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Mechanism of intracellular calcium ([Ca²⁺]_i) inhibition of lipolysis in human adipocytes ¹

BINGZHONG XUE, ANDREW G. GREENBERG^*, FREDERIC B. KRAEMER and MICHAEL B. ZEMEL²

Department of Nutrition, The University of Tennessee, Knoxville, Tennessee 37996, USA;
^* JM-USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, USA; and
Division of Endocrinology, Department of Medicine, Stanford University, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, USA

²Correspondence: The University of Tennessee, 1215 W. Cumberland Ave., Room 209, Knoxville, TN 37996-1900, USA. E-mail: mzemel{at}utk.edu

SPECIFIC AIMS

The present study was conducted to investigate the mechanism(s) responsible for the anti-lipolytic effect of intracellular calcium ([Ca²⁺]_i) in human adipocytes, thereby providing insight into the role of [Ca²⁺]_i in the development of obesity.

PRINCIPAL FINDINGS

1. Depolarization of human adipocytes by KCl causes significant increases in [Ca²⁺]_i, which greatly inhibits agonist-stimulated lipolysis
We used KCl as a depolarizing agent to stimulate [Ca²⁺]_i in human adipocytes. A dose-dependent increase in [Ca²⁺]_i was found, with 17.86 ± 1.65 nM, 87 ± 5.79 nM, and 158.71 ± 8.55 nM increases over baseline in response to 50, 80, and 100 mM KCl stimulation, respectively (P<0.05).

KCl (100 mM) also inhibited lipolysis induced by the following agonists (Fig. 1 ): isoproterenol, a ß-adrenergic receptor agonist; 8-cyclopentyl-1,3-dipropyl xanthine (DPCPX), a selective A1 adenosine receptor antagonist; forskolin, an adenylate cyclase activator; isobutyl methyl xanthine (IBMX), a phosphodiesterase (PDE) inhibitor; and 8-bromo-cAMP, a hydrolyzable cAMP analog that activates protein kinase A. However, lipolysis induced by dibutyryl cAMP, a nonhydrolyzable cAMP analog, was not inhibited by KCl. Insulin, which inhibits adipocyte lipolysis by activation of PDE, also inhibited 8-bromo-cAMP-induced lipolysis by 90% but was unable to inhibit dibutyryl cAMP-induced lipolysis. Our data suggest that KCl may exert its anti-lipolytic effect primarily via activation of PDE, similar to that of insulin.

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of KCl on human adipocyte [Ca2+]i (A, B) and forskolin-stimulated lipolysis (C). A) Human preadipocytes were plated and differentiated into mature adipocytes. Adipocytes were loaded with Fura-2/AM and [Ca2+]i was measured using a Fura-2 dual wavelength imaging system. The upper and lower panels represent fluorescent images of adipocyte [Ca2+]i before and after the addition of vehicle (H2O), 50, 80, and 100 mM KCl, respectively. B) Quantification of KCl-stimulated increase in [Ca2+]i. *P < 0.05 vs. control. C) Human adipocytes were treated with 0.1 µM forskolin and the indicated concentrations of KCl for 4 h. Glycerol release was measured as nmol/µg DNA and expressed as % of control. Data are expressed as mean ± SE for 6 experiments. *P < 0.05 vs. no KCl treatment.

2. The anti-lipolytic effects of KCl and insulin are mediated by the activation of adipocyte PDE 3B, albeit with different mechanisms
It has been reported that adipocytes possess several PDE isoform activities. In the present study, we also found that human adipocytes contain comparable PDE 3B and PDE 4 activity and less PDE 1 activity. Figure 2 demonstrates that KCl-inhibited lipolysis was reversed by the nonselective PDE inhibitor IBMX and the selective PDE 3B inhibitor cilostamide. However, the selective PDE 1 and 4 inhibitors 8-methoxymethyl IBMX and rolipram failed to reverse KCl’s effect. Cilostamide and its derivatives have been shown to selectively inhibit PDE3B activity and to block the anti-lipolytic effect of insulin. These data suggest that KCl’s anti-lipolytic effects are mediated by activation of PDE 3B, the same PDE isoform that mediates insulin’s anti-lipolytic effect.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of different agonists on the reversal of KCl inhibition of isoproterenol-stimulated lipolysis. Human adipocytes were treated with isoproterenol, KCl, and the indicated agonists for 4 h. Data are expressed as mean ± SE for 6 experiments. *P < 0.05 vs. control. P < 0.05 vs. KCl + isoproterenol. DPCPX: 8-cyclopentyl-1,3-dipropyl xanthine; IBMX: isobutyl methyl xanthine.

We then compared the anti-lipolytic effect of KCl with that of insulin. KCl (100 mM) and a maximum concentration of insulin (25 nM) exerted potent anti-lipolytic effects. However, KCl and insulin also exerted an additive anti-lipolytic effect. Whereas the PI-3 kinase inhibitor wortmannin completely reversed insulin’s anti-lipolytic effect, it only partially blocked KCl’s effect. These data suggest that KCl and insulin may activate PDE 3B via different mechanisms.

