Amylin/leptin synergy is absent in extreme obesity and not restored by calorie restriction-induced weight loss in rats.

OBJECTIVE: Co-administration of amylin and leptin induces synergistic and clinically meaningful (>10%) weight loss that is attenuated as the degree of obesity increases. We explored whether calorie restriction (CR) could restore amylin/leptin synergy in very obese rats. METHODS: Sprague Dawley rats on high-fat diet (696 ± 8 g, n = 72) were randomized to three cohorts (C1-C3). Rats in C1 were administered vehicle, rat amylin (50 µg kg-1 d-1), murine leptin (125 µg kg-1 d-1) or amylin and leptin for 28 days (n = 6 per group) via subcutaneous minipump. Simultaneously, C2 and C3 rats initiated CR. After moderate (12.4 ± 0.3%, 86.7 ± 2.8 g; C2) or severe (24.9 ± 0.3%, 172.7 ± 4.7 g; C3) weight loss, amylin and/or leptin was administered as described. RESULTS: In C1, leptin did not alter weight, and amylin induced 40.2 ± 6.1 g weight loss (-6.0 ± 0.9%), which was not enhanced by leptin (44.4 ± 4.9 g, -6.1 ± 0.8%). In C2, vehicle-treated (75.1 ± 7.8 g weight change from start of treatment, 1.1 ± 0.8% difference from start of pre-CR phase) and leptin-treated rats (68.6 ± 9.2 g, -1.3 ± 1.0%) rebounded to pre-restriction weight that was attenuated by amylin (29.2 ± 11.4 g, -6.2 ± 0.7%). Leptin did not enhance the effect of amylin (22.8 ± 11.7 g, -8.3 ± 1.5%). In C3, vehicle-treated and leptin-treated rats regained most of their weight (161.9 ± 11.8, -2.3 ± 0.8% and 144.6 ± 9.5 g, -2.3 ± 0.9%, respectively), which was attenuated by amylin (91.1 ± 16.8 g, -11.2 ± 0.7%), but not enhanced by leptin (83.0 ± 7.6 g, -10.7 ± 0.8%). CONCLUSIONS: Extreme obesity associated with leptin resistance perturbs amylin/leptin weight loss synergy in rats, which cannot be restored by pre-treatment weight loss.

Synergistic metabolic benefits of an exenatide analogue and cholecystokinin in diet-induced obese and leptin-deficient rodents.

AIM: To test the impact of cholecystokinin (CCK) plus either amylin or a glucagon-like peptide-1 receptor (GLP-1R) agonist on metabolic variables in diet-induced obese (DIO) rodents. METHODS: A stabilized acetylated version of CCK-8 (Ac-Y*-CCK-8), selective CCK1 receptor (CCK1R) or CCK2 receptor (CCK2R) agonists, amylin or the GLP-1R agonist and exenatide analogue AC3174 were administered in select combinations via continuous subcutaneous infusion to DIO rats for 14 days, or Lep(ob) /Lep(ob) mice for 28 days, and metabolic variables were assessed. RESULTS: Combined administration of Ac-Y*-CCK-8 with either amylin or AC3174 induced greater than additive weight loss in DIO rats, with the overall magnitude of effect being greater with AC3174 + Ac-Y*-CCK-8 treatment. Co-infusion of AC3174 with a specific CCK1R agonist, but not a CCK2R agonist, recapitulated the weight loss mediated by AC3174 + Ac-Y*-CCK-8 in DIO rats, suggesting that synergy is mediated by CCK1R activation. In a 4 × 4 full-factorial response surface methodology study in DIO rats, a synergistic interaction between AC3174 and the CCK1R-selective agonist on body weight and food intake was noted. Co-administration of AC3174 and the CCK1R-selective agonist to obese diabetic Lep(ob) /Lep(ob) mice elicited a significantly greater reduction in percentage of glycated haemoglobin and food intake relative to the sum effects of monotherapy groups. CONCLUSIONS: The anti-obesity and antidiabetic potential of combined GLP-1R and CCK1R agonism is an approach that warrants further investigation.

Polymorphisms of glutathione S-transferases (GST) and thymidylate synthase (TS)--novel predictors for response and survival in gastric cancer patients.

