Albert Waksman

Modifications of cellular lipids induce insulin resistance in cultured hepatoma cells

We altered the cellular lipid composition of an insulin sensitive rat hepatoma cell line through supplementation of the culture medium with linoleic acid (18:2) or 25-hydroxycholesterol, and we studied the effects on insulin stimulation of aminoacid transport system A and glycogen synthesis. The basal rate of sodium-dependent aminoisobutyric acid uptake was slightly reduced in hydroxysterol-treated cells and increased in 18:2-enriched cells. Maximal insulin stimulation of transport was decreased by about 40% in both 18:2 and 25-hydroxycholesterol modified cells, as compared to control cells. In addition to reduced responsiveness, the hydroxysterol-treated cells also showed a diminished sensitivity to insulin, as revealed by a right-shift of the dose-response curve leading to a ED50 of 1.2 X 10(-8) M (P less than 0.02), as compared to 2.45 X 10(-9) M in control cells and 2.13 X 10(-9) M in 18:2 enriched cells. Concerning glycogen synthesis, the basal rate was unaffected by 25-hydroxycholesterol supplementation and slightly reduced in cells enriched in 18:2. Maximal insulin stimulation of glycogen synthesis was reduced by about 40% in both types of lipid modified cells. 25-Hydroxycholesterol again provoked a decrease in sensitivity to insulin: the ED50 was enhanced to 4.9 X 10(-9) M (P less than 0.05), as compared to 1.25 X 10(-9) M in control cells and 1.57 X 10(-9) M in 18:2-supplemented cells. Taken together with the previously reported changes of insulin binding to lipid modified hepatoma cells (Bruneau et al. (1987) Biochim. Biophys. Acta 928, 287-296) our results demonstrate an influence of alterations of the cellular lipid composition on both binding and biological actions of insulin, leading to an insulin-resistant state. Divergences between insulin binding and action were obtained and it was suggested that post-binding events may be responsible for the observed changes. Our findings may be relevant to experimental and clinical states of insulin resistance.

Intramitochondrial intermembranal large amplitude protein movements: I. A possible novel aspect of membrane fluidity

Summary Large amplitude protein movements between mitochondrial inner membrane and intermembranal space have been studied. o 1 — Effects of exogenous Na+, K+, or NH4+ salts of aspartate, glutamate, acetate, pyruvate, β-hydroxybutyrate, 2-oxoglutarate, succinate, malate, fumarate and phosphate have been tested. 2 — Specificity of the influence of these substances on the protein release phenomenon have been studied by comparing the action of increasing concentrations of these substances and by polyacrylamide gel electrophoresis on the released proteins. 3 — Reversibility of the protein release has been shown to occur by realizing release binding cycles and by polyacrylamide gel analysis. 4 — A discussion excluding possible methodological artifacts and swelling as release vectors is developed. 5 — Possible functional consequences of the transitory presence of certain proteins in two submitochondrial compartments are presented.

Lipid composition in liver and brain of genetically obese (ob/ob), heterozygote (ob/+) and normal (+/+) mice

Lipid composition was studied in liver and brain of normal (+/+), heterozygote (ob/+) and obese (ob/ob) mice. It was found that this genetic defect is expressed differently in the lipid composition of these organs. Cholesterol is increased in liver but strongly decreased in brain of obese animals. Phosphatide fatty acid composition is modified in liver and not in brain. In contrast, phospholipids and total ganglioside sialic acid are affected similarly in both organs. Although clinically normal, heterozygote (ob/+) mice already show an abnormal lipid composition in liver and brain. The potential importance of these results is presented.

