Abstract
Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of long-chain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)-dephosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet beta-cell. ACC was quantitated in normal rat islets, human islets, and clonal beta-cells (HIT-15 or INS-1) using a [(14)C]bicarbonate fixation assay. In the beta-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations (<50 nmol/l) that inhibited protein phosphatase 2A (PP2A). Furthermore, pretreatment of the cytosolic fraction with anti-PP2A serum attenuated the glutamate- and magnesium-mediated activation of ACC, thereby suggesting that ACC may be regulated by an OKA-sensitive PP2A-like enzyme. Streptavidin-agarose chromatography studies have indicated that glutamate- and magnesium-mediated effects on ACC are attributable to activation of ACC's dephosphorylation; this suggests that the stimulatory effects of glutamate and magnesium on ACC might involve activation of an OKA-sensitive PP2A-like enzyme that dephosphorylates and activates ACC. In our study, 5-amino-imidazolecarboxamide (AICA) riboside, a stimulator of AMP kinase, significantly inhibited glucose-mediated activation of ACC and insulin secretion from isolated beta-cells. Together, our data provide evidence for a unique regulatory mechanism for the activation of ACC in the pancreatic beta-cell, leading to the generation of physiological signals that may be relevant for physiological insulin secretion.
Highlights
Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of longchain fatty acids, which have been implicated in physiological insulin secretion
We report that a glutamate-and magnesium-sensitive phosphatase 2A (PP2A)-like protein phosphatase dephosphorylates and activates ACC in the islet -cell, and that a stimulator of AMP kinase (e.g., 5-amino-imidazolecarboxamide [AICA] riboside) inhibits glucose-stimulated ACC activation and concomitant insulin secretion from isolated -cells
The stimulatory effects of glutamate and magnesium on ACC activity were demonstrable in the cytosolic fraction derived from normal rat (SD and Wistar) islets, human islets, and INS-1 cells
Summary
Glutamate- and magnesium-dependent stimulation of ACC activity in insulin-secreting cells. We determined the sensitivity of this factor to trypsin (see RESEARCH DESIGN AND METHODS) and observed that trypsin treatment of the membrane fraction markedly attenuated its inhibitory potency (36 Ϯ 8% inhibition in the presence of native membrane vs 68 Ϯ 18% in the presence of trypsin-treated membrane; P Ͻ 0.001; n ϭ 6 determinations in each case; additional data not shown) Together, these data suggest localization of a heat-resistant (and even heat-activated) trypsin-sensitive membrane-associated inhibitor of glutamate- and magnesium-activated ACC phosphatase in the pancreatic -cell. We observed that the ability of glutamate and magnesium to stimulate ACC activity was markedly reduced in the cytosolic fractions treated with either the PP2Ac antiserum (Ϫ57%) or OKA (Ϫ48%) (Fig. 5) These data provide additional evidence to suggest that glutamate- and magnesium-stimulated ACC activation might involve dephosphorylation of ACC catalyzed by an OKA-sensitive PP2A-like enzyme. These data show that inhibition of ACC activation results in the inhibition of glucose-stimulated insulin secretion, establishing a possible link between glucose-mediated ACC activation and insulin secretion; these results are compatible with those from extant studies [13,14,15] (see DISCUSSION)
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