MODULATION OF LIPOLYSIS IN RAT ADIPOCYTES BY CHOLESTEROL-DERIVED OXYSTEROLS

Abstract

The effects of several cholesterol-derived oxysterols on lipolysis in · rat adipocytes were studied. These sterols exhibited varying degrees of potency in enhancing epinephrine-induced lipolysis. The oxysterols tested had no lipolytic activity when present alone. Their relative potency in potentiating epinephrine-induced lipolysis is as follows : cholestanone > coprostanone = cholestanol. Cholesterol, in comparison, did not exhibit any potentiating effect. Cholestanone was selected to study its effect(s) on the ?-adrenergic receptor-adenylate cyclase- coupled pathway. The effects of several oxysterols on adipocyte ghost membrane fluidity was also investigated. Cholestanone could enhance and sustain the increase in intracellular cAMP levels induced by epinephrine in intact rat adipocytes. This effect was not due to inhibition of low Km phosphodiesterases because cholestanone did not inhibit the enzyme in cell-free assays. Therefore, protein kinase A activity (PKA ratio) was determined to see if the elevated cAMP level caused by cholestanone can preferentially activate the functional PKA pool that is involved in lipolysis. The results indicated that the cAMP pool measured in the presence of cholestanone and epinephrine could be a general cAMP pool as the PKA ratio measured after similar treatment did not show any significant changes compared to that due to epinephrine alone. Binding studies using [ 125I]cyanopindolol showed that cholestanone can increase the binding affinity of [ 125I]cyanopindolol to / ?-adrenergic receptors but does not affect the maximum binding. This increase in affinity could contribute to the increase in intracellular cAMP level and potentiation of / ?-adrenergic agonist-induced lipolysis. A qualitative analysis also showed that cholestanone could increase the binding of [ 3H]epinephrine to adipocyte ghost membranes. The effects of cholestanone on adenosine A1 receptors were investigated using theophylline and [R]-PIA. The results showed that cholestanone did not potentiate lipolysis by blocking these receptors. Membrane fluidity studies using the fluorescence polarization method and two polyene membrane probes, diphenylhexatriene (DPH) and trimethylaminophenylhexatriene (TMA-DPH) showed that cholestanone and cholesterol can partition into the membrane bilayer. In the presence of epinephrine, cholestanone was shown to partition more towards the bilayer core but cholesterol and the rest of the oxysterols tested could not. This selective partioning could have influenced the activity of the ?-adrenergic receptor-adenylate cyclase system and hence promoted lipolysis. Membrane phospholipid methylation studies also indicated that the oxysterols tested did not affect basal membrane phospholipid methylation. These results showed that the effects of these oxysterols are either rather specific or the membrane methyltransferases are not sensitive to the changes in the physical properties of the membrane caused by these oxysterols. However, more work needs to be done to confirm these assumptions. The location of the inserted sterols in the bilayer if known, will definitely strengthen the postulation that cholestanone can preferentially insert into the bilayer and affect the activity of the ?-adrenergic receptor-adenylate cyclase system. The influence of cholestanone on other lipolysis-related systems should also be investigated to provide an overall understanding of the role of oxysterols in modulating lipolysis.