Impairments in hepatocyte phosphoinositide metabolism in endotoxemia

The status of phospholipid metabolism and inositol lipids-mediated transmembrane signaling in rat hepatocytes was analyzed during chronic, nonlethal endotoxemia. Rats were infused intravenously (IV) with Escherichia coli endotoxin (ET) via subcutaneously implanted osmotic pumps at a rate of 0.1 mg/100 g bw/day. The experiments were performed after 30 hours of ET or sterile saline (NaCl) infusion, in hepatocytes prelabelled "in vitro" with 32P (15 microCi/mL) and further stimulated with vasopressin (VP, 0.23 mumol/L). Similar experiments were done with food-restricted animals, whose food intake was matched with the voluntary intake of ET-infused rats. Uptake of 32P label into phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) occurs rapidly in cells from pair-fed, saline and ET-infused animals, and reaches a plateau between 60 and 80 minutes of incubation. Labeling of phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) proceeds linearly after a ten-minute lag period for PI and 20 minutes for the two other lipids. The nutritional state greatly affects the distribution of 32P uptake into lipids, resulting in very low labeling of PA and PI and a high labeling of poly-PI as compared with control (taken from untreated rats) cells. In ET-v saline-infused rats, the labeling of PI and PE was depressed concomitantly with a proportional increase in the labeling of PIP and PC. The ability of VP to induce polyphosphoinositide (poly-PI) degradation in hepatocytes from saline-infused animals was similar to that observed in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased carnitine biosynthesis in rats with secondary biliary cirrhosis

Carnitine biosynthesis was investigated in rats with secondary biliary cirrhosis induced by bile duct ligation (BDL) for 4 weeks (n = 5) and in pair-fed, sham-operated control rats (n = 4). Control rats were pair-fed to BDL rats, and all rats were fed an artificial diet with negligible contents of carnitine, butyrobetaine, or trimethyllysine. Biosynthesis of carnitine and its precursors was determined by measuring their excretion in urine and accumulation in the body of the animals. Four weeks after BDL, total carnitine content was increased by 33% in livers from BDL rats when compared with control rats, but was unchanged in skeletal muscle and whole carcass. The plasma total carnitine concentration averaged 29.0 +/- 4.1 vs. 46.4 +/- 7.3 micromol/L in BDL rats and control rats, respectively. Urinary total carnitine excretion was reduced by 56% in BDL rats as compared with control rats. Carnitine biosynthesis was significantly decreased in BDL rats (0.45 +/- 0.19 vs. 0.93 +/- 0.08 micromol/100 g body weight/d in BDL and control rats, respectively). The tissue content of free and protein-linked trimethyllysine, a carnitine precursor, and trimethyllysine plasma concentrations were not different between BDL and control rats. However, urinary trimethyllysine excretion was increased 5-fold in BDL rats and approximated glomerular filtration. In contrast, urinary excretion of butyrobetaine, the direct carnitine precursor, was decreased by 40% in BDL rats as compared with control rats. Trimethyllysine biosynthesis was not different, but butyrobetaine biosynthesis was decreased by 51% in BDL as compared with control rats. In conclusion, carnitine biosynthesis is decreased in BDL rats as a result of a defect in the conversion of trimethyllysine to butyrobetaine.     

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.     

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes.

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.     

Inositol affects the intracellular turnover of pulmonary surfactant phospholipids in the rat

Rats, given a diet supplemented with 20% inositol, had threefold increased plasma inositol concentrations. The pool size of their alveolar surfactant fraction and their lamellar body fraction were the same as in the control rats and differences in phospholipid composition of the surfactant fractions were mainly restricted to changes in the percentages phosphatidylinositol (PI) and phosphatidylglycerol (PG). The change in phospholipid composition did not affect the pressure-volume relationship of the lungs. The labeling of phosphatidylcholine (PC), saturated phosphatidylcholine (SPC) or PI in the alveolar lavage fraction was the same for both groups, whereas labeling of alveolar PG was delayed in the inositol-fed rats. The specific activity-time relationships of the lamellar body phospholipids differed significantly between the control and inositol-fed rats and the differences in disappearance rate of the label from these fractions suggest that approximately 25-30% of the lamellar body material in inositol-fed rats is directed to a third, intracellular pool. We conclude that an increase in PI and a concomitant decrease in PG content of surfactant do not affect the clearance of alveolar surfactant, but enlarge the turnover of the lamellar body fraction because of intracellular degradation.     

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.     

G protein-coupled receptor internalization signaling is required for cardioprotection in ischemic preconditioning

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gbetagamma-sequestering peptide, betaARKct (TG betaARKct mice), to test whether the protection of PC is Gbetagamma-dependent. To test the role of G(i) protein, we used wild-type mice pretreated with the G(i) inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG betaARKct mice. To determine the mechanism of Gbetagamma-induced protection in PC, we investigated one of the downstream targets of Gbetagamma, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG betaARKct hearts; therefore, we investigated other targets of Gbetagamma. Recent studies suggest a role for Gbetagamma in GPCR internalization. We found that betaARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kgamma, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kgamma blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.     

