Unsaturated fatty acyl-CoA inhibition of cholesterol synthesis in vitro.

1. The influence of saturated and unsaturated fatty acids and fatty acyl coenzyme A thioesters on cholesterol synthesis in vitro has been studied in a rat liver post-mitochondrial supernatant system. 100 micronM free fatty acids do not influence in vitro cholesterol synthesis. Various fatty acyl-CoA thioesters at 10--100 microntm inhibit [14C]acetate incorporation into digitonin-precipitable sterols, the more unsaturated derivatives causing the greatest inhibition. 10 micronM arachidonoyl-CoA inhibits [14C]acetate incorporation into sterols 17% and 50 micronM inhibits 55%. [14C]Acetyl-CoA incorporation into sterols is similarly inhibited but [14C]mevalonate incorporation is not inhibited. Thus, the inhibition may be on the rate-controlling step of cholesterol synthesis, the conversion of beta-hydroxy-beta-methylglutaryl-CoA to mevalonate. Unsaturated fatty acyl-CoA thioesters may be important in regulating cholesterol synthesis. 2. Studies were undertaken to determine if the previously observed inhibition of cholesterol synthesis by thyroxine in vitro may relate to the thyroxine stimulation of fatty acid desaturation. 50 micronM thyroxine causes a preferential incorporation of [14C]acetate into unsaturated fatty acids while inhibiting acetate incorporation into sterols. However, a sufficient increase in unsaturated fatty acyl-CoA thioesters to account for the thyroxine inhibition of cholesterol synthesis was not demonstrated.

Fatty acyl-CoA inhibition of beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

The influence of the fatty acyl-CoA thioesters on rat liver microsomal hydroxymethylglutaryl-CoA reductase activity was tested in vitro to determine if the previously demonstrated inhibition of [14C]acetate incorporation into cholesterol is due to inhibition of this rate limiting step in cholesterol synthesis. The polyunsaturated fatty acyl-CoA thioesters caused the greatest inhibition of enzyme activity, 50 micron arachidonoyl-CoA inhibiting 67% and 5 micron inhibiting 22%. 50 micron linoleoyl-CoA inhibited 56% with the more saturated thioesters causing less inhibition. 50--100 micron free fatty acids, free CoA, cholesterol esters, phospholipids, carnitine derivatives, prostaglandins and non-specific detergents caused little or no inhibition of enzyme activity. Kinetic studies revealed the inhibition to be noncompetitive with respect to hydroxymethylglutaryl-CoA with a Ki for arachidonoyl CoA of 3.10 micron. Fatty acyl-CoA inhibition of in vitro cholesterol synthesis is due to inhibition of hydroxymethylglutaryl-CoA reductase activity. Variation in intracellular concentrations of fatty acyl-CoA thioesters may signficantly alter cholesterol synthesis.

Fatty acid desaturation in experimental diabetes mellitus.

Microsomal fatty acid desaturation is defective in streptozotocin-induced experimental diabetes. This defect is correctable by insulin treatment. The electron transport chain needed for microsomal fatty acid desaturation was studied in liver microsomes of streptozotocin diabetic rats, and the defect was localized to the terminal desaturase enzyme. Cytochrome b5 levels were elevated in the face of decreased fatty acid desaturation and returned to normal after 48 h of insulin treatment; 2 U of regular insulin every 6 h for 24 h repaired the fatty acid desaturation defect, while 0.5 U failed to correct the defect. Both the delta 6 and delta 9 desaturase defects (linoleic acid and stearoyl-CoA desaturation) required similar amounts of insulin and periods of time for correction, although these are different enzymes. This is consistent with the desaturation defect being due to a protein synthetic effect. Diabetic rats treated twice daily with injections of 4 U of NPH insulin showed a "super" repair of their desaturase defect by 48 h: delta 9 desaturase activity increased eight times over control activity, while delta 6 desaturase activity increased two and one-half times over control activity. This, together with the fact that delta 6 desaturase activity in diabetes (64% of control) is altered less than is delta 9 desaturase activity (22% of control), indicates that delta 6 desaturase enzyme activity is less responsive to insulin than is delta 9 desaturase enzyme activity. The physiologic significance of altered fatty acid desaturation in diabetes mellitus is unknown.