Reversible intramitochondrial release of protein related to unsaturated fatty acids of membranes

Abstract The influence of temperature on intramitochondrial protein and enzyme release was studied in control and “lipid-deficient” rat liver mitochondria and in synaptosomal and “cell body” mitochondria of rat brain. (i) The fatty acid composition of the phospolipid fraction was shown to be different in control and lipid-deficient preparations. (ii) Arrhenius curves for temperature-dependent release of protein showed breaks. (iii) When comparing control to lipid-free rat liver mitochondria and cell body to synaptosomal rat brain mitochondria, shifts in the breaks in the Arrhenius plots were observed for release of aspartate aminotransferase, protein and malate dehydrogenase. (iv) Intramitochondrial temperature-dependent, succinate-induced protein release was also studied in rat liver mitochondria which had previously undergone a succinate-induced release and rebinding cycle. These mitochondria showed a temperature-dependent protein release identical to that of untreated mitochondria.

Succinate transport inhibition by valproate in rat renal mitochondria

Sodium valproate is an antiepileptic drug which may have side effects on different organs. Its mechanism of action, as yet unclear, may involve an effect on membranes. One possibility, an effect on mitochondrial membranes via an inhibition of oxidative phosphorylation, has been studied here by observing the transmembranal transport of a respiratory substrate, succinate, in rat kidney mitochondria incubated with valproate, or from rats injected with valproate. Succinate transport was inhibited in both conditions, which suggests that the effect was probably due to a direct effect of valproate rather than to an action of a valproate metabolite. For the valproate-incubated mitochondria, inhibition, described by a bell-shaped curve, started at a valproate concentration of 10(-7) M and was maximum at valproate 10(-5)M. Valproates effect on mitochondrial transmembranal succinate transport can be compared to other evidence for membranal actions of valproate, actions which may clarify certain therapeutic or toxic properties of this drug.

Intramitochondrial release and binding of mitochondrial aspartate aminotransferase and malate dehydrogenase in the presence and absence of monovalent and bivalent cations

Abstract Intramitochondrial release and binding of mitochondrial aspartats aminotransferase and malate dehydrogenase was shown to be controlled by a sucrose-cation-sucrose cycle in vitro . The effect of ion concentration on the aspartate aminotransferase release suggests distinct modes of action for bivalent and monovalent cations.

Intramitochondrial release and binding of mitochondrial aspartate aminotransferase and malate dehydrogenase in the presence and absence of exogenous succinate

Abstract Differential shuttling of aspartate aminotransferase and malate dehydrogenase between submitochondrial fractions was shown to occur. This phenomenon is dependent upon the nature of the extramitochondrial compartment.

Are soluble and membrane-bound rat brain acetylcholinesterase different ?

Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and >250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AchE possess hydrophobic domain(s) different from the 20 kD peptide already described.

Seasonal variation of the composition of membrane lipids in liver mitochondria of the hibernator cricetus cricetus relation to intramitochondrial intermembranal protein movement

Abstract 1. 1. European hamster ( Cricetus cricetus ) raised under constant temperature conditions show a seasonal variation in the profiles of the structural fatty acids of the liver mitochondrial membrane. In these animals there are but small differences between sleeping and aroused animals in winter. 2. 2. The most important variation in lipid composition occurs in the prehibernating phase—at the end of summer and the beginning of autumn. 3. 3. However, there is no apparent coordinated relation between the seasonal variation of the composition of the fatty acids and the membrane “fluidity” as expressed by the break in the Arrhenius curve for protein release in intermembranal space of the liver mitochondria. 4. 4. This break occurred at a higher temperature in active animals in winter (arousedl than in summer. 5. 5. No general correlation could be found between the breaks in the Arrhenius curves and the variations of the different fatty acid species during the seasonal cycle, except for the most polyunsaturated fatty acid (docosahexanoic acid) where an excellent inverse correlation was observed. 6. 6. Our results suggest that the more fluid parts of the lipidic leaflet of the mitochondrial membrane are those more specifically involved in such phenomena as succinate induced intramitochondrial protein movement and that the changes in composition of the mitochondrial lipids are a possible adaptative advantages for the hibernator.

Release and binding of proteins and enzymes with isolated inner mitochondrial membranes

Abstract Extramitochondrial substances trigger specific and reversible release of proteins and enzymes from inner membranal compartment towards intermembranal space. This shuttling phenomenon has been shown to occur with isolated inner membranes.