholipids from the mesenteric vascular bed indicated that proportions of stearic (18:0), linoleic (18:2n-6) and dihomo-gamma-linolenic (20:3n-6) acids were higher whereas those of oleic (18:1n-9) was lower in diabetic rats than those in the controls. Cholesterol feeding had no significant effect on the levels of arachidonic acid (20:4n-6) in the controls but it significantly decreased those in diabetic rats. It is suggested that cholesterol feeding lowers the release of PGI2 from the mesenteric vascular bed possibly because of a reduced level of arachidonic acid, the major precursor for prostaglandin synthesis. This could be due to an impairment of delta-5 desaturase.     

Hereditary defect of hepatobiliary cysteinyl leukotriene elimination in mutant rats with defective hepatic anion excretion

Hepatobiliary and renal elimination of cysteinyl leukotrienes were investigated in a mutant rat strain with a hereditary defect in the hepatobiliary excretion of conjugated bilirubin, dibromosulfophthalein and ouabain. After intravenous injection of [3H]leukotriene C4, the initial half-life of radioactivity circulating in blood was 79 +/- 15 sec (S.D.) in transport mutant rats as compared to 31 +/- 6 sec (S.D.) in normal Wistar rats. The intrahepatic leukotriene radioactivity was increased 5-fold after 1 hr in mutant rats, while the biliary elimination of [3H]leukotrienes was reduced to 1.8% of control. In normal rats, 77 +/- 7% (S.D.) of the administered leukotriene radioactivity were recovered in bile within 1 hr. The total recovery of radioactivity from bile, urine, liver, intestine, stomach, kidneys, muscular system and blood 1 hr after intravenous [3H]leukotriene C4 was 89 +/- 6% (S.D.) in normal rats and 46 +/- 4% (S.D.) in transport mutants. Enterohepatic circulation was studied after intraduodenal administration of N-acetyl-[3H]leukotriene E4, a major cysteinyl leukotriene metabolite in rat bile. In transport mutants, hepatobiliary elimination of the intestinally absorbed [3H]leukotriene was reduced to 5%, whereas urinary excretion was not significantly affected. [3H]Leukotriene metabolites in bile, liver and urine were separated by reversed-phase high-performance liquid chromatography. The proportion of N-acetyl-[3H]leukotriene E4 relative to polar leukotriene metabolites was higher in the bile of transport mutants as compared to control Wistar rats when analyzed within 30 to 60 min after intravenous injection of [3H]leukotriene C4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red blood cell and plasma fatty acid composition in diabetes mellitus

Arachidonic acid deficiency and increased linoleic acid levels have been a consistent finding in a variety of tissues in experimental diabetes. To determine if patients with type II non-insulin-dependent diabetes show changes in red blood cell and plasma fatty acid composition, a group of non-insulin-dependent diabetic patients were studied prior to and following treatment with glyburide which substantially improved their diabetic control. Red blood cell and plasma fatty acid composition was compared with that of a group of nondiabetic subjects and to red cell fatty acid composition in normal and streptozotocin diabetic rats. The diabetic patients had no changes in linoleic or arachidonic acid levels prior to treatment and no changes following glyburide therapy. These studies and the available literature suggest to us that either more severe diabetes is required to produce the fatty acid abnormalities described in the diabetic rat or that there is a fundamental species difference in the mechanism of diabetes or in fatty acid metabolism between the human and the rat which allows the human diabetic to more easily maintain normal tissue fatty acid composition.     

Catabolism of branched-chain amino acids by diaphragm muscles of fasted and diabetic rats

In vitro catabolism of branched-chain amino acids, leucine and valine, was investigated using diaphragm muscles from normal, streptozotocin-diabetic and overnight fasted rats. Oxidation and transamination of [1-14C] branched-chain amino acids were both stimulated to a similar extent by diabetes or fasting, when diaphragms were incubated with glucose. Transamination of leucine and valine was increased when diaphragms were incubated with pyruvate; stimulation of transamination was greatest in diaphragms from diabetic rats. Leucine and valine oxidation by control diaphragms was inhibited by pyruvate while it was unchanged or slightly stimulated in diaphragms from fasted or diabetic rats. Thus diaphragms from diabetic rats oxidized two to threefold more branched-chain amino acids than controls when they were incubated with pyruvate. The specific radioactivity of extracellular alpha-ketoisocaproate (KIC; the product of leucine transamination) produced by diaphragms incubated with [14C]leucine was similar for all groups (fed, fasted, or diabetic) in the presence or absence of pyruvate. Oxidation of [1-14C]KIC by diaphragms from fasted or diabetic rats, incubated with glucose, was the same or less than KIC oxidation by control diaphragms. Incubation with pyruvate inhibited KIC oxidation by control diaphragms to a significantly greater degree than that by diaphragms from diabetic or fasted rats. These data suggest the following Flux through branched-chain amino acid transaminase is limited by the availability of amino group acceptors in diaphragms from normal and overnight fasted rats, and to a greater extent in diaphragms from diabetic rats. Flux through the transaminase may be a major determinant of accelerated branched-chain amino acid oxidation by diaphragms in fasting and diabetes. In diaphragms of fasted and diabetic rats, flux through the branched-chain alpha-ketoacid dehydrogenase complex is resistant to inhibition by pyruvate, which is normally observed in controls.     

