Prostaglandin‐sensitive adenylyl cyclase of cultured preadipocytes and mature adipocytes of the rat: Probable role of G_i in determination of stimulatory or inhibitory action

Abstract

Preadipocytes of rats were obtained from the stromal-vascular fraction of collagenase-digested perirenal fat pads and grown in serum-containing medium. By day 8 of culture the cells reached confluence and by 12 days were lipid-laden. The adenylyl cyclase of the plasma membranes was compared to that of mature fat cells. Unlike the membranes from adipocytes, the preadipocytes showed adenylyl cyclase activity that was stimulated by GTP. Stimulation of preadipocyte membranes by Gpp(NH)p, NaF, and forskolin was comparable to that of membranes from adipocytes, but the response to epinephrine and isoproterenol was minimal (approximately 1.5-fold for preadipocytes vs. 4-5-fold for adipocytes). In contrast, GTP-dependent stimulation of adenylyl cyclase of preadipocytes by PGE1 was nearly 8-fold. Stimulation occurred even in the presence of both GTP and 140 mM NaCl, a condition that leads to inhibition by PGE1 of adenylyl cyclase in membranes of adipocytes. Other characteristics of the adenylyl cyclase of preadipocyte membranes that differ from those of adipocytes include lack of inhibition by GTP of forskolin-activated activity, and, following treatment with pertussis toxin, enhanced stimulation by PGE1. ADP-ribosylation of Gi and Gs with pertussis and cholera toxins, respectively, indicated that the membranes of preadipocytes contained only 5-11% of the Gi of adipocytes and a much lower ratio of Gi:Gs. These findings suggest that cultured preadipocytes have an incompletely developed Gi pathway that may account for the stimulatory effect of prostaglandins on the adenylyl cyclase of these cells as opposed to the inhibitory action of PG in mature fat cells.

Role of guanine nucleotide regulatory proteins in insulin stimulation of glucose transport in rat adipocytes. Influence of bacterial toxins

“…Sensitivity of a biological response to pertussis or cholera toxin does not serve as direct proof that a Gprotein is involved in the signal-transmission sequence for that response, and this caution must be kept in mind while drawing conclusions. However, this finding can be suggestive, and there are a number of situations where G-protein involvement initially indicated by pertussistoxin sensitivity in intact cells has been confirmed by other criteria in more purified systems [1,6,[43][44][45][46][47][48][49][50]. Ref-…”

“…3) suggests that control of insulin coupling efficiency in adipocytes is largely independent of cyclic AMP. The influence of cholera toxin on adenylate cyclase and lipolysis has been extensively studied in adipocytes [44,51,52], but little is known about cholera-toxin effects on insulin action in these cells to compare with the work in liver [5] and muscle cells [40]. Adrenergic agents can regulate insulin sensitivity [12,36], and Lonnroth et al…”