Effect of diabetes and insulin replacement on the lipid properties of hepatic smooth endoplasmic reticulum.

This study is a characterization of the lipid properties of the smooth and rough endoplasmic reticulum (SER, RER) of liver from streptozotocin-induced diabetic rats. A significant decrease in membrane microviscosity was observed in the SER but not the RER of diabetic rats when compared to that of normal controls. This decrease in SER membrane microviscosity correlated with a decrease in cholesterol/phospholipid ratio of these membranes that could be accounted for solely by a change in the membrane cholesterol content. Changes in phospholipid fatty acyl chain composition were also observed in the SER membranes but these changes were small when compared to the large change in cholesterol content observed. Insulin treatment for only one day did not significantly alter the microviscosity of the SER but after 2, 4 and 6 days of treatment both membrane microviscosity and membrane cholesterol content were restored to values similar to those for normal animals. No significant changes in the RER lipid composition were observed. It is well known that increases in glucose-6-Pase (G-6-Pase) activity of liver ER membranes are associated with diabetic onset. An increase in the specific activity of G-6-Pase was observed in both SER and RER membrane preparations, although the observed increase in the SER membrane is higher. The changes in the G-6-Pase activity of the SER membranes were correlated with the alterations in the microviscosity and lipid composition of these membranes. It is postulated that lipid properties of the SER membranes may contribute to the regulation of G-6-Pase activity in that membrane.

Dynamic lipid changes in rapidly proliferating hepatic smooth endoplasmic reticulum during acute dexamethasone treatment of adrenalectomized rats.

The regulation by the cell of subcellular membrane components is dependent on a highly complex balance of nutritional, hormonal and metabolic events. We have characterized the lipid components of the endoplasmic reticulum (ER) of the liver of adrenalectomized (ADX) rats and the response of these membrane components to glucocorticoid administration. Membrane microviscosity as measured by fluorescence depolarization of 1,6-diphenylhexatriene (DPH) was measured and correlated with lipid composition and content of the membranes. In the ADX rat, a significant increase in membrane microviscosity of the smooth endoplasmic reticulum (SER) was observed and this was accompanied by an increase in the cholesterol content/mg protein and a decrease in the phospholipid content/mg protein. A change in the fatty acyl chain composition is observed with a significant increase in the mole percentage of arachidonic acid (20:4) and an accompanying decrease in saturated fatty acids. Within 2-6 hr of dexamethasone administration, a decrease in membrane microviscosity is observed that returns this value to one similar to that for normal control animals. Both the cholesterol and the phospholipid contents/mg protein are likewise restored to levels similar to that for control animals beginning at the 2-hr time point. The arachidonic acid and saturated fatty acid content of the constituent phospholipids do not begin to return to values similar to those for control animals until 6 hr after dexamethasone administration. From these experiments, we can conclude that glucocorticoids play a significant regulatory role in determining the lipid properties of rat hepatic microsomal membranes.

The dietary regulation of stearyl coenzyme A desaturase activity and membrane fluidity in the rat aorta.

Numerous studies have demonstrated that alterations in membrane composition or fluidity are often associated with alterations in the properties of membrane-bound enzymes. In order to obtain membranes of varying fluidity, rats were fed diets that were either fat-free or supplemented with 15% safflower oil, and two properties associated with aorta and liver microsomal membranes were selected for study: stearyl CoA desaturase activity, and fluidity as monitored by fatty acid composition and microviscosity (measured by fluorescence depolarization). If fluidity directly modulates desaturase activity, one would predict that a low fluidity would stimulate the desaturase activity. Ten times more desaturase activity is present in aorta microsomes from rats on a fat-free diet than in microsomes from rats on a safflower oil supplemented diet. However, on the fat-free diet, these aorta microsomes were more fluid than those of rats fed safflower oil supplemented diet. The fluidity of liver microsomal membranes was not altered in response to diet, despite significant changes in desaturase enzyme content. The contrasting evidence presented here suggests that no correlation exists between desaturase enzyme activity and membrane fluidity in the two tissues studies. We have demonstrated that the aorta has appreciable capacity to desaturate stearyl CoA and that dietary manipulation causes significant changes in aorta membrane fluidity that may be of sufficient magnitude to affect the overall metaboism of aorta cells.

Regulation of S-adenosylmethionine synthetase in Escherichia coli.

Addition of methionine to the growth medium of Escherichia coli K-12 leads to a reduction in the specific activity of S-adenosylmethionine (SAM) synthetase. Thus the enzyme appears to be repressible rather than inducible. Mutant strains (probably metJ(-)) are constitutive for SAM synthetase as well as for the methionine biosynthetic enzymes, suggesting that the regulatory systems for these enzymes have at least some elements in common. Cells grown to stationary phase in complete medium, which have low specific activities of the enzymes, were routinely used for derepression experiments. The lag in growth and derepression when these cells are incubated in minimal medium is shortened by threonine. Ethionine, norleucine, and alpha-methylmethionine are poor substrates or nonsubstrates for SAM synthetase and are ineffective repressors. Selenomethionine, a better substrate for SAM synthetase than methionine, is also slightly more effective at repression than methionine. Although SAM is considered to be a likely candidate for the corepressor in the control of the methionine biosynthetic enzymes, addition of SAM to the growth medium does not cause repression. Measurement of SAM uptake shows that too little is taken into the cells to have a significant effect, even if it were active in the control system.

A comparison of the effects of NN'-dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enrgy-linked reactions in mitochondria and submitochondrial particles.

1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.