Effects of chronic endotoxaemia on oxygen consumption at different ambient temperatures in the unanaesthetised rat

Oxygen consumption has been measured at different ambient temperatures at intervals during the intravenous infusion of endotoxin (1 mg/kg.day-1) from a subcutaneously implanted osmotic minipump in unanaesthetised rats. On day 1 of the infusion oxygen consumption was elevated at ambient temperatures of 10, 28, and 31 degrees C but not at 20 degrees C, compared with pair-fed saline-infused controls. There was a significant negative correlation between oxygen consumption on days 1 and 3 and environmental temperature (10, 20, and 28 degrees C) in both groups, but the regression line describing the relation in endotoxin-infused rats was displaced above that for the saline-infused control without a change in slope. The "minimal observed" oxygen consumption, which is taken as an estimate of basal metabolic rate, was elevated by the infusion of endotoxin. The endotoxin-induced increase in "minimal observed" oxygen consumption was removed by indomethacin (5 mg/kg.sc) on day 1 of the infusion but was ineffective on days 3 and 7.     

Platelet phospholipids in liver cirrhosis

In the present study we have investigated and also compared the biochemical nature of the platelet phospholipids in patients with portal liver cirrhosis and in normals. Normal platelets contained 10.88 +/- 0.83 microgram of phospholipid phosphorus per 10(9) platelets whereas the cirrhosis platelets contained 7.63 +/- 1.29 microgramP/10(9) platelets. In cirrhosis there was a 29.87% decrease of phospholipids compared to normal. However the percentage distribution of phospholipids in cirrhosis was similar to normal. But each phospholipid value in cirrhosis was found lower then normal. Estimated as microgram phosphorus per 10(9) platelets decreased amounts of phosphotidyl inositol (PI) (0.29 +/- 0.16), phosphotidyl serine (PS) (0.49 +/- 0.12), phosphotidyl ethanolamine (PE) (2.0 +/- 0.43) and spingomyelin (SPH) (1.42 +/- 0.31) and phosphotidyl choline (PC) (3.25 +/- 0.62) and total lipid phosphorus (7.63 +/- 1.29) were found in the patient group.     

Endogenous hypertriglyceridemia in a nonobese rat model: plasma lipoproteins and dietary sensitivity

Sprague-Dawley male rats from the Ivanovas-Sieve colony (IVA-SIV) show higher plasma triglyceride levels compared to the standard Charles River (CR) rats. The triglyceride enrichment occurs primarily in the very-low-density lipoproteins (VLDL), otherwise of a normal % composition, suggesting that the number of particles is increased, rather than their triglyceride (TG) content. High-density lipoprotein (HDL) particles, corresponding to HDL1, appear to be increased in the IVA-SIV rats, as confirmed by rate-zonal ultracentrifugation. The apoprotein composition of isolated lipoproteins (apo B content and isoelectric focusing pattern), does not differ in the two strains. The IVA-SIV rats are remarkably more TG inducible, compared to the standard CR. This can be shown with fructose loading and with a cholesterol-cholic acid diet. The plasma TG increase, after Triton administration, indicative of the VLDL-TG production, is fourfold higher in the IVA-SIV, compared to the CR rats. These findings provide evidence for some similarities between the IVA-SIV rat and human endogenous hypertriglyceridemia, and suggest an increased TG biosynthesis in this model.     

Energy restriction dilutes the changes related to dietary fat type in membrane phospholipid fatty acid composition in rats

To investigate liver cell membrane phospholipid (PL) fatty acid (FA) composition in response to the consumption of different types of dietary fat and graded levels of energy intake, rats were fed for 10 weeks on a diet containing either fish oil, safflower oil, or beef tallow. Within each dietary fat group, subgroups were either provided free access to food or energy-restricted to 85% or 70% of the ad libitum intake by reducing the dietary carbohydrate content while keeping other macronutrient intakes constant. Higher (P < .05) proportions of docosahexaenoic acid, linoleic acid, and monounsaturated FA were observed in the membrane PL of the fish oil, safflower oil, and beef tallow groups, respectively, resembling the FA composition in the diets. However, such modifications of dietary FA composition in, membrane PL FA were influenced by body energy status. The higher docosahexaenoic acid and total n-3 FA content in phosphatidylcholine (PC), sphingomyelin (SPH), and phosphatidylserine (PS) of the ad libitum fish oil group compared with the other dietary groups no longer existed when energy supply was restricted. Therefore, reducing energy intake tended to dilute the changes of membrane PL FA composition occurring as a function of dietary FA composition. These data suggest that the influence of dietary fat type on cellular structure and perhaps function becomes increasingly important with progressively positive energy balance.     

