Chronic exposure to GLP-1 increases GLP-1 synthesis and release in a pancreatic alpha cell line (α-TC1): evidence of a direct effect of GLP-1 on pancreatic alpha cells.

Incretin therapies, which are used to treat diabetic patients, cause a chronic supra-physiological increase in GLP-1 circulating levels. It is still unclear how the resulting high hormone concentrations may affect pancreatic alpha cells. The present study was designed to investigate the effects of chronic exposure to high GLP-1 levels on a cultured pancreatic alpha cell line.α-TC1-6 cell line was cultured in the presence or absence of GLP-1 (100 nmol/l) for up to 72 h. In our model GLP-1 receptor (GLP-1R) was measured. After the cells were exposed to GLP-1 the levels of glucagon secretion were measured. Because GLP-1 acts on intracellular cAMP production, the function of GLP-1R was studied. We also investigated the effects of chronic GLP-1 exposure on the cAMP/MAPK pathway, Pax6 levels, the expression of prohormone convertases (PCs), glucagon gene (Gcg) and protein expression, glucagon and GLP-1 production.In our model, we were able to detect GLP-1R. After GLP-1 exposure we found a reduction in glucagon secretion. During further investigation of the function of GLP-1R, we found an activation of the cAMP/MAPK/Pax6 pathway and an increase of Gcg gene and protein expression. Furthermore we observed a significant increase in PC1/3 protein expression, GLP-1 intracellular content and GLP-1 secretion.Our data indicate that the chronic exposure of pancreatic alpha cells to GLP-1 increases the ability of these cells to produce and release GLP-1. This phenomenon occurs through the stimulation of the transcription factor Pax6 and the increased expression of the protein convertase PC1/3

Molecular and cognitive signatures of ageing partially restored through synthetic delivery of IL2 to the brain.

Funder: EC|ERC|HORIZON EUROPE European Research Council (ERC); Id: http://dx.doi.org/10.13039/100010663Funder: EC|European Research Council (ERC); Id: http://dx.doi.org/10.13039/501100000781Cognitive decline is a common pathological outcome during aging, with an ill-defined molecular and cellular basis. In recent years, the concept of inflammaging, defined as a low-grade inflammation increasing with age, has emerged. Infiltrating T cells accumulate in the brain with age and may contribute to the amplification of inflammatory cascades and disruptions to the neurogenic niche observed with age. Recently, a small resident population of regulatory T cells has been identified in the brain, and the capacity of IL2-mediated expansion of this population to counter neuroinflammatory disease has been demonstrated. Here, we test a brain-specific IL2 delivery system for the prevention of neurological decline in aging mice. We identify the molecular hallmarks of aging in the brain glial compartments and identify partial restoration of this signature through IL2 treatment. At a behavioral level, brain IL2 delivery prevented the age-induced defect in spatial learning, without improving the general decline in motor skill or arousal. These results identify immune modulation as a potential path to preserving cognitive function for healthy aging

Western blot analysis for PC1/3 and PC2 protein expression in alpha cells (TC1-6).

<p>Western blot analysis of active PC1/3 (A) and PC2 (B) protein expression in control cells and in cells cultured for 72 hours in the presence of GLP-1 (100 nmol/l). The upper sides of the panels show representative Western Blots for active PC1/3 or active PC2. The lower sides of the panels show the densitometric analysis (means from five different Western Blot experiments). The data are expressed as the means ± SE. ** p<0.01 <i>vs</i>. control group.</p

GLP-1 receptor expression in alpha cells (TC1-6 and InR1G9) and in beta cells (TC1).