To evaluate the predictive value of a panel of gene polymorphisms involved in metabolism of 5-FU and cisplatin on clinical outcome in advanced gastric cancer patients. A total of 52 patients were enrolled in this study. DNA was extracted from paraffin-embedded tumour specimen. Genotypes were determined using PCR-RFLP. Median survival time was 6.0 months (95% CI 3.9;8.1). Overall response rate was 26%. Patients possessing the glutathione S-transferase P1-105 Valine/Valine (GSTP1-105VV) genotype showed a response rate of 67% compared to 21% in patients harbouring at least one GSTP1-105 Isoleucine (GSTP1-105I) allele (P=0.038). GSTP1-105VV patients demonstrated a significant superior median survival time of 15.0 months (95% CI 7.8;22.0) compared to 6.0 months (95% CI 5.1;7.0) in patients with at least one GSTP1-105I allele (P=0.037). Patients possessing a favourable thymidylate synthase (TS) genotype (2R/2R, 2R/3RC, 3RC/3RC) experienced a superior survival time of 10.2 months (95% CI 5.1;15.3) compared to 6.0 months (95% CI 5.0;7.0) in patients with unfavourable TS genotypes (P=0.099). Patients harbouring the GSTP1-105II genotype and one of the unfavourable TS genotypes showed an inferior median survival time of 6.0 months (95% CI 3.9;8.1) compared to 11 months (95% CI 6,23;15,77) in patients with either GSTP1-105VV or a favourable TS genotype (P=0.044). Testing for TS and GSTP1 polymorphisms may allow identification of gastric cancer patients who will benefit from 5-FU/cisplatin chemotherapy, sparing others the side effects of this chemotherapy.

Characteristic pattern of genetic aberrations in ovarian granulosa cell tumors.

The cytogenetic abnormalities of granulosa cell tumors (GCT) of the ovary are only partially known. Up to now, mainly numerical chromosomal aberrations have been described. Therefore we performed a comprehensive study on paraffin-embedded material of 20 GCT (17 adult, 3 juvenile; patient age between 16 and 78 y) combining comparative genomic hybridization (CGH); fluorescence in situ hybridization (FISH) using DNA-specific probes for chromosome 12, 17, 22, and X; DNA cytometry; and immunohistochemistry (inhibin, p53, Ki67). By DNA cytometry, 16 of 20 tumors (80%) were diploid. However, 6 of 16 diploid tumors (37%) showed aberrations by FISH. FISH revealed monosomy 22 in 8/18 cases (40%); trisomy 12 in 5/20 (25%); monosomy X in 2/20 (10%); and loss of chromosome 17 in one case (5%). The main findings by CGH were gains of chromosomes 12 (6 cases, 33%) and 14 (6 cases, 33%) and losses of chromosomes 22 (7 cases, 35%) and X (1 case, 5%), mostly comprising whole chromosomes or chromosome arms. Inhibin and p53 were expressed in 100% and 95% of the tumors, respectively. The Ki67 index ranged from 0% to 61%. Neither immunohistochemistry, nor DNA cytometry and molecular genetic analysis, provided statistically significant prognostic information. In summary, our study reveals a distinctive pattern of cytogenetic alterations in GCT. Our observations confirm earlier reports that trisomy 12 and 14 are frequent aberrations; however, monosomy 22 seemingly is even more prevalent.

FISH analysis of gene aberrations (MYC, CCND1, ERBB2, RB, and AR) in advanced prostatic carcinomas before and after androgen deprivation therapy.

Genetic mechanisms leading to androgen-independent growth in advanced prostatic carcinomas (PC) are still poorly understood. Analysis of genes potentially involved in the regulation of tumor cell proliferation and apoptosis might confer better insight into this process and might lead to improved therapeutic strategies. Fluorescence in situ hybridization (FISH) analysis of dissociated nuclei with DNA probes for MYC (8q24)/#8, cyclin D1 gene (CCND1; 11q13)/#11, ERBB2 (17q13)/#17, the androgen receptor gene (AR; Xq12)/#X, and the retinoblastoma gene (RB; 13q14) was applied to formalin-fixed tissue from 63 patients with advanced PC after androgen deprivation therapy (ADT); matched tumor tissue before ADT was also available in 22 of these cases. The cut-points used were: "increased copy number," > or = 30% of all nuclei with increased FISH signals (centromere and/or gene); "amplification," > or = 15% of nuclei with "increased gene copy number." CCND1 and MYC gene "amplifications" were present before ADT in 25% and 33% of the cases, respectively; the frequency of these "amplifications" increased to 37% and 57% after ADT. Loss of the RB gene was nearly four times more frequent after ADT than before therapy (22% versus 6%). AR and ERBB2 gene "amplifications" occurred only after ADT in 36% and 30% of cases, respectively. With the exception of the AR gene, the copy number increase was low. After treatment, MYC and AR gene "amplifications" correlated with the proliferation rate (Ki-67/MIB1 index; p = 0.01 and p = 0.04), whereas ERBB2 "amplifications" were associated with increased apoptotic index (PCD/TUNEL; p = 0.016). However, no correlation between FISH results and clinical follow-up could be established. FISH analysis of genes putatively involved in PC progression revealed characteristic patterns of aberrations in advanced PC before and after ADT. Distinct changes in gene copy number before and after therapy suggests possible involvement of these genes in the escape from androgen control.