Triglyceride removal from very low density lipoproteins in vivo as a function of their triglyceride content.

Rabbit very low density lipoproteins (VLDL) were fractionated in 3 subfractions differing in density and triglyceride (TG) content. The fraction of total weight represented by triglycerides was: VLDL1 TG = 71%, VLDL2 TG = 62%, VLDL3 TG = 55%. No other difference in composition was observed. After intravenous administration of radioglycerol all subfractions were labelled and the distribution of radioactivity in their glycerolipids was the same. In 5 rabbits, a biologically labelled triglyceride-rich VLDL1 subfraction preparation was administered and the specific activity of triglyceride was determined in all 3 subfractions in serial samples. A precursor--product relationship between VLDL1 TG and VLDL TG in other subfractions was not observed. In another set of experiments the disappearance rates of VLDL TG from subfractions were determined in recipient rabbits. The half-lives were: VLDL1 TG = 9.5 +/- 1.3 min, VLDL2 TG : 16.0 +/- 1.3 min, VLDL3 TG: 26.9 +/- 1.3 min. It is concluded that triglyceride removal from a VLDL subfraction varies inversely with its triglyceride content. A role for partial VLDL lipolysis in this metabolic heterogeneity was not established.     

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P₂. There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P₂ binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P₂ and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.     

Effects of solvent/detergent-treated plasma and fresh-frozen plasma on haemostasis and fibrinolysis in complex coagulopathy following open-heart surgery

BACKGROUND AND OBJECTIVES: Solvent/detergent-treated plasma (SDP) contains markedly lower protein S (PS) and plasmin inhibitor (PI) activity than standard fresh-frozen plasma (FFP). It has also been reported that SDP contains no alpha(1)-antitrypsin. Despite the lack of clinical data, it is suspected that SDP may be less effective than FFP in the treatment of complex coagulopathies. We therefore conducted a prospective trial to study the impact of SDP and FFP on haemostasis and fibrinolysis in complex coagulopathy after open-heart surgery. MATERIALS AND METHODS: Patients received either 600 ml of SDP (n = 36) or 600 ml of FFP (n = 31) at an infusion rate of 30 ml/min. The following parameters were measured before treatment and 60 min after termination of plasma infusion: prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, factor VIII, antithrombin, protein C (PC), free PS and PS activity, prothrombin fragments F1+2 (F1+2), D-dimers (DD), fibrinogen degradation products (FDP), plasmin-plasmin inhibitor complexes (PPI), plasminogen, PI and alpha(1)-antitrypsin. RESULTS: The rise in fibrinogen, factor VIII, antithrombin, PC, free PS, alpha(1)-antitrypsin and plasminogen, and the decrease in PT and APTT, did not significantly differ between the two study arms. However, PS activity did not increase after SDP infusion but did show a significant elevation after infusion with FFP. PI declined significantly after SDP and remained uninfluenced by FFP. Neither SDP nor FFP had any significant influence on F1+2, DD or FDP. However, a significant decrease in PPI levels caused by both types of plasma indicated a reduction in hyperfibrinolysis. Clinical haemostasis evaluation revealed no significant difference between the two treatment regimens. No adverse reactions were observed. CONCLUSION: With the exception of PS and PI, SDP and FFP improved haemostasis and fibrinolysis to a similar degree. The clinical significance of these findings has to be determined in patients with severe acquired PS and PI deficiency requiring plasma transfusions.     

Frequency of natural coagulation inhibitor (antithrombin III, protein C and protein S) deficiencies in Japanese patients with spontaneous deep vein thrombosis.

One hundred and thirteen consecutive Japanese patients with deep venous thrombosis (DVT) were studied for the incidences of antithrombin III (AT-III), protein C (PC) and protein S (PS) deficiencies, and the results were compared with those of normal subjects. Ten of the 392 normal Japanese subjects were found with PS deficiency (n = 8, 2.02%) or PC deficiency (n = 2, 0.5%). PS deficiencies comprised type I (1/8, 12.5%), type 11 (4/8, 50%), and type III (3/8, 37.5%). All PC deficiencies were type I. Among patients with DVT, 32 (28.3%) were deficient in AT-III, PC and PS. These patients consisted of two AT-III deficiency (1.77%), nine PC deficiency (7.96%), 20 PS deficiency (17.7%), and one combined deficiency of PC and PS (0.88%). Both of the patients with AT-III deficiency were classified as type II, all those with PC deficiency as type I, and those with PS deficiency as type I in 25% (5/20), type II in 55% (11/20) and type III in 20% (4/20). The frequency of PC and PS deficiencies in patients with DVT were 15.6 and 7.38 times the control population frequency, respectively, and this difference was statistically significant (P < 0.05). These data suggest that the Japanese population has a high frequency of PC and PS deficiencies. We recommend that PS activity should be measured for screening of thrombosis since type II deficiency accounted for approximately 50% of PS deficiency cases in both patients and the normal group in the Japanese.     

