Insulin receptor signaling and glucagon-like peptide 1 effects on pancreatic beta cells

Glucagon-like peptide-1 (GLP-1) is a potent gluco-incretin hormone, which plays a central role on pancreatic beta cell proliferation, survival and insulin secreting activity and whose analogs are used for treating hyperglycemia in type 2 diabetes mellitus. Notably, abnormal insulin signaling affects all the above-mentioned aspects on pancreatic beta cells. The aim of our study was to investigate whether the protective effects of GLP1-1 on beta cells are affected by altered insulin receptor signaling. To this end, several effects of GLP-1 were studied in INS-1E rat beta cells transfected either with an inhibitor of insulin receptor function (i.e., the Ectonucleotide Pyrophosphatase Phosphodiesterase 1, ENPP1), or with insulin receptor small interfering RNA, as well as in control cells. Crucial experiments were carried out also in a second cell line, namely the βTC-1 mouse beta cells. Our data indicate that in insulin secreting beta cells in which either ENPP1 was up-regulated or insulin receptor was down-regulated, GLP-1 positive effects on several pancreatic beta cell activities, including glucose-induced insulin secretion, cell proliferation and cell survival, were strongly reduced. Further studies are needed to understand whether such a scenario occurs also in humans and, if so, if it plays a role of clinical relevance in diabetic patients with poor responsiveness to GLP-1 related treatments

ENPP1 Affects Insulin Action and Secretion: Evidences from In Vitro Studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser473, ERK1/2-Thr202/Tyr204 and GSK3-beta Ser9 phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser473, ERK1/2-Thr202/Tyr204 and GSK3-beta Ser9 in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities

Synthesis of pyrazolo[4,3-c][1,2,6]benzothiadiazocine,a new ring system as potential COX inhibitor

Derivatives of the new ring system 1,4-dihydropyrazolo[4,3-c][1,2,6] benzothiadiazocin-11(10H)one 5,5-dioxide were synthesized in five or six steps in 57-66% overall yields and tested as COX inhibitors

A Facile Synthesis of Deaza-Analogues of the Bisindole Marine Alkaloid Topsentin

molecule

GLP-1 fails to rescue apoptosis in ENPP1 transfected cells.

<p>Apoptosis was assessed by caspase 3/7 activity, in INS-1E (panel A) and βTC-1 (panel B) cells, either transfected with neo or ENPP-1 gene variant. Cells were treated with staurosporine (0.25 μmol/l, 2 hours) in the absence or presence of 100 nmol/l GLP-1. Treatment with GLP-1 significantly decreased apoptosis induced by staurosporine in INS-1E-neo and in βTC-1-neo cells but not in INS-1E-ENPP1 and in βTC-1-ENPP1 cells. Values are expressed as means ± SD in INS-1E (n = 7) in βTC-1 (n = 8) independent experiments, each comprising three wells. (* p<0.05 compared with untreated INS-1E-neo or βTC-1-neo cells; § p<0.005 compared with INS-1E-neo or βTC-1-neo cells treated with staurosporine; ° p<0.05 compared with untreated INS-1E-ENPP1 or βTC-1-ENPP1; # p< 0.05 compared with INS-1E-neo or βTC-1-neo cells treated with staurosporine; $ p< 0.05 compared with INS-1E-neo or βTC-1-neo cells exposed to GLP1).</p

GLP-1 effects on phosphorylation of Akt and ERK 1/2.

<p>Phosphorylation of Akt and p-Akt/Akt ratio (panel A) and ERK 1/2 and the p-ERK/ERK ratio (panel B) was evaluated by western blot analysis in INS-1E. Cells were serum-starved for 12 hours and then incubated (2 hours at 37°C) in the presence of BSA or GLP-1 (100 nmol/l). Values are means ± S.D. of four independent experiments. (* p<0.05 compared with BSA treated INS-1E-neo cells; $ vs INS-1E neo GLP-1 100 nM).</p

GLP-1 fails to increase cell proliferation in ENPP-1 transfected cells.

<p>Proliferation was assessed by BrdU incorporation in INS-1E (panel A) and βTC-1 (panel B) cells, either transfected with neo or ENPP-1 gene variant. Cells were starved for 12 hours and then incubated for 48 hours in the absence or presence of 100 nmol/l GLP-1. Values are means ± S.D. of four independent experiments. * p<0.05 for INS-1E and for βTC-1 compared with untreated INS-1E-neo or βTC-1-neo cells.</p

GLP-1 effects on insulin secretion, proliferation and apoptosis are impaired in insulin receptor knockdown cells.

<p>Panel A. Insulin secretion in INS-1E cells, transfected with insulin receptor siRNA. Cells were incubated with 2.8 or 16.6 mmol/l glucose in the absence or presence of GLP-1 (100 nmol/l). Values are expressed as means ± SD of three independent experiments (*p<0.05 vs. scramble cells exposed to 2.8 mmol/l glucose; ** p<0.05 vs. scramble cells exposed to 2.8 mmol/l glucose plus 100 nmol/l GLP-1; #vs. scramble cells exposed to 16.6 mmol/l glucose; °p<0.05 vs. siRNA cells exposed to 2.8 mmol/l glucose; °° siRNA cells exposed to 2.8 mmol/l glucose plus 100 nmol/l GLP-1). Panel B. Cell proliferation, assessed by BrdU incorporation, in INS-1E cells transfected with insulin receptor siRNA. Cells were switched to serum free medium for 12 hours and then incubated for 48 hours in the absence or presence of GLP-1 (100 nmol/l). Values are expressed as means ± SD of four independent experiments, each comprising three wells (ç p<0.05 vs. untreated scramble cells). Panel C. Apoptosis, assessed by caspase 3/7 activity, in INS-1E cells transfected with insulin receptor siRNA. Cells were treated with staurosporine (0.25μmol/l, 2 hours) in the absence or presence of 100 nmol/l GLP-1. Treatment with GLP-1 significantly decreased apoptosis induced by staurosporine in scramble control cells but not in siRNA transfected cells. Values are expressed as means ± SD of nine independent experiments, each comprising three wells. (§ p<0.05 vs. untreated scramble or siRNA transfected cells; # p<0.05 vs. scramble cells treated with staurosporine in the absence of GLP-1).</p