Kisspeptin and gamma amino butyric acid (GABA), synthesized in the central nervous system, are critical for reproduction. Both are also expressed in peripheral organs/tissues critical to metabolic control (liver/pancreas/adipose). Many kisspeptin neurons coexpress GABAB receptors (GABABR) and GABA controls kisspeptin expression and secretion. We developed a unique mouse lacking GABABR exclusively from kisspeptin cells/neurons (Kiss1-GABAB1KO) to evaluate the impact on metabolism/reproduction. We confirmed selective deletion of GABABR from Kiss1 cells in the anteroventral periventricular nucleus/periventricular nucleus continuum (AVPV/PeN; immunofluorescence and PCR) and arcuate nucleus (ARC), medial amygdala (MeA), pituitary, liver and testes (PCR). Young Kiss1-GABAB1KO males were fertile, with normal LH and testosterone. Kiss1 expression was similar between genotypes in AVPV/PeN, ARC, MeA, bed nucleus of the stria terminalis (BNST) and peripheral organs (testis, liver, pituitary). Kiss1-GABAB1KO males presented higher fasted glycemia and insulin levels, an impaired response to a glucose overload, reduced insulin sensitivity and marked insulin resistance. Interestingly, when Kiss1-GABAB1KO males got older (9-months old) their body weight (BW) increased, in part due to an increase in white adipose tissue (WAT). Old Kiss1-GABAB1KO males showed higher fasted insulin, increased pancreatic insulin content, insulin resistance and a significant decrease pancreatic kisspeptin levels. In sum, lack of GABABR specifically in Kiss1 cells severely impacts glucose homeostasis in male mice, reinforcing kisspeptin involvement in metabolic regulation. These alterations in glucose homeostasis worsened with aging. We highlight the impact of GABA through GABABR in the regulation of the pancreas kisspeptin system in contrast to liver kisspeptin that was not affected.

We have demonstrated that oligodeoxynucleotide IMT504 promotes significant improvement in the diabetic condition in diverse animal models. Based on these results, here we evaluated whether these effects observed in vivo could be due to direct effects on β‐cells. We demonstrate by immunofluorescence that IMT504 enters the cell and locates in cytoplasm where it induces GSK‐3β phosphorylation that inactivates this kinase. As GSK‐3β tags Pdx1 for proteasomal degradation, by inactivating GSK‐3β, IMT504 induces an increase in Pdx1 protein levels, demonstrated by Western blotting. Concomitantly, an increase in Ins2 and Pdx1 gene transcription was observed, with no significant increase in insulin content or secretion. Enhanced Pdx1 is promising since it is a key transcription factor for insulin synthesis and is also described as an essential factor for the maintenance β‐cell phenotype and function. Dose‐dependent inhibition of H2O2‐induced apoptosis determined by ELISA as well as decreased expression of Bax was also observed. These results were confirmed in another β‐cell line, beta‐TC‐6 cells, in which a cytokine mix induced apoptosis that was reversed by IMT504. In addition, an inhibitor of IMT504 entrance into cells abrogated the effect IMT504. Based on these results we conclude that the β‐cell recovery observed in vivo may include direct effects of IMT504 on β‐cells, by maintaining their identity/phenotype and protecting them from oxidative stress and cytokine‐induced apoptosis. Thus, this work positions IMT504 as a promising option in the framework of the search of new therapies for type I diabetes treatment.

We have previously demonstrated that treatment with IMT504 promotes significant improvement in the diabetic condition in diverse animal models. We have also shown effects on gene expression on freshly isolated islets from diabetic IMT504-treated animals. Based on these results, here we evaluated if the effects of IMT504 observed in vivo were due to direct effects on beta cells. In particular we studied cell viability, enzyme activation and gene expression. A murine beta cell line (MIN6B1) was used. Cells were cultured in DMEM with 20 mM glucose, 15% SFB, 71 uM βmercaptoethanol. Cell viability was analized by MTS: cells were stimulated for 24 or 48 h with 0 (C), 2 (IMT2), 4 (IMT4) and 8 ug/ml (IMT8) of IMT504 in DMEM, 20 mM glucose, 2% SFB, 71 uM βmercaptoethanol. Gene expression of Pdx1 , Ins2 , Ins1 and Mafa was analized by qPCR, using cyclophilin as housekeeping gene. Phosphorylation of proteins of interest was analyzed by Western Blot. Cells were stimulated for 24 and 48 h with IMT504 in DMEM, 20 mM glucose, 0.5% BSA, 71 uM βmercaptoethanol. Enzyme phosphorylaton was also assayed at short time stimulation periods i.e. 0, 5, 15 ,30 and 60 min. For gene expression the dosis of IMT504 used were 0 (C), 0.4 (IMT0.4), 2.2 (IMT2.2) and 4 (IMT4) ug/ml; and for Western Blot were 0 (C), 2 (IMT2), 4 (IMT) and 8 (IMT8) ug/ml. No differences in cell viability were observed at the time points studied (ANOVA for repeated measures: NS). Expression of Pdx1 and Ins2 was significantly increased by 48 h stimulation with IMT504 [ANOVA for repeated measures: Pdx1 (A.U.): C=0.93±0.05; IMT0.4=0.80±0.06; IMT2.2=1.04±0.09; IMT4=1.37±0.09 p<0.05, IMT4 different from C and IMT0.4; Ins2 (A.U.): C=0.99±0.04; IMT0.4=1.04±0.06; IMT2.2=1.13±0.06; IMT4=1.52±0.08 p<0.05, IMT4 different from C and IMT0.4], while no changes in Ins1 and Mafa were observed (NS). pGSK3 β /GSK3 β ratio (A.U.) and pAkt/Akt ratio (A.U.) were calculated and informed as fold increase with regard to 0 min. At 60 min, pGSK3 β /GSK3 β ratio was increased compared to 0 min (ANOVA with repeated measures: pGSK3 β /GSK3 β ratio (A.U.) 0 min=1, 5 min=1.15±0.06, 15 min=1.20±0.05; 30 min=1.54±0.14; 60 min=2.04±0.17, 60 min different from 0 min, p<0.03). No significant differences were observed in pAkt/Akt ratio (ANOVA with repeated measures, NS). For 24 and 48 hs we found no significant differences between groups. Our results demonstrate a direct effect of IMT504 on gene expression in beta cells and suggest that IMT504 could exert its actions on beta cells through a pathway that includes GSK3 β phosphorylation. Further studies must be done to dilucidate their implications on beta cell function ...