Decreased incorporation of long-chain fatty acids into erythrocyte phospholipids of STZ-D rats.

We studied the mechanisms for the altered fatty acid composition in erythrocytes (RBCs) derived from streptozocin-induced diabetic (STZ-D) rats. After 3-wk duration of diabetes, blood glucose, plasma triglyceride, and plasma free-fatty acid levels were all significantly increased. In the diabetic platelet-poor plasma (PPP), the most significant increases in free fatty acids were stearate, linoleate, eicosatrienoate (n-6), and docosahexaenoate (n-3). Fatty acid composition of RBC phospholipids was also altered, with significant decreases in arachidonate, docosatetraenoate (n-6), and docosapentaenoate (n-6) and increases in linoleate and docosahexaenoate. Insulin treatment of the diabetic rats resulted in normalization of docosapentaenoate, arachidonate, and linoleate levels in RBC phospholipids but not of docosahexaenoate or docosatetraenoate levels. The incorporation of [5,6,8,9,11,12,14,15-3H]arachidonate into diabetic RBC phospholipids was significantly decreased compared with the corresponding control RBC, regardless of the incubation medium used, which was the PPP derived either from the control or diabetic rats. Therefore, the decreased incorporation of [5,6,8,9,11,12,14,15-3H]arachidonate into diabetic RBC phospholipids was independent of the altered lipid composition of the PPP incubation media. Furthermore, the decreased incorporation was not specific for arachidonate, because the incorporation of other long-chain fatty acids such as [9,10-3H]oleate, [1-14C]palmitate, [2-14C]eicosatrienoate (n-6), and [1-14C]linoleate into RBC phospholipids was also comparably decreased. More important, the decreased fatty acid incorporations were reversed by insulin treatment of the diabetic rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Side effects resulting from the use of growth hormone and insulin-like growth factor-I as combined therapy to frail elderly patients.

BACKGROUND: The objective of this study was to examine the relationship between serum IGF-I concentration and the incidence of side effects of therapy with recombinant human growth hormone (rhGH) and recombinant human insulin-like growth factor-I (rhIGF-I). METHODS: Thirteen high-risk, undernourished elderly males were started on a 15-day course of rhGH and rhIGF-I by subcutaneous injection. The dose of rhGH was held constant at .0125 mg/kg/day, whereas the dose of rhIGF-I was increased in a stepwise fashion from 10 micrograms/kg to the targeted dose of 40 micrograms/kg twice a day. RESULTS: Nine subjects completed the protocol and reached the full target dose of both hormones. Fluid retention, gynecomastia, and orthostatic hypotension were the most common complications. The hormone injections increased the serum concentration of IGF-I (from 72.7 +/- 40.9 to 483.7 +/- 251.4 eta g/ml, p = .001) and IGFBP-3 (from 1.82 +/- 0.66 to 2.72 +/- 1.18 mg/L, p = .012), and decreased serum albumin (from 34.3 +/- 5.5 to 31.4 +/- 4.6 g/L, p = .009). The magnitude of the initial increase in the serum IGF-I concentration was a powerful risk factor for severe orthostatic hypotension, diffuse myalgias, and drug-induced hepatitis. There was no association between the serum IGF-I concentration and fluid retention or gynecomastia. CONCLUSIONS: Treatment of the undernourished frail elderly with the anabolic agents rhGH and rhIGF-I at the specified dosages may produce undesirable side effects including fluid retention, gynecomastia, and orthostatic hypotension. Although these agents hold therapeutic promise, they must be used with caution in this high-risk population.

Comparison of the effects of salbutamol and clenbuterol on skeletal muscle mass and carcass composition in senescent rats.

