Role of tyrosine phosphorylation in leptin activation of ATP‐sensitive K⁺ channels in the rat insulinoma cell line CRI‐G1

Abstract

Using whole‐cell and cell‐attached recording configurations, the role of phosphorylation in leptin activation of ATP‐sensitive K+ (KATP) channels was examined in the rat CRI‐G1 insulinoma cell line. Whole‐cell current clamp recordings demonstrated that, following dialysis with the non‐hydrolysable ATP analogue 5′‐adenylylimidodiphosphate (AMP‐PNP; 3‐5 mM), the leptin‐induced hyperpolarization and increase in K+ conductance were completely inhibited. Under current clamp conditions, application of the broad‐spectrum protein kinase inhibitor H‐7 (10 μM) had no effect on the resting membrane potential or slope conductance of CRI‐G1 insulinoma cells and did not occlude the actions of leptin. Application of the tyrosine kinase inhibitors genistein (10 μM), tyrphostin B42 (10 μM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell‐attached recordings, the presence of tyrphostin B42 (10 μM) in the pipette solution activated tolbutamide‐sensitive KATP channels in CRI‐G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. The serine/threonine‐specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 μM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole‐cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 μM) prevented the actions of both leptin and tyrphostin B42. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3‐kinase (PI 3‐kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.

Leptin Suppression of Insulin Secretion and Gene Expression in Human Pancreatic Islets: Implications for the Development of Adipogenic Diabetes Mellitus

“…These studies in human islets further add to a mounting body of evidence showing that leptin suppresses insulin secretion by pancreatic β-cells (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)32) and does so by activating the ATP-sensitive K + channel on β-cells (21,27,30). Further, these studies augment the available evidence that leptin inhibits not only insulin secretion, but also the expression of the proinsulin gene.…”

“…Germane to the studies undertaken and described herein is that leptin also acts directly on receptors in pancreatic β-cells to suppress insulin secretion in rodents (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Leptin suppresses insulin secretion, insulin messenger ribonucleic acid (mRNA) levels, and transcription from the rat insulin promoter (30a) in isolated islets, perfused pancreata of mice and rats, and rats studied in vivo.…”

Leptin Effects on Pancreatic β-Cell Gene Expression and Function

“…Consequently, activation of K ATP channels in pancreatic ␤-cells by leptin reduces cytosolic calcium concentration, and this fall can be overcome by coincubation with 20 mmol/l glucose and GLP-1 (10 nmol/l). Although the molecular mechanism by which leptin activates K ATP channels is not fully understood, studies indicate that phosphorylation or dephosphorylation of proteins may be involved (51), and treatment of insulinoma HIT-T15 cells with murine leptin results in a threefold activation of phosphatidylinositol (PI) 3-kinase (21). Phosphodiesterase 3B, which reduces the cellular content of cAMP, is also activated by leptin, and leptin (1-5 nmol/l) suppressed the elevation of cAMP induced by GLP-1 in HIT-T15 cells (5 nmol/l) (21).…”

“…The different functional, molecular, and gene regulatory effects of leptin involving insulin secretion (8,18 -28, 30,31,33-40,42-44,51-61), pancreatic ␤-cell gene expression (19,20,31,38,62,63), and signal transduction (21,22,27,38,51,54,64) are summarized in Table 1.…”

Physiological and Pharmacological Characteristics of Quisqualic Acid-Induced K+-Current Response in the Ganglion Cells of Aplysia.

“…Recently, it has been reported that stimulation of the mGluR 1 receptor produces the opening of a cationic channel directly by activation of Src-type of protein tyrosine kinase without activation of the trimeric G protein [37]. Furthermore, stimulation of a certain type of cytokine receptor family, such as leptin receptor, can activate a protein phosphatase to open the K ATP channel without the activation of trimeric G protein [38].…”