Further characterization of a model of chronic endotoxemia in the rat: adenine nucleotide content in liver

Male Sprague-Dawley rats (320-380 g) were treated with Escherichia coli endotoxin (ET) either acutely (3 and 4.5 mg/100 g b.w.,) or chronically (0.1 mg/100 g b.w./24 h, i.v. from subcutaneously implanted osmotic pumps). Control rats received only sterile saline. At 6 h after injection and after 30, 54, 126, and 198 h of continuous ET infusion, the animals were sacrificed, and adenine nucleotide content in liver was assayed. Acute ET treatment with either dose produced a slight decrease in ATP content (15% and 11% with 3 mg and 4.5 mg ET/100 g b.w., respectively) associated with an increase of ADP and AMP content and a fall of the energy charge (by 9.2% and 6.4%, with the two ET doses, respectively). Chronic ET treatment did not affect either of the measured parameters. However, in both saline- and ET-infused rats a decrease of ATP content paralleled by an increased level of ADP and AMP and by a fall of energy charge were seen when compared to acutely saline-injected rats. Such changes can be attributed to the surgical trauma associated with pump implantation. It is concluded that the energy status of the liver during endotoxemia cannot account for metabolic disturbances associated with this pathophysiological state.     

Chronic ethanol drinking and food deprivation affect rat hypothalamic-pituitary-thyroid axis and TRH in septum

Because chronic ethanol ingestion may perturb thyroid function, we evaluated the effect of 4-wk of oral 10% ethanol ingestion on the hypothalamic-pituitary-thyroid (HPT) axis and septal thyrotropin-releasing hormone (TRH) in 200-g male Wistar rats. Animals were divided into three groups: absolute control receiving tap water and food ad libitum; ethanol group receiving food ad libitum and 10% ethanol as the sole source of drinking fluid; pair-fed group receiving tap water and an amount of food corresponding to the consumption of ethanol group. After 4-wk of treatment, the body weight of the ethanol group was 7% and of the pair-fed rats 19% lower than that of the absolute controls. Both chronic ethanol treatment and food deprivation produced a decrease in plasma thyroid-stimulating hormone (TSH). Pair-fed rats also had a lower plasma T₃. Type I iodothyronine 5′-deiodinase activity in the liver was increased in the pair-fed and even more in the ethanol-treated group. The content and secretion in vitro of TRH from the hypothalamic paraventricular nucleus and median eminence were unchanged. TRH content in the septum was increased in both the ethanol and pair-fed groups. TRH secretion from the septum in vitro was lower in the pair-fed, but unchanged in the ethanol group. These data suggest that 4-wk of peroral ethanol intake affects thyroid function mostly at the extrahypothalamic level and that there is a contribution of concomitant food deprivation. Both ethanol treatment and food deprivation increased TRH content in the septum.     

Effect of growth hormone administration to hypophysectomized rats on muscle lipid metabolism.

This study was undertaken to determine whether increased oxidation of intracellular muscle lipids could explain the impaired carbohydrate metabolism of that tissue following growth hormone (GH) administration. Pieces of diaphragm from either hypophysectomized (hypox) rats injected for 10 days with saline or 1 mg bovine GH or animals of similar weight with intact pituitary glands were studied. In hypox rats, GH administration increased both weight gain and plasma glucose concentrations at sacrifice and impaired glucose uptake by diaphragms in vitro. Tissue triglyceride (TG) content was markedly decreased in hypox muscle compared to diaphragms removed from animals with intact pituitary glands. GH administration lowered TG levels even further. Initial phospholipid (PL) content was similar in all groups and fell significantly during the second hour of incubation only in hypox muscle. GH administration had no effect on PL changes. The differences in TG- and PL-specific activities strongly suggested that most of the C¹⁴O₂ produced in the second hour of incubation was derived from TG. Approximately 25% of diaphragm TG was oxidized to CO₂ in all three groups of animals. Changes in lipid-specific activities during the second hour indicated that in tissue from hypox animals, TG-fatty acids were converted to PL-fatty acids, whereas no such exchange occurred in muscle from intact rats. In conclusion: (1) enhanced oxidation of intracellular muscle lipids does not explain the effect of GH on carbohydrate metabolism; (2) diaphragm TG content is markedly decreased in hypox animals and is not secondary to GH deficiency; (3) diaphragms incubated in vitro will utilize PL for energy only if TG levels are low; and (4) as PI content falls, some TG-fatty acids are shifted into PL, possibly in an attempt to maintain the important “structural-functional elements” of muscle.