18 research outputs found
Isoform-specific insulin receptor signaling involves different plasma membrane domains
In pancreatic β-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11−) and B-type (Ex11+), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform–specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12–amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion
Selective insulin signaling in the pancreatic beta-cell via the two insulin receptor isoforms
Insulin exhibits pleiotropic effects that are tissue- as well as
development-dependent. However, the mechanisms by which insulin gains
selective effects are poorly understood. Selectivity in insulin signaling
is currently discussed as the result of the activation of specific signal
transduction pathways. This may be gained by activating specific adapter
proteins, such as IRS proteins and Shc, that 'channel' the insulin signal
in a more defined way by specifically interacting with downstream located
effector proteins. The insulin receptor (IR), the first step in these
cascades, exists in two isoforms as a result of alternative mRNA splicing
of the11th exon of the pro-receptor transcript. IR-A lacks whereas IR-B
contains the respective sequence coding for 12 amino acids in the
C-terminus of the a-chain of the receptor. Studies on general and
tissue-specific IR knockout models have demonstrated that a defect
IR-mediated insulin signaling leads to a type 2 diabetes-like phenotype.
However, these knockouts do not discriminate between the two IR isoforms.
Besides their different affinity for insulin, differences in kinase
activity as well as internalization and recycling for IR-A and IR-B have
been described. These data implied differences in the function of either
IR isoform. Although all cell types express both isoforms to a various
degree, little is known about the mechanisms that underlie IR
isoform-specific signaling and their biological importance remains
obscure.
Besides the classical insulin target tissues liver, muscle and fat,
recent research disclosed the pancreatic P-cell as an important target
for pleiotropic insulin action, here involving signal transduction
through IR and IGF-I receptors. The overall objective of the present
thesis work was to test the hypothesis that the two IR isoforms
contribute to selective insulin signaling. Specifically, we aimed to
investigate the molecular mechanisms that allow simultaneous and
selective transcriptional activation of three model genes encoding
insulin, beta-cell glucokinase (betaGK) and c-fos by insulin signal
transduction via the two IR isoforms in the pancreatic P-cell.
We show here that insulin activates the transcription of these three
genes by different mechanisms. Insulin activates transcription of its own
gene by signaling via IR-A and IRS/P13K la/mTOR/p70s6k. In contrast,
betaGK and c-fos genes are activated by insulin signaling via IR-B but
employing different signaling cascades. While insulin-stimulated betaGK
promoter up-regulation requires the integrity of the IR-B NPEY-motif and
signaling via PI3K-C2alphaPDK1/PKB, c-fos gene activation needs the
intact YTHM-motif and signaling via P13K la/p52-Shc/MEK1/ERK1/2. Studying
the molecular mechanisms that underlie the selective signaling via IR-A
versus IR-B, we found that both IR-A-mediated insulin and IRB-mediated
betaGK promoter activation are not dependent on IR isoform-specific
differences in internalization but on their spatial segregation in the
plasma membrane. Our data demonstrate that localization and function of
the two receptor types depend on the 12 amino acids encoded by exon 11.
Moreover, our data suggest that selective activation of the insulin and
betaGK promoters occurs by signaling from non-caveolae plasma membrane
micro-domains that are differently sensitive towards cholesterol
depletion. Analyzing the mechanisms that allow activation of selective
signaling cascades downstream of IR-B, we found that insulin activates
the betaGK promoter from membrane-standing IR-B, while c-fos promoter
activation is dependent on clathrin-mediated IR-B endocytosis.