The disappearance from the circulation of chylomicrons obtained from control and diabetic rats

Previous studies have demonstrated that cholesterol synthesis is increased in the small intestine of diabetic animals and that there is an increased transport of this newly synthesized cholesterol from the small intestine into the circulation. Chylomicrons play an important role in the transport of cholesterol from the small intestine into the circulation, and the present study compared the rates of disappearance from the circulation and the fate of chylomicron cholesterol obtained from control and diabetic animals when administered to either intact control or diabetic rats. Thoracic duct lymph was collected from normal and diabetic rats after the administration of [14C]cholesterol and [3H]vitamin A1. The labeled chylomicrons were isolated by centrifugation and then administered to either control or diabetic rats. The major observation of this study is that chylomicron-associated sterols obtained from diabetic animals were cleared from the circulation in a normal manner. If one compares the rate of disappearance of either [3H]vitamin A1 or [14C] cholesterol-labeled normal chylomicrons administered to control animals with that of labeled diabetic chylomicrons administered to diabetic animals, the half-times in the circulation are almost identical (control: [3H]vitamin A1 t 1/2, 3.6 min; [14C]cholesterol t 1/2, 5.7 min; diabetic: [3H]vitamin A1 t 1/2, 3.5 min; [14C]cholesterol t 1/2, 4.4 min). In both experimental situations the rapid disappearance of [14C]cholesterol was associated with the appearance of [14C]cholesterol in the liver. Very little [14C]cholesterol was present in tissues other than liver, indicating that the rapid removal of labeled chylomicron remnants from the circulation was accounted for by hepatic uptake. These results demonstrate that in diabetic animals chylomicron-associated sterols are cleared from the circulation normally and that the bulk of intestinally derived cholesterol carried in the chylomicron lipoprotein fraction is rapidly delivered to the liver, where it could potentially influence lipoprotein metabolism.     

Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart

In this study, we examined phosphoinositide metabolism during ischemia and reperfusion using an isolated and perfused rat heart. When myocardial phosphoinositides were prelabeled with [3H]inositol, reperfusion after 30 minutes of normothermic global ischemia resulted in significant accumulations of radiolabeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly in the heart reperfused with [3H]inositol after 30 minutes of ischemia compared with that perfused with [3H]inositol after 30 minutes of nonischemic perfusion. However, isotopic incorporation of [3H]glycerol into diacylglycerol, phosphatidic acid, and all of the three phosphoinositides was diminished in the reperfused hearts. Reperfusion of the ischemic heart prelabeled with [14C]arachidonic acid resulted in significant increases in [14C]diacylglycerol and [14C]phosphatidic acid. The enhanced accumulations of [3H]inositol phosphates during reperfusion were not affected by treatment with prazosin plus atropine or indomethacin, but were inhibited by hypoxic reperfusion, reperfusion with Ca2+-free buffer, or by mepacrine. These results suggest that myocardial reperfusion stimulates phosphodiesteratic breakdown and turnover of phosphoinositides, and increased Ca2+ influx caused by reperfusion may be involved in the mechanism of stimulation of phosphatidylinositol-specific phospholipase C activity in the rat heart.     

Quantitative importance of the 25-hydroxylation pathway for bile acid biosynthesis in the rat

During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. In the present study, we have quantitated the relative importance of these two pathways in vivo by measuring production of [14C] acetone from [14C]-26-cholesterol. Four days after intraperitoneal injection of 20 to 40 muCi [14C]-26-cholesterol and 1 day after beginning a constant intravenous infusion of unlabeled acetone at 25 mumoles per kg per min, 6 male and 2 female Sprague-Dawley rats underwent breath collections. Expired acetone was trapped and purified as the 2,4-dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied times the acetone infusion rate to calculate production of [14C]acetone. [14C] Acetone production averaged 1.7% of total release of 14C from [14C]-26-cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C] acetone production from [14C]isopropanol averaged 111% of the [14C]isopropanol infusion rate. We conclude that, in the normal rat, the 25-hydroxylation pathway accounts for less than 2% of bile acid synthesis.