Aging decreases skeletal muscle mass and strength, making elderly subjects particularly vulnerable to catabolic effects of age-related diseases. Clenbuterol, a muscle anabolic beta 2-adrenergic agonist, has reduced or restored skeletal muscle losses in experimental catabolic states. However, the doses of clenbuterol used to prevent or reverse muscle wasting in most animal models have exceeded the estimated safe dose in man. Recently, another beta 2-adrenergic agonist, salbuamol (albuterol), has been shown to increase muscle weight and protein content in young rats at a dose similar to that used clinically. In contrast to clenbuterol, salbutamol is currently approved for human use as a bronchodilator in the United States. This study has compared the muscle and protein anabolic effects of salbutamol at a clinically relevant dose with those of clenbuterol at a dose typically used in animal models of muscle wasting. Salbutamol and clenbuterol were administered by implanted osmotic minipumps to Fisher-344 rats aged 3 and 24 months at doses of 1.03 mg and 600 micrograms per kilogram per 24 hours for 3 weeks. The weights of five hindlimb muscles, as well as carcass protein and fat content, were determined. Salbutamol and clenbuterol increased combined hindlimb muscle weight 19% and 28% in young rats, with 19% and 25% increases in old rats. Similarly, these drugs increased gastrocnemius weight and protein content 19% and 24% in young rats, with 19% and 23% increases in old rats. Salbutamol and clenbuterol increased carcass protein content 20% and 30% in young rats, with 12% and 21% increases in old rats.(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.

Altered fatty acid composition in the plasma, platelets, and aorta of the streptozotocin-induced diabetic rat.

Decreased arachidonate levels have been described in various tissues of the streptozotocin-induced diabetic rat. However, reported arachidonate changes in platelets from diabetic patients have been variable. In this communication, we describe experiments that indicate that in the short-term streptozotocin diabetic rat (2 to 3 weeks), the fatty acid composition of plasma and red blood cell lipids was altered but remained unchanged in platelet and aorta phospholipids. The altered fatty acid composition of the diabetic red blood cells and plasma cholesterol esters and phospholipids was similar to that previously found in the diabetic liver. However, in long-term diabetes (6 weeks), the phospholipid fatty acid composition of the platelet and aorta became significantly altered. Thus, in the 6-week diabetic platelet, there were increases of linoleate, dihomo-gamma-linolenate, docosapentaenoate (C22:5n-3), and docosahexaenoate, and decreases of oleate, arachidonate, and docosatetraenoate. In the aorta, there were increases of linoleate, eicosapentaenoate, and docosahexaenoate, and decreases of arachidonate, docosatetraenoate, and docosapentaenoate (C22:5n-6). Results from these experiments indicate that the fatty acid composition of plasma and red blood cell lipids was altered in short-term diabetes (2 to 3 weeks), but that of platelet and aorta phospholipids was not changed until more prolonged diabetes was present. Insulin treatment of the diabetic rat increased the levels of palmitoleate and oleate and decreased the levels of linoleate in platelet and aorta lipids from insulin-treated diabetic rats, suggesting an overcorrection of diminished delta 9 and delta 6 fatty acid desaturation as compared with the nondiabetic control.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of experimental hyperthyroidism on protein turnover in skeletal and cardiac muscle.

Since experimental hyperthyroidism reduces skeletal muscle mass while simultaneously increasing cardiac muscle mass, the effect of hyperthyroidism on muscle protein degradation was compared in skeletal and cardiac muscle. Pulse-labeling studies using (3H) leucine and (14C) carboxyl labeled aspartate and glutamate were carried out. Hyperthyroidism caused a 25%-29% increase in protein breakdown in both sarcoplasmic and myofibrillar fractions of skeletal muscle. Increased muscle protein degradation may be a major factor in the development of skeletal muscle wasting and weakness in hyperthyroidism. In contrast, protein breakdown appeared to be reduced 22% in the sarcoplasmic fraction of hyperthyroid heart muscle and was unchanged in the myofibrillar fraction. Possible reasons for the contrasting effects of hyperthyroidism on skeletal and cardiac muscle include increased sensitivity of the hyperthyroid heart to catecholamines, increased cardiac work caused by the hemodynamic effects of hyperthyroidism, and a different direct effect of thyroid hormone at the nuclear level in cardiac as opposed to skeletal muscle.

