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. 1997 Jun;46(6):1087-93.
doi: 10.2337/diab.46.6.1087.

Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells

T J Kieffer  1 , R S Heller, C A Leech, G G Holz, J F Habener
Affiliations

Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells

T J Kieffer et al. Diabetes. 1997 Jun.

Abstract

In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that beta-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting beta-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the beta-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of [Ca2+]i in ob/ob beta-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob beta-cells demonstrated that leptin activated ATP-sensitive potassium channels (K(ATP)) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of K(ATP). Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the beta-cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM.

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Figures

FIG. 1
FIG. 1
A: RT-PCR of rat hypothalamic and islet cDNA using a specific primer pair to amplify a 376-bp region of the Ob-Rb. The preparation of the cDNA was either in the presence (RT+) or absence (RT−) of reverse transcriptase. B: autoradiogram of a Southern blot of the above gel probed with a 32P-labeled oligo with a complementary sequence internal to the PCR primers. C: RT-PCR for Ob-Rb in tumor-derived cell lines representative of islet β-cells (INS-1), δ-cells (RIN-B2), and α-cells (INR1G-9); RT-PCR of α-cell (INR1G-9) cDNA using a specific primer pair to amplify a 307-bp region of glucagon.
FIG. 2
FIG. 2
A: binding of purified boiled (left panel) or nonboiled (right panel) Cy3-leptin to dispersed rat islet cells. B: combined Cy3-leptin binding and immunofluorescence of dispersed rat islet cells. Both columns show the same fields of view, either under a DTAF filter to allow hormone detection (green, left column) or under a Cy3 filter to allow detection of the Cy3-leptin (orange, right column). Hormone immunoreactive cells are indicated as either positive (solid arrows) or negative (hollow arrows) for Cy3-leptin binding. C: dual immunofluorescence of dispersed rat islet cells for hormone immunoreactivity (green, left column) and Ob-R immunoreactivity (orange, right column).
FIG. 3
FIG. 3
A: simultaneous perforated patch recordings of membrane potential (top trace) and fura-2 measurement of [Ca2+]i (lower trace) from a normal mouse β-cell bathed in SES containing 5.5 mmol/l glucose. A 3-min application of 100 ng/ml leptin is indicated by the bar. B: measurement of [Ca2+]i from an ob/ob mouse β-cell bathed in SES containing 5.5 mmol/l glucose. The introduction of 100 ng/ml leptin is indicated by the lower bar. The contents of the bath were switched to a solution containing 100 ng/ml leptin, 20 mmol/l glucose, and 10 nmol/l GLP-I, as indicated by the step of the upper bar. C: recordings from an ob/ob mouse β-cell bathed in SES containing 5.5 mmol/l glucose and voltage clamped at −70 mV using the perforated patch technique. The pipette potential was shifted by ±10 mV (1 s duration) to monitor the membrane conductance. Leptin (100 ng/ml) and tolbutamide (100 μmol/l) application (indicated by bars) were from puffer pipettes placed close to the cell. D: single channel records in an on-cell patch from an ob/ob mouse β-cell bathed in SES containing 5.5 mmol/l glucose. The currents shown in each panel are continuous stretches of data. Control currents are shown in the left panel, and currents beginning 4 min after the start of a 3-min pulse of leptin (100 ng/ml) are shown in the right panel. Channel openings are shown as downward current deflections indicated by the opening of 1, 2, or 3 channels from a closed state (0). E: between 10 and 13 min after the start of the leptin pulse, the patch pipette was stepped to different potentials to obtain the single-channel current-voltage relationship to measure the conductance of the channel. Single-channel current amplitudes at the different potentials were obtained from Gaussian fits to all-points histograms of digitized current records in D. F: the mean value for open probability of KATP plotted against time (in minutes) from six on-cell patches from ob/ob mouse β-cells at a pipette potential of 0 mV.
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