<p>(A) Full length <i>Glp1r</i> transcript expression as analysed by RT-PCR in α-TC1-6 and in InR1G9 cells. (B) <i>Glp1r</i> gene expression as analysed by Real Time PCR (means of five experiments) in beta cells (β-TC1 cells as positive control) and alpha cells (TC1-6); data are expressed as (2^−ΔΔCt) considering as 1 the <i>Glp1r</i> gene expression in alpha cells (TC1-6) and using mouse <i>actin beta (Actb)</i> as housekeeping gene. (C) Representative Western Blot of GLP-1R protein expression in β-TC1 cells and in TC1-6 cells. (D) Densitometric analysis of GLP-1R protein expression in β-TC1 cells and in TC1-6 cells. The data are the means of five different experiments ± SE. (E) GLP-1R (<i>red</i>) immunoreactivity in TC1-6 cells. Hoechst 33258 was used to visualize nuclei (<i>blue</i>) and phalloidin to stain the cytoskeloton (<i>green</i>). Squares (upper side) indicate the areas shown at higher magnification (lower side).</p

miR-296-3p, miR-298-5p and their downstream networks are causally involved in the higher resistance of mammalian pancreatic α cells to cytokine-induced apoptosis as compared to β cells

<p>Abstract</p> <p>Background</p> <p>The molecular bases of mammalian pancreatic α cells higher resistance than β to proinflammatory cytokines are very poorly defined. MicroRNAs are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus.</p> <p>Results</p> <p>Through high-throughput real-time PCR, we analyzed the steady state microRNA transcriptome of murine pancreatic α (αTC1-6) and β (βTC1) cells: their comparison demonstrated significant differences. We also characterized the alterations of αTC1-6 cells microRNA transcriptome after treatment with proinflammatory cytokines. We focused our study on two microRNAs, miR-296-3p and miR-298-5p, which were: (1) specifically expressed at steady state in αTC1-6, but not in βTC1 or INS-1 cells; (2) significantly downregulated in αTC1-6 cells after treatment with cytokines in comparison to untreated controls. These microRNAs share more targets than expected by chance and were co-expressed in αTC1-6 during a 6–48 h time course treatment with cytokines. The genes encoding them are physically clustered in the murine and human genome. By exploiting specific microRNA mimics, we demonstrated that experimental upregulation of miR-296-3p and miR-298-5p raised the propensity to apoptosis of transfected and cytokine-treated αTC1-6 cells with respect to αTC1-6 cells, treated with cytokines after transfection with scramble molecules. Both microRNAs control the expression of IGF1Rβ, its downstream targets phospho-IRS-1 and phospho-ERK, and TNFα. Our computational analysis suggests that MAFB (a transcription factor exclusively expressed in pancreatic α cells within adult rodent islets of Langerhans) controls the expression of miR-296-3p and miR-298-5p.</p> <p>Conclusions</p> <p>Altogether, high-throughput microRNA profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of miR-296-3p and miR-298-5p, coupled to upregulation of their targets as IGF1Rβ and TNFα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines.</p

Glucagon secretion and glucagon content in alpha cells (TC1-6).

<p>(A) Glucagon secretion in α-TC1 cells. The glucagon levels were measured in cells cultured with or without GLP-1 for 72 hours, washed and cultured for two more hours in KRB (glucose 25 mmol/l) in the presence or absence of GLP-1 (100 nmol/l) and/or insulin (10⁻⁹ M) for five minutes. The data are expressed as fmol/µg protein/hour; the means ± standard error (SE), n = 5; * p<0.05; ** p<0.01; ***p<0.001, using one-way ANOVA followed by Bonferroni test. (B) The intracellular glucagon content in α-TC1 cells. The cells were cultured in DMEM, with or without GLP-1 (100 nmol/l) for 72 hours, washed and cultured for two hours in KRB (glucose 25 mmol/l) in the presence or absence of GLP-1 (100 nmol/l) and/or insulin (10⁻⁹ M) for the last five minutes. The cells were then lysed in 0.1 M HCl and assayed for glucagon content. The data are expressed as ng/ml; means ± SE, n = 5.</p

GLP-1 (active and total) quantification in cells (TC1-6) chronically exposed to GLP-1, Exendin-4 or Exendin-9.