3. KCl inhibits isoproterenol stimulated increase in cAMP level as well as phosphorylation of hormone-sensitive lipase in human adipocytes
Isoproterenol (5 nM) induced a fivefold increase in cAMP level (P<0.001) and a significant increase in adipocyte hormone-sensitive lipase (HSL) phosphorylation in human adipocyte phosphoproteins immunoprecipitated with anti-HSL antibody. However, this was blocked by the addition of KCl and insulin.

DISCUSSION

In our previous study, we demonstrated that agouti protein, a murine obesity gene product, inhibited adipocyte lipolysis via an increase in [Ca²⁺]_i. Adipose tissue from obese agouti mutants exhibited a reduced lipolytic rate in response to ß-adrenergic receptor agonists and PDE inhibitors whereas the response to dibutyryl cAMP was normal. This suggests that increasing [Ca²⁺]_i by agouti may affect adipocyte cAMP levels, leading to inhibition of lipolysis.

In the present study, KCl was used to stimulate [Ca²⁺]_i by depolarization and was shown to exert potent anti-lipolytic effects. KCl treatment inhibited lipolysis induced by ß-adrenergic receptor activation, adenylate cyclase activation, PDE inhibition, and a hydrolyzable cAMP analog. However, lipolysis induced by dibutyryl cAMP, a nonhydrolyzable cAMP analog, was not affected by KCl, suggesting that this effect of KCl may be mediated primarily by the activation of PDE. Consistent with this, KCl treatment significantly prevented isoproterenol-stimulated cAMP production in human adipocytes. This also resulted in a net decrease in HSL phosphorylation and, consequently, inhibition of lipolysis.

More than 10 PDE isoforms have been discovered and characterized. Several studies have shown that adipocytes possess several PDE isoform activities; a low K_m, cGMP-inhibitable PDE isoform (PDE 3B), the PDE isoform that mediates insulin’s anti-lipolytic activity, accounts for the major PDE activity. A cAMP-specific PDE isoform (PDE 4) is the major cytosolic PDE isoform. The adipocyte cytosolic fraction also contains distinct Ca²⁺/calmodulin- and cGMP-stimulated PDE activity (PDE 1 and 2, respectively), albeit to a less extent.

We found that human adipocytes contain comparable PDE 3B and PDE 4 activity and less PDE 1 activity. KCl inhibition of isoproterenol-stimulated lipolysis was reversed only by the selective PDE 3B inhibitor cilostamide, suggesting that the anti-lipolytic effect of KCl is mediated by activation of PDE 3B, the same PDE isoform that mediates insulin’s anti-lipolytic effect. However, we showed that the anti-lipolytic effects of KCl and insulin are additive. PI-3 kinase inhibition by wortmannin, which has been shown to be critical in insulin-induced activation of PDE 3B, completely abolished insulin’s anti-lipolytic effect. However, it only partially blocked KCl’s effect. These data indicate a different regulatory effect of insulin and KCl on PDE 3B.

In the case of insulin, receptor activation leads to the activation of PI-3 kinase. The generation of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate by PI-3 kinase results in recruitment and phosphorylation of the serine-threonine kinase protein kinase B (PKB) by 3'-phosphoinositide-dependent kinase-1 (PDK1). PKB then phosphorylates and activates PDE 3B, leading to a reduction in cAMP and inhibition of lipolysis. However, it is not clear which pathway leads to KCl’s activation of PDE 3B. As new members of the PDE family have been identified recently, it is possible that KCl will activate PDE isoforms (other than PDE 3B) that are not yet well characterized.

In conclusion, we demonstrated that KCl increases [Ca²⁺]_i via depolarization, which exhibits potent anti-lipolytic effects in human adipocytes. This effect is mediated primarily by activation of adipocyte PDE and a reduction in cAMP levels, leading to a decrease in HSL phosphorylation and inhibition of lipolysis.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic illustration of mechanisms of the anti-lipolytic effect of [Ca2+]i. Increasing [Ca2+]i by KCl leads to the activation of adipocyte PDE 3B via a mechanism different from that of insulin. This results in a reduction in cAMP levels, leading to a decrease in PKA activity, a decrease in HSL phosphorylation, and, consequently, inhibition of lipolysis. A1AR: A1 adenosine receptor; AC: adenylate cyclase; IRS: insulin receptor substrate; PI-3 kinase: phosphoinositide 3 kinase; PKB: protein kinase B; PDE 3B: phosphodiesterase 3B; HSL: hormone-sensitive lipase; PKA-C: protein kinase A catalytic subunit; PKA-R: protein kinase A regulatory subunit.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0278fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.01-0278fje

This Article Full Text (PDF) All Versions of this Article: 15/13/2527 01-0278fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by XUE, B. Articles by ZEMEL, M. B. Search for Related Content PubMed PubMed Citation Articles by XUE, B. Articles by ZEMEL, M. B.