Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient ob/ob mice

Abstract

Leptin controls feeding behavior and insulin secretion from pancreatic beta-cells. Insulin stimulates the production of leptin, thereby establishing an adipoinsular axis. Earlier we identified leptin receptors on pancreatic beta-cells and showed leptin-mediated inhibition of insulin secretion by activation of ATP-sensitive potassium channels. Here we examine transcriptional effects of leptin on the promoter of the rat insulin I gene in rodent beta-cells. A fall in levels of preproinsulin mRNA is detected in vivo in islets of ob/ob mice 24 h after a single injection of leptin, in isolated ob/ob islets treated with leptin in vitro and in the beta-cell line INS-1 on leptin exposure when preproinsulin mRNA expression is stimulated by 25 mM glucose or 10 nM glucagon-like peptide 1. Under these conditions, transcriptional activity of -410 bp of the rat insulin I promoter is inhibited by leptin, whereas transactivation of a 5'-deleted promoter (-307 bp) is not. The -307 sequence contains the known glucose-responsive control elements (E2:A3/4). Constitutive activation of ATP-sensitive potassium channels by diazoxide does not alter leptin inhibition of preproinsulin mRNA levels. Distinct protein-DNA complexes appear on the rat insulin I promoter sequences located between -307 and -410 with nuclear extracts from ob/ob islets in response to leptin, including a signal transducer and activator of transcription (STAT)5b binding site. These results indicate that leptin inhibits transcription of the preproinsulin gene by altering transcription factor binding to sequences upstream from the elements (307 bp) that confer glucose responsivity to the rat insulin I gene promoter. Thus leptin exerts inhibitory effects on both insulin secretion and insulin gene expression in pancreatic beta-cells, but by different cellular mechanisms.

Figure 1

Leptin reduces serum insulin levels and preproinsulin mRNA expression in pancreatic islets of ob/ob mice. (A) Effects of a single intraperitoneal injection with leptin (1 μg/g body weight) or vehicle (n = 6) on plasma insulin and serum glucose concentrations in ob/ob mice. ∗, P < 0.05. (B) Semiquantitative RT-PCR for preproinsulin expression in pancreatic islets from leptin- or vehicle-injected ob/ob mice (Left, in vivo) and islets from noninjected ob/ob mice incubated in culture with leptin (6.25 nM) for 24 h (Right, in vitro). Densitometric values are derived from two independent experiments, each performed in triplicate. ∗, P < 0.05. PCR amplification was performed for 18 cycles, and linearity of the assay with respect to amplification kinetics and relative cDNA input is demonstrated (bottom graphs). Values are means ± SD.

Figure 2

Regulation of preproinsulin mRNA expression by leptin in INS-1 β-cells. (A) Southern detection of OB-RB RT-PCR products in INS-1 cells and rat hypothalamus (positive control). (B) Representative Northern blot for rat preproinsulin mRNA in INS-1 cells treated for 6 h and 16 h with leptin (0.6 or 6.25 nM) or vehicle at 25 mM glucose. (C) Representative Northern blot for rat preproinsulin mRNA in INS-1 cells treated for 16 h with 6.25 nM leptin or vehicle at 11.1 mM glucose in the absence or presence of 10 nM GLP-1. For each study condition, the experiments have been performed at least twice.

Figure 3

Direct gene regulatory effects of leptin on the rat insulin I promoter in INS-1 cells are independent from K_ATP channel activation. (A) DNA constructs of the rat insulin I gene promoter used for luciferase reporter gene assays. Major known regulatory elements are indicated. INS1A, INS1B, and INSTAT denote the location of the oligonucleotides used in electrophoretic mobility-shift analysis. (B) Indicated reporter gene constructs were transfected into INS-1 cells and cells treated with leptin (6.25 nM) or vehicle at 5.6 mM and 25 mM glucose. ∗, P < 0.05. (C) Indicated reporter gene constructs were transfected into INS-1 cells and cells treated with leptin (6.25 nM) or vehicle at 11.1 mM glucose in the presence and absence of 10 nM GLP-1. ∗, P < 0.05. (D) Representative Northern blot for rat preproinsulin mRNA in INS-1 cells treated with 6.25 nM leptin or vehicle at 25 mM glucose in the presence or absence of 100 μM diazoxide. The results shown are representative of three independently performed experiments. (E) −410rINS-1 was transfected into INS-1 cells and cells treated with leptin (6.25 nM) or vehicle at 5.6 mM and 25 mM glucose in the presence and absence of 100 μM diazoxide. Means ± SD.

Figure 4

Leptin induces formation of distinct DNA binding complexes in extracts of ob/ob pancreatic islets on upstream sequences of the rat insulin I promoter. Pancreatic islets of ob/ob mice were treated with 6.25 nM leptin or vehicle and nuclear extracts analyzed by electrophoretic mobility-shift analysis on rat insulin I promoter sequences. Probes were (A) INSTAT and (B) INS-1A and INS-1B (see Fig. 3A). In A antisera to STAT1, 3, and 5b were used to identify specific DNA-binding proteins. STAT5b antiserum shows an interaction by retardation (supershift) of the mobility of the STAT5b complex. A, B, C, D, E, F, STAT5b, specific complexes; ∗, nonspecific complex; FP, free probe; NE, nuclear extract; L, leptin-treated extract; V, vehicle-treated extract; Comp, ×100 excess of unlabeled competitor; mutINSTAT, competitor with mutated STAT binding site.