<p>Panel (A) shows the total intracellular GLP-1 content of α-TC1-6 cells cultured in the presence or absence of GLP-1 (100 nmol/l), Exendin-4 (100 nmol/l), Exendin-9 (100 nmol/l) or Exendin-9 (100 nmol/l) in co-presence of GLP-1 (100 nmol/l) for 72 hours. The data are expressed as means ± SE. ** p<0.01, using one-way ANOVA followed by Bonferroni test. Panel (B) shows the acute secretion of active GLP-1 by α-TC1-6 cells cultured in the presence or absence of GLP-1 (100 nmol/l), Exendin-4 (100 nmol/l), Exendin-9 (100 nmol/l) or Exendin-9 (100 nmol/l) in co-presence of GLP-1 (100 nmol/l) for 72 hours. After culture, the cells were washed in GLP-1-free Krebs-Ringer buffer and incubated for two more hours in Krebs-Ringer buffer containing 16.7 mmol/l of glucose and 0.5% BSA (pH 7.4) in the absence of GLP-1 (100 nmol/l) Exendin-4 (100 nmol/l) or Exendin-9 (100 nmol/l). The data are expressed as the means ± SE. ** p<0.01, *** p<0.001 using one-way ANOVA followed by Bonferroni test.</p

Western blot analysis for PC1/3 and PC2 protein expression in alpha cells (TC1-6).

<p>Western blot analysis of active PC1/3 (A) and PC2 (B) protein expression in control cells and in cells cultured for 72 hours in the presence of GLP-1 (100 nmol/l). The upper sides of the panels show representative Western Blots for active PC1/3 or active PC2. The lower sides of the panels show the densitometric analysis (means from five different Western Blot experiments). The data are expressed as the means ± SE. ** p<0.01 <i>vs</i>. control group.</p

Western blot analysis for Erk 44/42^MAPK phosphorylation in alpha cells (TC1-6 and InR1G9).

<p>The upper side of panel (A) shows a representative Western blot for Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) and for actin in TC1-6 cells: control cells (line 1), in cells cultured in the presence of GLP-1 (100 nmol/l) for 72 hours (line 2), in cells treated with adenylyl cyclase activator (Forskolin 50 nmol/l) for 72 hours (line 3), in cells treated with GLP-1 (100 nmol/l) and an adenylyl cyclase inhibitor (KH7 25 µmol/l) for 72 hours (line 4) and in cells cultured for 72 hours in the co-presence of GLP-1 (100 nmol/l) and Exendin-9 (100 nmol/l) (line 5). The lower side shows the densitometric analysis from five different experiments. The data are expressed as the means ± SE. *** p<0.001 <i>vs</i>. control groups, using one-way ANOVA followed by Bonferroni test. (B) Western blot analysis for Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) and actin in InR1G9 cells cultured in the presence or absence of GLP-1 (100 nmol/l) for 72 hours, washed and acutely stimulated with insulin (10⁻⁹ M) for the last five minutes in the presence or absence of GLP-1 (100 nmol/l).</p

Real-time PCR analysis and Western blot analysis for <i>Pax6</i> and <i>glucagon (Gcg)</i> gene and protein expression in alpha cells (TC1-6).

<p>Panel (A) shows the expression of <i>Pax 6</i> as determined by Real-Time PCR analysis (means from five different experiments) in control cells and in cells cultured for 72 hours in the presence of GLP-1 (100 nmol/l) alone or in combination with Exendin-9 (100 nmol/l). Panel (B) shows a representative Western Blot for the Pax6 protein expression in control cells and in cells cultured for 72 hours in the presence of GLP-1 (100 nmol/l) alone or in combination with Exendin-9 (100 nmol/l); Panel (C) shows the densitometric analysis (means from five different Western Blot experiments). The data are expressed as means ± SE. ** p<0.01, using one-way ANOVA followed by Bonferroni test. Panel (D) shows the expression of the <i>glucagon</i> (<i>Gcg</i>) gene as determined by Real-Time PCR analysis (means from five different experiments) in control cells and in cells cultured for 72 hours in the presence of GLP-1 (100 nmol/l) alone or in combination with Exendin-9 (100 nmol/l). Panel (E) shows a representative Western Blot for proglucagon protein expression in control cells and in cells cultured for 72 hours in the presence of GLP-1 (100 nmol/l) alone or in combination with Exendin-9 (100 nmol/l). Panel (F) shows the densitometric analysis (means from five different Western Blot experiments). The data are expressed as the means ± SE. * p<0.05; ** p<0.01, using one-way ANOVA followed by Bonferroni test.</p