Effects of clenbuterol on skeletal muscle mass, body composition, and recovery from surgical stress in senescent rats.

Aging decreases skeletal muscle mass and strength, which may be exacerbated by age-related diseases. There is a need for therapeutic agents to prevent or restore loss of skeletal muscle in elderly subjects with muscle wasting disorders. Clenbuterol, a beta 2-adrenergic agonist, dramatically increases skeletal muscle mass in young animals and partially prevents or restores muscle loss in experimental models of muscle wasting. However, the protein anabolic and fat catabolic effects of clenbuterol have not been studied in senescent animals. To determine whether this drug has potential for preventing or repairing muscle loss in elderly subjects, we have examined its effects in young and old rats. Clenbuterol was administered by implanted osmotic minipumps to Fischer-344 rats ages 3, 12, and 23 months, at a dose of 1.5 mg/kg/24 h for 3 weeks. The weights of five hindlimb muscles and carcass protein and fat content were determined. Clenbuterol treatment increased the weight of skeletal muscles 22% to 39% in 3-month-old rats, 19% to 35% in 12-month-old rats, and 22% to 25% in 23-month-old animals. Likewise, clenbuterol increased carcass protein content 19% in 3-month-old rats, 16% in 12-month-old rats, and 24% in 23-month-old animals. Conversely, the drug reduced carcass fat content 36% in 3-month-old rats, 32% in 12-month-old rats, and 38% in 23-month-old rats. Therefore, clenbuterol had similar anabolic and catabolic effects in all age groups. In addition, clenbuterol stimulated recovery of skeletal muscle protein lost following pump implantation in senescent rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of thyroid hormone on metabolic adaptation to fasting.

The acute effect of triiodothyronine (T3) on mobilization of fat and protein energy stores has been measured in five fasting, normal men. Fasting subjects were chosen for this study to amplify catabolic effects occurring during brief thyroid hormone treatment. Subjects were fasted for 72 hr on two occasions with admintration of T3, 150 mug every 12 hr, for 72 hr before and during the second fast. Plasma beta hydroxybutyrate, acetoacetate, and free fatty acid levels as well as ketone, creatine, and urea excretion were measured during control and T3 fasts. T3 enhances catabolism of protein stores as indicated by the doubling of urea excretion during the T3 fasts. Likewise, creatine excretion is increased six to ninefold during the T3 fasts. Catabolism of fat stores is enhanced during the T3 fasts as shown by increased plasma free fatty acid and ketone levels, and increased ketone excretion. Brief T3 treatment for 3 days augments the expected protein and fat catabolism of starvation without causing subjective changes of hyperthyroidism. Much of the catabolic expression of hyperthyroidism may simply reflect inadequate caloric intake to fuel energy requiring processes stimulated by thyroid hormone such as cell membrane sodium pumping and protein synthesis.

Modifications of platelet phospholipid fatty acid composition in streptozocin-induced diabetic rats.

Increased thromboxane A2 (TXA2) generation by platelets has been reported both in diabetic patients and streptozocin-induced diabetic rats. This increase is in contrast to the decreased prostacyclin (PGI2) synthesis by endothelial cells in diabetes. An imbalance in the ratio of TXA2/PGI2 has been implicated in increased platelet aggregation and a high incidence of vascular disease in human diabetes. The mechanism for this imbalance, however, remains elusive. In a previous study from our laboratory, we reported unchanged arachidonic acid levels in platelet membrane phospholipids of 3-week diabetic rats, but a decreased arachidonic acid level in platelet membrane phospholipids of 6-week diabetic rats. In the present communication, we report the role of enzymes that are involved in remodeling arachidonic acid levels of platelet membrane phospholipids in both 3- and 6-week diabetic rats. No alterations were observed in the activities of arachidonoyl-CoA synthetase, acyl-CoA: lysophosphatidylcholine acyltransferase, or phospholipase A2 in platelets from both 3- and 6-week diabetic rats. However, both increased uptake and incorporation of [14C]arachidonic acid into platelets were observed in the diabetic platelet-rich plasma. In conclusion, increased TXA2 formation in diabetic platelets is not due to alterations in the activities of enzymes involved in the incorporation into or release of arachidonate from the diabetic platelet membrane phospholipid, but may be due to increased efficiency of uptake, incorporation or possibly redistribution of this fatty acid among phospholipid classes in diabetic platelets.

