Raphaël Roduit

Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells.

Aims/hypothesis. Glucagon-like peptide-1 is a potent glucoincretin hormone and a potentially important drug in the treatment of Type II (non-insulin-dependent) diabetes mellitus. We have investigated whether it acts as a growth factor in beta (INS-1)-cells and have studied the signalling pathways and transcription factors implicated in this process. Methods. Cell proliferation was assessed by tritiated thymidine incorporation measurements. We have examined the action of glucagon-like peptide-1 on the enzymatic activity of phosphatidylinositol 3-kinase. The DNA binding activity of transcription factors was investigated by electrophoretic mobility shift assay. Measurements of mRNA were done using the northern technique. Results. Glucagon-like peptide-1 caused an increase in tritiated thymidine incorporation in beta (INS-1)-cells and phosphatidylinositol 3-kinase activity in a dose-dependent manner non-additively with glucose. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294 002 blocked the effects of glucagon-like peptide-1 on DNA synthesis. Transcription factor pancreatic and duodenal homebox gene 1 (PDX-1) DNA binding activity was increased by glucagon-like peptide-1 at 3 or 11 mmol/l glucose and the phosphatidylinositol 3-kinase inhibitor LY294 002 suppressed the action of glucagon-like peptide-1 on PDX-1 DNA binding activity. Glucagon-like peptide-1 and glucose alone did not change activating protein-1 DNA binding activity. They synergised, however, to increase the activity of activating protein-1. Glucagon-like peptide-1 also increased the expression of PDX-1, glucose transporter 2, glucokinase and insulin mRNAs. Finally, glucagon-like peptide-1 increased the incorporation of tritiated thymidine in isolated rat islets. Conclusion/interpretation. The results suggest that glucagon-like peptide-1 may act as a growth factor for the beta cell by a phosphatidylinositol 3-kinase mediated event. Glucagon-like peptide-1 could also regulate the expression of the insulin gene and genes encoding enzymes implicated in glucose transport and metabolism through the phosphatidylinositol 3-kinase/PDX-1 transduction signalling pathway. [Diabetologia (1999) 42: 856–864]

Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside.

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH4)2SO4 precipitation, 2–3-fold increases in the V max of MCD and a 40% decrease in its K m for malonyl-CoA (190versus 119 μm) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.

Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic β cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitro cultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the α subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased (∼55 percent) in the presence of palmitic acid, but increased (∼180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic β cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.

Glucose and leptin induce apoptosis in human {beta}-cells and impair glucose-stimulated insulin secretion through activation of c-Jun N-terminal kinases

c‐Jun N‐terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK sig naling is involved in insulin resistance and β‐ cell secre tory function and survival. Chronic high glucose con centrations and leptin induce interleukin‐1 Vβ (IL‐1β) secretion from pancreatic islets, an event that is possi bly causal in promoting β‐ cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce P‐cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK‐binding domain of IB1/JIP‐1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose‐induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL‐1 β‐ converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein‐1 binding site, was up‐regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose‐ and leptin‐induced caspase 1 and JNK inhibition prevented this up‐regulation. Therefore, JNK inhibition may protect β‐ cells from the deleterious effects of high glucose and leptin in diabetes.— Maedler, K., Schulthess, F. T., Bielman, C., Berney, T., Bonny, C., Prentki, M., Donath, M. Y., Roduit, R. Glucose and leptin induce apoptosis in human β‐ cells and impair glucose‐stimulated insulin secretion through activation of c‐Jun N‐terminal kinases. FASEB J. 22, 1905–1913 (2008)

Inhibition of glucose-induced insulin secretion by long-term preexposure of pancreatic islets to leptin

Here we evaluated the effect of leptin on glucose‐induced insulin secretion by normal rat pancreatic islets. We show in perifusion experiments that leptin had no acute effect on the secretory activity of β‐cells. However, following preexposure to leptin a pronounced time‐ and dose‐dependent inhibition of both first and second phases of secretion was observed. Maximum inhibition was obtained at 24 h and with 100 nM leptin. This inhibition did not involve a decrease in cellular insulin content. It was also not observed with islets from fa/fa rats. Leptin thus inhibits insulin secretion by a mechanism which requires long‐term preexposure to the hormone and which may involve alteration in β‐cell gene expression.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family

NR2E3, a photoreceptor‐specific nuclear receptor (PNR), represses cone‐specific genes and activates several rod‐specific genes. In humans, mutations in NR2E3 have been associated with the recessively‐inherited enhanced short‐wavelength sensitive S‐cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP‐linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS‐like phenotype, which in 1 of the 2 cases was strikingly “milder” than the patients carrying the p.G56R mutation alone. Impaired repression of cone‐specific genes by the corepressors atrophin‐1 (dentatorubral‐pallidoluysian atrophy [DRPLA] gene product) and atrophin‐2 (arginine‐glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed. Hum Mutat 0,1–10, 2008.

A unique set of SH3-SH3 interactions controls IB1 homodimerization.

Islet‐brain 1 (IB1 or JIP‐1) is a scaffold protein that interacts with components of the c‐Jun N‐terminal kinase (JNK) signal‐transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic β‐cells, where it controls expression of several insulin‐secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)–SH3 interactions. X‐ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP‐mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1‐dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose‐dependent insulin secretion in pancreatic β‐cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.

Lipoprotein Lipase and Leptin Are Accumulated in Different Secretory Compartments in Rat Adipocytes

Adipose cells produce and secrete several physiologically important proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for the accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients, and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low density secretory vesicles. Insulin-sensitive Glut4 vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of “downstream” secretory vesicles.

The loss of GLUT2 expression in the pancreatic β-cells of diabetic db/db mice is associated with an impaired DNA-binding activity of islet-specific trans-acting factors

GLUT2 expression is reduced in the pancreatic beta-cells of several diabetic animals. The transcriptional control of the gene in beta-cells involves at least two islet-specific DNA-binding proteins, GTIIa and PDX-1, which also transactivates the insulin, somatostatin and glucokinase genes. In this report, we assessed the DNA-binding activities of GTIIa and PDX-1 to their respective cis-elements of the GLUT2 promoter using nuclear extracts prepared from pancreatic islets of 12 week old db/db diabetic mice. We show that the decreased GLUT2 mRNA expression correlates with a decrease of the GTIIa DNA-binding activity, whereas the PDX-1 binding activity is increased. In these diabetic animals, insulin mRNA expression remains normal. The adjunction of dexamethasone to isolated pancreatic islets, a treatment previously shown to decrease PDX-1 expression in the insulin-secreting HIT-T15 cells, has no effect on the GTIIa and PDX-1 DNA-binding activities. These data suggest that the decreased activity of GTIIa, in contrast to PDX-1, may be a major initial step in the development of the beta-cell dysfunction in this model of diabetes.

Malonyl‐CoA decarboxylase is present in the cytosolic, mitochondrial and peroxisomal compartments of rat hepatocytes

A role for cytosolic malonyl‐CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl‐CoA levels and of hepatic fatty acid oxidation.