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

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

DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in a Genetic Mouse Model of Alport Syndrome

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3-/- mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3-/- mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population

MOESM2 of Selective pharmacological inhibition of DDR1 prevents experimentally-induced glomerulonephritis in prevention and therapeutic regime

Additional file 2. Additional tables