In conclusion, the results of the present thesis work clearly demonstrate
that spatial segregation of selective signaling pathways originating from
IR-A and IR-B allows the simultaneous activation of discrete signaling
cascades that lead to specific insulin effects
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Selective gene activation by spatial segregation of insulin receptor B signaling
ABSTRACT
Insulin exerts pleiotropic effects at the cellular level. Signaling via the two isoforms of the insulin receptor (IR) may explain the activation of different signaling cascades, while it remains to be explored how selectivity is achieved when utilizing the same IR isoform. We now demonstrate that insulin‐stimulated transcription of c‐fos and glucokinase genes is activated simultaneously in the insulin‐producing β‐cell via IR‐B localized in different cellular compartments. Insulin activates the glucokinase gene from plasma membrane‐standing IR‐B, while c‐fos gene activation is dependent on clathrin‐mediated IR‐B‐endocytosis and signaling from early endosomes. Moreover, glucokinase gene up‐regulation requires the integrity of the jux‐tamembrane IR‐B NPEY‐motif and signaling via PI3K‐C2α‐like/PDK1/PKB, while c‐fos gene activation requires the intact C‐terminal YTHM‐motif and signaling via PI3K Ia/Shc/MEK1/ERK. By using IR‐B as an example it is thus possible to demonstrate how spatial segregation allows simultaneous and selective signaling via the same receptor isoform in the same cell.—Uhles S., Moede T., Leibiger B., Berggren P.‐O., and Leibiger I. B. Selective gene activation by spatial segregation of insulin receptor B signaling. FASEB J. 21, 1609–1621 (2007
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Insulin-feedback via PI3K-C2 alpha activated PKB alpha/Akt1 is required for glucose-stimulated insulin secretion
Phosphatidylinositide 3-kinases (PI3Ks) play central roles in insulin signal transduction. While the contribution of class Ia PI3K members has been extensively studied, the role of class II members remains poorly understood. The diverse actions of class II PI3K-C2 alpha have been attributed to its lipid product PI(3) P. By applying pharmacological inhibitors, transient overexpression and small-interfering RNA-based knockdown of PI3K and PKB/Akt isoforms, together with PI-lipid profiling and live-cell confocal and total internal reflection fluorescence microscopy, we now demonstrate that in response to insulin, PI3K-C2 alpha generates PI(3,4) P-2, which allows the selective activation of PKB alpha/Akt1. Knockdown of PI3K-C2 alpha expression and subsequent reduction of PKB alpha/Akt1 activity in the pancreatic beta-cell impaired glucose-stimulated insulin release, at least in part, due to reduced glucokinase expression and increased AS160 activity. Hence, our results identify signal transduction via PI3K-C2 alpha as a novel pathway whereby insulin activates PKB/Akt and thus discloses PI3K-C2 alpha as a potential drugable target in type 2 diabetes. The high degree of codistribution of PI3K-C2 alpha and PKB alpha/Akt1 with insulin receptor B type, but not A type, in the same plasma membrane microdomains lends further support to the concept that selectivity in insulin signaling is achieved by the spatial segregation of signaling events.-Leibiger, B., Moede, T., Uhles, S., Barker, C. J., Creveaux, M., Domin, J., Berggren, P.-O., Leibiger, I. B. Insulin-feedback via PI3K-C2 alpha activated PKB alpha/Akt1 is required for glucose-stimulated insulin secretion. FASEB J. 24, 1824-1837 (2010). www.fasebj.or
DDR1 role in fibrosis and its pharmacological targeting
International audienceDiscoidin domain receptor1 (DDR1) is a collagen activated receptor tyrosine kinase and an attractive anti-fibrotic target. Its expression is mainly limited to epithelial cells located in several organs including skin, kidney, liver and lung. DDR1's biology is elusive, with unknown downstream activation pathways; however, it may act as a mediator of the stromal-epithelial interaction, potentially controlling the activation state of the resident quiescent fibroblasts. Increased expression of DDR1 has been documented in several types of cancer and fibrotic conditions including skin hypertrophic scars, idiopathic pulmonary fibrosis, cirrhotic liver and renal fibrosis. The present review article focuses on: a) detailing the evidence for a role of DDR1 as an anti-fibrotic target in different organs, b) clarifying DDR1 tissue distribution in healthy and diseased tissues as well as c) exploring DDR1 protective mode of action based on literature evidence and co-authors experience; d) detailing pharmacological efforts attempted to drug this subtle anti-fibrotic target to date
Incretin-like effects of small molecule trace amine-associated receptor 1 agonists
Objective: Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight.
Methods: We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice.
Results: TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls.
Conclusions: We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity
Selective pharmacological inhibition of DDR1 prevents experimentally-induced glomerulonephritis in prevention and therapeutic regime
Abstract Background Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. Methods The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. Results DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. Conclusions Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium
Selective pharmacological inhibition of DDR1 prevents experimentally-induced glomerulonephritis in prevention and therapeutic regime
Abstract Background Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. Methods The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. Results DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. Conclusions Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium
Removal of Ca 2+ Channel β 3 Subunit Enhances Ca 2+ Oscillation Frequency and Insulin Exocytosis
An oscillatory increase in pancreatic β cell cytoplasmic free Ca
2+ concentration, [Ca
2+]
i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca
2+ channel β
3 subunit in the molecular regulation of these [Ca
2+]
i oscillations has now been clarified by using β
3 subunit-deficient β cells. β
3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca
2+]
i oscillation frequency in β cells lacking the β
3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP
3) and increased Ca
2+ mobilization from intracellular stores. Hence, the β
3 subunit negatively modulated InsP
3-induced Ca
2+ release, which is not paralleled by any effect on the voltage-gated L type Ca
2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the β
3 subunit in the β cell may constitute the basis for a novel diabetes therapy