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

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.Bleomycin‐induced lung injury promotes transient fibrosis accompanied by increased capillary density in young mice. In contrast, persistent fibrosis, capillary rarefaction, loss of endothelial cell identity, and reduction of Nos3 are observed in aged mice. eNOS/NO signal is an important driver of fibroblast quiescence and fibrosis resolution, that is lost with aging. Lung vascular bed plays a critical role during lung repair and fibrosis resolution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156458/2/acel13196_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156458/1/acel13196.pd

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

Pericytes in Microvessels: From “Mural” Function to Brain and Retina Regeneration

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFβ, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine

Molecular Mechanisms Involved in Escherichia coli K1 and Haemophilus influenzae Type a Blood-Brain Barrier Impairment

Blood Brain Barrier (BBB) is a protective barrier against possible neurotoxic molecules, such as proteins or endogenous metabolites, xenobiotics derived from the environment or ingested with the food. At the same time, BBB selectively allows the cross of ions and nutrients from blood into the CNS by active transport. BBB is made of brain microvascular endothelial cells, characterized by the presence of tight inter-endothelial junctions and the lack of fenestrations, and pericytes, the cells closest to brain endothelial cells with which they share a common basement membrane. Bacterial meningitis is a common and severe Central Nervous System (CNS) infection, caused by different pathogens, including Escherichia coli K1 (E. coli K1) and Haemophilus influenzae type a (Hia). During E. coli K1 meningitis, CNS cells release pro-inflammatory mediators, while the bacterium promotes the association between Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) and p85 subunit of PI3K. All these events lead to increase of BBB permeability and bacterial invasion of microvascular endothelial cells. It is still unknown if the emergency of invasive Hia disease is due to the serotype replacement after the wide use of Hib vaccine for child immunization. Despite Hia is now recognized as a pathogen responsible of disease comparable to Hib, no in vitro studies are conducted to clarify the mechanisms by which Hia is able to cross BBB, leading to meningitis. Aim of the study was to understand the molecular mechanisms through which E. coli K1 (first study) and Hia (second study) are able to cross BBB and to cause meningitis. Results of first showed that, after E. coli K1 infection, endothelial cells secrete VEGF, which target VEGFR-1 on the membrane of adjacent pericytes and determine their leak, leading to increased BBB permeability and allowing bacterial invasion. The second study demonstrated the correlation between the adenosine signaling pathway and BBB breakdown, highlighting the modulatory role of A2A and A2B adenosine receptors. In conclusion, association of an antibiotic therapy with a drug able to block the VEGFR-1 on pericytes could represent a novel strategy against E. coli K1 meningitis, while further studies on involvement of pericital adenosine receptors activation in Hia infection could be promising for the development of new pharmaceutical targets

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 insulin secretion in ENPP-1 transfected cells.

<p>INS-1E (panel A) and βTC-1 (panel B) cells, either transfected with neomicine (neo) or ENPP1 gene variant, were exposed to 100 nmol/l GLP-1 (60 min, 37°C) and glucose induced insulin secretion measured. Values are expressed as means ± S.D. of five independent experiments; *p<0.05 compared to neo-transfected cells at 2.8 mmol/l glucose; **p<0.05 compared to neo cells at 16.6 mmol/l glucose.</p

Mesenchymal Cells in The Lung: Evolving Concepts and Their Role in Fibrosis

Mesenchymal cells in the lung are crucial during development, but also contribute to the pathogenesis of fibrotic disorders, including idiopathic pulmonary fibrosis (IPF), the most common and deadly form of fibrotic interstitial lung diseases. Originally thought to behave as supporting cells for the lung epithelium and endothelium with a singular function of producing basement membrane, mesenchymal cells encompass a variety of cell types, including resident fibroblasts, lipofibroblasts, myofibroblasts, smooth muscle cells, and pericytes, which all occupy different anatomic locations and exhibit diverse homeostatic functions in the lung. During injury, each of these subtypes demonstrate remarkable plasticity and undergo varying capacity to proliferate and differentiate into activated myofibroblasts. Therefore, these cells secrete high levels of extracellular matrix (ECM) proteins and inflammatory cytokines, which contribute to tissue repair, or in pathologic situations, scarring and fibrosis. Whereas epithelial damage is considered the initial trigger that leads to lung injury, lung mesenchymal cells are recognized as the ultimate effector of fibrosis and attempts to better understand the different functions and actions of each mesenchymal cell subtype will lead to a better understanding of why fibrosis develops and how to better target it for future therapy. This review summarizes current findings related to various lung mesenchymal cells as well as signaling pathways, and their contribution to the pathogenesis of pulmonary fibrosis

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