Kenneth P. Strickland

Fatty acid oxidation by skeletal muscle mithochondria in duchenne dystrophy

Abstract In a study of palmitate oxidation by skeletal muscle mitochondria in Duchenne muscular dystrophy, it was found that in cases with a family history of sex-linked recessive inheritance palmitate oxidation was markedly reduced whereas in isolated cases the values were essentially normal. Reduced values of palmitate oxidation were also observed in the definite female carriers o the sex-linked dystrophic gene. These findings suggest that isolated cases may have a different disorder from the cases of familial Duchenne muscular dytrophy.

Triglyceride metabolism in skeletal muscle from normal and dystrophic mice.

Abstract 1. 1. Optimal conditions were established and measurements were made of the capacity of homogenates of skeletal muscle from normal and dystrophic mice (Strain 129) to incorporate [1- 14 C]palmitate or sn -[1- 14 C ]glycerol-3- P into triglycerides and phospholipids. 2. 2. sn -[1- 14 C ]Glycerol-3- P was incorporated into the triglycerides and phospholipids to about the same extent in both normal and dystrophic muscle. Similarly no difference was observed in the incorporation of [1- 14 C]palmitate into the phospholipids. The incorporation of [l- 14 C]palmitate into the triglycerides showed a slight increase in the muscle of dystrophic mice. The data obtained were consistent with the view that no quantitative differences appear to exist in the de novo synthesis of triglycerides and phospholipids in normal and dystrophic muscle. Suggestive evidence was obtained of an increased exchange of free fatty acid with the fatty acid of triglycerides in dystrophic muscle. 3. 3. The capacity of skeletal muscle homogenates from normal and dystrophic mice (Strain 129) to carry out the hydrolysis of tributyrin and tripalmitin has been studied. 4. 4. Evidence based on studies with inhibitors suggested the possible existence of two different lipases which are responsible for the hydrolysis of these two substrates. One is termed a “short-chain triglyceride lipase” which catalyses the hydrolysis of tributyrin and the second, a “long-chain triglyceride lipase” which catalyses the hydrolysis of tripalmitin. 5. 5. The long-chain triglyceride lipase (“tripalmitinase”) had an optimal pH of 6.5. Previous treatment of muscle preparation with the non-ionic detergent, Cutscum, was necessary. The addition of albumin had no effect. The enzyme activity was shown to sediment in the 90000 × g pellet. 6. 6. Skeletal muscle from dystrophic mice was found to have significantly elevated short-chain (125 %) and long-chain (minimal value of 52 %) triglyceride lipase activities compared to the activities of the corresponding littermate controls.

Insulin binding to myotonic dystrophy fibroblasts.

Insulin receptor binding was examined in cultured skin fibroblasts from 10 myotonic dystrophy patients and 10 age- and sex-matched control subjects. The conditions for insulin binding to fibroblasts were optimal and employed HEPES binding buffer, pH 8.0 at 15 degrees C for 5 h. These conditions correspond to those previously employed with monocytes from MyD subjects. The normalized initial insulin binding capacity showed a decrease of 62% from 5.04 +/- 0.28% of the total labeled insulin added/mg protein in the control to 1.93 +/- 0.13% in the myotonic dystrophy group (P less than 0.01) due mainly to a marked reduction in high affinity receptors or in receptor affinity. The addition of 1.0 ng/ml of unlabeled insulin produced significant decreases to 3.80 +/- 0.25% in the control group and 1.24 +/- 0.09% in the MyD group. The results are similar to previously reported findings with monocytes from myotonic dystrophy patients and suggest that a surface membrane defect exists in this disease. However, the conditions that have been employed in the binding procedures in all of the studies, while optimal, are performed at a high pH and low temperature and could have an important bearing on the interpretation of a membrane disorder.