Altered microsomal phospholipid composition in the streptozotocin diabetic rat.

Streptozotocin diabetes in the rat alters liver microsomal membrane fatty acid composition. The present study was undertaken to determine if such changes in fatty acid composition were due to changes in the amount of individual phosphoglycerides or to disproportionate changes in fatty acid composition in any of the individual phosphoglycerides. The diabetic animals showed a small increase in total microsomal phospholipid, which is due to a selective increase in the phosphatidylethanolamine fraction. The changes in fatty acid composition in the total lipid extract (decreased palmitoleic, oleic and arachidonic acids and increased linoleic and docosahexaenoic acids) from the diabetic animals were present in both the major phosphoglycerides, phosphatidylcholine and phosphatidylethanolamine, with very little change in fatty acid composition in the phosphatidylserine and inositol fraction. Further studies are necessary to delineate the cause of the abnormal membrane phospholipid composition in the diabetic animal.

Effects of streptozotocin-induced diabetes on phosphoglyceride metabolism of the rat liver.

We have studied the effect of streptozotocin (SZ)-induced diabetes on fatty acyltransferase and phospholipase enzyme activities involved in the synthesis and degradation of rat liver phosphoglycerides. Neither mitochondrial nor microsomal acyl-CoA:glycerol 3-phosphate acyltransferase (GPAT) activity was altered, although insulin treatment stimulated mitochondrial GPAT activity. However, microsomal acyl-CoA:1-acylglycerol 3-phosphate acyltransferase (1-acyl-GPAT) activity increased (24-33 per cent, p less than 0.01) in the diabetic animals using 3 different acyl-CoA donors: palmitoyl-CoA, oleoyl-CoA and linoleoyl-CoA. SZ-induced diabetes also increased acyl-CoA;1-acylglycerol 3-phosphorylcholine acyltransferase (GPCAT) activity (38-45 per cent, p less than 0.01) with 3 different acyl-CoA donors: oleoyl-CoA, linoleoyl-CoA and arachidonoyl-CoA. 1-acyl-GPAT and GPCAT activity returned to normal with insulin treatment. In contrast to the increased activity of the microsomal fatty acyl-transferases 1-acyl-GPAT and GPCAT, SZ-induced diabetes decreased mitochondrial phospholipase A2 activity and lysophospholipase activity (49-70 per cent, p less than 0.01). Insulin treatment of the diabetic rats corrected the decreased lysophospholipase and stimulated phospholipase A2 activity 35 per cent higher than controls. Since microsomal 1-acyl-GPAT and GPCAT are known to have higher activity toward unsaturated fatty acyl-CoA donors, the increased GPCAT activity coupled with the decreased lysophospholipase activity and the increased 1-acyl-GPAT activity in diabetes would tend to increase the formation of newly synthesized phospholipids containing unsaturated fatty acids. This mechanism plus the decreased fatty acid desaturase (4) may be the factors which alter the fatty acid composition of phosphoglycerides in diabetic rat liver microsomes.

Influence of hypo- and hyperthyroidism on rat liver glycerophospholipid metabolism.

