Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus

Abstract

Previously we demonstrated the expression of the long form of the leptin receptor in rodent pancreatic beta-cells and an inhibition of insulin secretion by leptin via activation of ATP-sensitive potassium channels. Here we examine pancreatic islets isolated from pancreata of human donors for their responses to leptin. The presence of leptin receptors on islet beta-cells was demonstrated by double fluorescence confocal microscopy after binding of a fluorescent derivative of human leptin (Cy3-leptin). Leptin (6.25 nM) suppressed insulin secretion of normal islets by 20% at 5.6 mM glucose. Intracellular calcium responses to 16.7 mM glucose were rapidly reduced by leptin. Proinsulin messenger ribonucleic acid expression in islets was inhibited by leptin at 11.1 mM, but not at 5.6 mM glucose. Leptin also reduced proinsulin messenger ribonucleic acid levels that were increased in islets by treatment with 10 nM glucagon-like peptide-1 in the presence of either 5.6 or 11.1 mM glucose. These findings demonstrate direct suppressive effects of leptin on insulin-producing beta-cells in human islets at the levels of both stimulus-secretion coupling and gene expression. The findings also further indicate the existence of an adipoinsular axis in humans in which insulin stimulates leptin production in adipocytes and leptin inhibits the production of insulin in beta-cells. We suggest that dysregulation of the adipoinsular axis in obese individuals due to defective leptin reception by beta-cells may result in chronic hyperinsulinemia and may contribute to the pathogenesis of adipogenic diabetes.

Fig. 1

Leptin binds to human pancreatic β-cells. Human leptin labeled with the fluorochrome Cy3 (red fluorescence) was incubated for 30 min with dispersed human islet cells that had been cultured overnight. The cells were costained with an antiserum to insulin and a secondary antibody coupled to fluorescein isothiocyanate (green fluorescence). Shown is a typical β-cell in phase contrast (upper left), stained with insulin antiserum (upper right), with Cy3-labeled leptin (lower left), and with insulin and Cy3-leptin images superimposed (lower right).

Fig. 2

Leptin suppresses proinsulin mRNA levels in human islets in high (11.1 mM), but not normal (5.6 mM), concentrations of glucose. Aliquots of human islets were cultured for 48 h in the absence (A; vehicle) or the presence (B) of 6.25 nM human leptin. RNA concentrations were determined by semiquantitative RT-PCR as previously described (51). Actin mRNA served as a control. The bar graphs represent the relative amounts of RT-PCR products determined by fluorescence densitometry (see Materials and Methods). Values are the mean ± SEM (n = 3). The densitometric value for 5.6 mM glucose at 6 h was arbitrarily set at 1.0.

Fig. 3

Leptin suppresses GLP-1-stimulated proinsulin mRNA levels in human islets in the presence of both normal (5.6 mM) and high (11.1 mM) glucose. Human islets were incubated for 16 h in the presence of normal (5.6 mM) or high (11.1 mM) glucose with and without 6.25 nM leptin and 10 nM GLP-1. The amounts of proinsulin and actin mRNAs were evaluated as described in Fig. 2 and Materials and Methods.

Fig. 4

Leptin inhibits Ca²⁺ influx induced by 16.7 mM glucose in human β-cells. Two fura-2-loaded human β-cells were initially bathed in standard extracellular saline (SES containing 16.7 mM glucose) and exhibited irregular spikes of [Ca²⁺]_i resulting from influx during Ca²⁺-dependent action potentials. A stable baseline of [Ca²⁺]_i in 16.6 mM glucose was established visually and recorded for 5 min before lowering glucose to 5.6 mM and subsequent application of leptin. The bathing solution was then exchanged for 5.6 mM glucose SES, and this led to the inhibition of spiking activity, consistent with hyperpolarization of the β-cell membrane as the glucose concentration was reduced. Reintroduction of 16.7 mM glucose-SES plus 6.25 nM human leptin then led to an initial restoration of spiking activity that was immediately inhibited in the presence of leptin at this high glucose concentration. The upper and lower panels show two representative β-cell responses to leptin of eight cells so studied.