Studies of phosphate transport in Escherichia Coli. I. Reexamination of the effect of osmotic and cold shock on phosphate uptake and some attempts to restore uptake with phosphate binding protein

1. The first stage of osmotic shock caused a slight reduction in the primary uptake of inorganic orthophosphate (Pi) in AB3311 cells of Escherichia coli which normally exhibit a biphasic type of phosphate uptake. The second stage of osmotic shock resulted in a marked reduction (a total of 80-85%) in the primary uptake phase and a lesser reduction (50%) in the secondary uptake. When osmotically shocked cells are allowed to recover in a phosphate-free, but otherwise complete medium sufficient repair occurs in the shocked cells to overcome growth lags and to restore the above losses in phosphate uptake almost to normal after 90-180 min of recovery. 2. Extensive investigation was made of the more mild cold shock procedure which involves the rapid disperion of Escherichia coli cells into 80 vol. of water at 2 degrees C. The most consistent cold shock effects, as evidenced by reductions in phosphate uptake, were obtained in cells after they were washed in appropriate buffered salts media, suspended in a minimal volume of water and shocked from 37 degrees C rather than 24 degrees C. Less severe shocks were obtained after washing in imidazole/salts/glucose or Tris/salts/glucose media than in NaCl/Tris. 3. A number of attempts were made to restore by the addition of phosphate binding protein the reduction in phosphate uptake of E. coli AB3311 cells caused by a variety of cold shocks. In no instance was good restoration of phosphate uptake achieved. Qualitatively, it appeared that a better restoration of uptake occurred in unstarved and starved cells washed in imidazole/salts/glucose where the cold shock effect was less severe.

The in vivo incorporation of labelled acetate into the cholesterol and fatty acids of tissues of dystrophic mice

Abstract The in vivo incorporation of acetate-2- 14 C into cholesterol and fatty acid of tissues of dystrophic mice and their littermate controls was determined. The greatest active incorporation of the acetate is observed with liver followed by kidney, muscle, and brain. Comparison of the radioactivites in the whole tissue and the perchloric-acid-soluble fraction for dystrophic mice with those for the controls showed no appreciable differences. When the mice were not starved, both the specific activities and the relative activities (specific activity of dystrophic tissue divided by specific activity of control tissue) of cholesterol and fatty acid were significanctly reduced in liver, kidney, and muscle of the dystrophic animals. Brain and kidney of starved mice showed a reduced relative activity in cholesterol and fatty acid. No changes were observed in liver and muscle. Incorporation into cholesterol and fatty acid, on a whole muscle basis, is in general reduced for dystrophic muscle. Under the conditions studied ( in vivo incorporation for 1 hour into tissues of starved and nonstarved animals), the results obtained do not provide evidence of an enhanced incorporation of acetate-2- 14 C into cholesterol and fatty acids in dystrophic muscle.

Glucose transport and oxidation in adipose tissue of patients with myotonic dystrophy

The effect of insulin on the transport of 2-deoxyglucose and the oxidation of glucose in chopped adipose tissue was investigated in 14 myotonic dystrophy (MyD) patients and 28 age and size-matched control subjects. The transport of 0.55 mM 2-deoxyglucose was measured over 3 min at 37 degrees C both with and without 32 ng/ml of insulin. Oxidation was determined at 37 degrees C for 90 min by the measurement of 14CO2 released from a system containing 0.55 mM glucose with and without 50 ng/ml of insulin. Basal 2-deoxyglucose transport was not reduced in MyD subjects but insulin-stimulated 2-deoxyglucose transport in MyD was significantly less at 0.512 +/- 0.220 nmole compared to control subjects with 0.906 +/- 0.160 nmole/100 mg tissue/3 min (P less than 0.02). Both the basal and insulin-stimulated glucose oxidation were significantly less in the MyD group. Insulin-stimulated oxidation was 2.92 +/- 0.21 nmole in the control subjects compared to 2.20 +/- 0.27 nmole/100 mg tissue/90 min in the MyD cases (P less than 0.02). Similar findings were obtained when calculations were based on nmoles of 2-deoxyglucose transport and glucose oxidation/100 mg lipid. The findings indicate that both glucose transport and oxidation are impaired in MyD.

Palmityl-CoA synthetase activity in the muscle of dystrophic mice

Abstract Palmityl-CoA synthetase activity (acid CoA ligase (AMP), E C 6.2.1.3.) was determined using the radioassay method. The rate of formation of palmityl-CoA under the optimal conditions established was 20 nmoles per mg protein per min for mitochondria and 5.8 nmoles for the 9000 × g supernatant. The activity of palmityl-CoA synthetase in mitochondria from skeletal muscle of dystrophic mice was not significantly different from that obtained in normal littermate controls, whereas the activity of this enzyme in the 9000 × g supernatant fraction from dystrophic muscle preparation was found to be significantly higher than for the corresponding controls. It is concluded that the previously observed decrease in palmitate-1- 14 C oxidation in dystrophic muscle mitochondria was not due to a defect in the activation of palmitic acid.