The effects of hyper- and hypothyroidism on enzyme activities involved in phospholipid metabolism in the rat liver were studied. Hyperthyroidism significantly decreases activities of both microsomal acyl-CoA:glycero-3-phosphate acyltransferase (GPAT) (34%, p less than 0.01) and microsomal acyl-CoA:1-acylglycero-3-phosphocholine acyltransferase (GPCAT) (28-33%, p less than 0.01). This may contribute to the decreased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hyperthyroidism. Mitochondrial GPAT, phospholipase A2 and cytosol lysophospholipase are unaffected by hyperthyroidism. In contrast, hypothyroidism stimulates mitochondrial GPAT (38%, p less than 0.01) and microsomal GPCAT (14-19%) activities but decreases both mitochondrial phospholipase A2 (36%, p less than 0.01) and cytosol lysophospholipase (56%, p less than 0.01) activities. The increased GPCAT activity may contribute to the increased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hypothyroidism. Triiodothyronine (T3) treatment of the hypothyroid rat (25 micrograms/100 g body weight/day for four days) corrected phospholipase A2 and lysophospholipase activities to the level of the control rat, but failed to correct the increased mitochondrial GPAT activity and not only corrected but lowered GPCAT activity to the level of the hyperthyroid rat.

Effects of streptozotocin-induced diabetes on microsomal long-chain fatty acyl-CoA synthetase and hydrolase.

Streptozotocin-induced diabetes significantly decreased rat liver microsomal long-chain fatty acyl-CoA (LCA-CoA) hydrolase. The decrease was observed using either palmitoyl-CoA (35 per cent, p less than 0.01) or oleoyl-CoA (23 per cent, p less than 0.01) as the substrate for the enzyme. Under the same conditions, diabetes did not significantly alter activity of LCA-CoA synthetase. Daily subcutaneous injections of protamine zinc insulin (10-12 units/day) into the diabetic rats returned their blood glucose to normal but only partially corrected the LCA-CoA hydrolase activity and did not effect LCA-CoA synthetase activity. The decreased LCA-CoA hydrolase and the unchanged LCA-CoA synthetase activities in the diabetic rat liver were interpreted as factors that may contribute to elevation of fatty acyl-CoA levels in the diabetic liver.

Effects of dietary fats on fatty acid composition and delta 5 desaturase in normal and diabetic rats.

We have studied the effect of various diets on the phospholipid fatty acid composition and in vitro delta 5 desaturase activity of hepatic microsomes derived either from the normal or streptozotocin-induced diabetic rat. The diets studied were the standard rat chow diet and a basal fat-free diet supplemented either with 20 percent saturated fat, 20 percent unsaturated fat, or 20 percent menhaden oil. Phospholipid fatty acid composition analysis revealed that the normal rat fed the saturated fat or menhaden oil diet had significantly decreased arachidonate levels, consistent with decreased delta 5 desaturase activities and decreased 18:2n-6 intake. On the contrary, the unsaturated fat diet decreased dihomo-gamma-linolenate and increased arachidonate levels, without increased delta 5 desaturase activity. Streptozotocin-induced diabetes resulted in decreased arachidonate and delta 5 desaturase activity. The unsaturated fat diet fed to the diabetic rat also failed to correct this decreased delta 5 desaturase activity. The unsaturated fatty acids in this diet also displaced a substantial amount of n-3 fatty acids in both normal and diabetic microsomes, due to the competition between these two fatty acid families for incorporation into the membrane phospholipids. Conversely, the menhaden oil diet fed to the normal and diabetic rats displaced n-6 fatty acids, reduced delta 5 desaturase activity, and enhanced 22:6n-3 incorporation into diabetic microsomes.

Effect of anabolic hormones and insulin-like growth factor-I on muscle mass and strength in elderly persons.

The secretion of growth hormone and testosterone declines with age. Because these hormones have prominent anabolic effects on skeletal muscle, part of the age-related decrease in muscle mass and strength have been attributed to declining levels of these hormones; however, growth hormone replacement in healthy elderly people has produced side effects but no functional benefit. In contrast, short-term growth hormone and insulin-like growth factor 1 treatment have reduced protein loss and improved function during acute catabolic states. Although testosterone replacement has increased lean body mass and improved the sense of well being, functional benefit has not been clearly demonstrated, and the risks of long-term replacement have not been defined.

My spastic patients.

Managing patients with severe cerebral palsy can be very challenging due to communication difficulties and unusual presentations. The tribulations and rewards of a general practitioner dealing with these patients are detailed in this article.