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OBJECTIVE: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IR(C860S)) showed a defective insulin internalization without affecting insulin binding and IR autophosphorylation. Moreover, this mutation reduces insulin receptor substrate (IRS)-1 tyrosine phosphorylation and insulin-induced metabolic and mitogenic effects. Altogether, these observations support a role of the extracellular domain of IR beta-subunit in insulin and receptor intracellular targeting as well as in insulin signaling. DESIGN AND METHODS: This study assesses in more details the effect of IR(C860S) mutation on the trafficking of the insulin-IR complex. In particular, IR internalization, phosphorylation, dissociation and recycling, as well as insulin degradation and retroendocytosis have been investigated in CHO cells overexpressing either wild type (CHO-IR(WT)) or mutated IRs. RESULTS: the C860S mutation significantly decreases IR internalization both insulin stimulated and constitutive. In spite of a similar dissociation of internalized insulin-IR complex, recycling of internalized IR was significantly faster (half life (t(1/2)): 21 min vs 40 min, P<0.001) and more extensive (P<0.01) for IR(C860S) than for IR(WT). On the other hand, insulin degradation and retroendocytosis were superimposable in both cell lines. As expected, insulin-induced phosphorylation was similar in both IRs, however dephosphorylation was much more rapid and was greater (P<0.01) in CHO-IR(WT) as compared with CHO-IR(C860S) cells. CONCLUSIONS: Transmembrane and intracellular domain of IR seem to be determinants for IR internalization. Now we report that Cys 860 in the IR beta-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.

OBJECTIVE: The objective of the study was to evaluate the expression and functional activity of Peroxisome proliferator-activated receptor (PPAR) gamma in pituitary adenomas from 14 consecutive acromegalic patients and to establish its role in apoptosis. SUBJECTS AND METHODS: Fourteen consecutive acromegalic patients were enrolled in the study. Wistar-Furth rats were used for in vivo studies. Expression of PPARgamma was evaluated by RT-PCR and Western blot. Apoptosis and cell cycle were assessed by FACS analysis. The effects of PPARgamma ligands on transcriptional regulation of GH gene were evaluated by RT-PCR and electromobility shift assay. RESULTS: PPARgamma was expressed in all human GH-secreting adenoma (GH-oma), in normal pituitary tissue samples (39+/-24% and 78+/-5% of immunostained nuclei respectively; P<0.0002; ANOVA), and in rat GH-secreting (GH3) cells. A PPRE-containing reporter plasmid transfected into GH3 cells was activated by ciglitazone or rosiglitazone (TZDs), indicating that PPARgamma was functionally active. Treatment of GH3 cells with TZDs increased apoptosis in a dose-dependent manner (P=0.0003) and arrested cell proliferation, reducing the number of cells in the S-phase (P<0.0001 vs untreated cells). TZDs increased the expression of TRAIL, leaving unaffected that of p53 and Bax. TZDs reduced GH concentrations in the culture media from 43.7+/-5.4 ng/ml to 2.1+/-0.3 ng/ml (P<0.0001) and in cell extracts (P<0.004). PPARgamma-RXRalpha heterodimers bound to GH promoter, inhibiting its activity and reducing GH mRNA levels (1.8 x 10(6) vs 5.7 x 10(6) transcripts respectively vs untreated cells; P<0.002). Subcutaneous GH-oma developed in rats injected with GH3 cells; tumor growth increased in placebo-treated rats and to a lesser extent in TZDs-treated animals (24.1+/-2.0 g, and 14.8+/-4.2 g respectively, P<0.03). Serum GH concentrations were lower in TZDs-treated rats than in controls (871+/-67 ng/ml vs 1.309+/-238 ng/ml; P<0.05). CONCLUSIONS: The results of this study indicate that PPARgamma controls GH transcription and secretion as well as apoptosis and growth of GH-oma; thus, TZDs have the potential of a useful tool in the complex therapeutic management of acromegalic patients.