Glucose-dependent insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice.

A role for the gastro-intestinal tract in controlling bone remodeling is suspected since serum levels of bone remodeling markers are affected rapidly after a meal. Glucose-dependent insulinotropic polypeptide (GIP) represents a suitable candidate in mediating this effect. The aim of the present study was to investigate the effect of total inhibition of GIP signaling on trabecular bone volume, microarchitecture and quality. We used GIP receptor (GIPR) knockout mice and investigated trabecular bone volume and microarchitecture by microCT and histomorphometry. GIPR-deficient animals at 16 weeks of age presented with a significant (20%) increase in trabecular bone mass accompanied by an increase (17%) in trabecular number. In addition, the number of osteoclasts and bone formation rate was significantly reduced and augmented, respectively in these animals when compared with wild-type littermates. These modifications of trabecular bone microarchitecture are linked to a remodeling in the expression pattern of adipokines in the GIPR-deficient mice. On the other hand, despite significant enhancement in bone volume, intrinsic mechanical properties of the bone matrix was reduced as well as the distribution of bone mineral density and the ratio of mature/immature collagen cross-links. Taken together, these results indicate an increase in trabecular bone volume in GIPR KO animals associated with a reduction in bone quality.

GLUT2 and the incretin receptors are involved in glucose-induced incretin secretion.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins secreted in response to oral glucose ingestion by intestinal L and K cells, respectively. The molecular mechanisms responsible for intestinal cell glucose sensing are unknown but could be related to those described for beta-cells, brain and hepatoportal sensors. We determined the role of GLUT2, GLP-1 or GIP receptors in glucose-induced incretins secretion, in the corresponding knockout mice. GLP-1 secretion was reduced in all mutant mice, while GIP secretion did not require GLUT2. Intestinal GLP-1 content was reduced only in GIP and GLUT2 receptors knockout mice suggesting that this impairment could contribute to the phenotype. Intestinal GIP content was similar in all mice studied. Furthermore, the impaired incretins secretion was associated with a reduced glucose-stimulated insulin secretion and an impaired glucose tolerance in all mice. In conclusion, both incretins secretion depends on mechanisms involving their own receptors and GLP-1 further requires GLUT2.

In vivo and in vitro effects of somatostatin and insulin on glucagon release in a human glucagonoma.

Inhibition of pancreatic glucagon secretion has been reported to be mediated by glucose, insulin and somatostatin. As no human pancreatic alpha-cell lines are available to study in vitro the relative importance of insulin and glucose in the control of pancreatic glucagon release, we investigated a patient presenting with a malignant glucagonoma who underwent surgical resection of the tumour. Functional somatostatin receptors were present as octreotide administration decreased basal glucagon and insulin secretion by 52 and 74%, respectively. The removed tumour was immunohistochemically positive for glucagon, chromogranin A and pancreatic polypeptide but negative for insulin, gastrin and somatostatin. The glucagonoma cells were also isolated and cultured in vitro. Incubation experiments revealed that change from high (10 mM) to low (1 mM) glucose concentration was unable to stimulate glucagon secretion. A dose-dependent inhibition of glucagon release by insulin was however, observed at low glucose concentration. These findings demonstrate that insulin could inhibit glucagon secretion in vitro in the absence of elevated glucose concentrations. These data suggest, as observed in vivo and in vitro in several animal studies, that glucopenia-induced glucagon secretion in humans is not mediated by a direct effect of low glucose on alpha-cells but possibly by a reduction of insulin-mediated alpha-cell suppression and/or an indirect neuronal stimulation of glucagon release.

Role of retroviral restriction factors in the interferon-α-mediated suppression of HIV-1 in vivo.

The antiviral potency of the cytokine IFN-α has been long appreciated but remains poorly understood. A number of studies have suggested that induction of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) and bone marrow stromal cell antigen 2 (BST-2/tetherin/CD317) retroviral restriction factors underlies the IFN-α-mediated suppression of HIV-1 replication in vitro. We sought to characterize the as-yet-undefined relationship between IFN-α treatment, retroviral restriction factors, and HIV-1 in vivo. APOBEC3G, APOBEC3F, and BST-2 expression levels were measured in HIV/hepatitis C virus (HCV)-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated IFN-α/ribavirin (IFN-α/riba) combination therapy. IFN-α/riba therapy decreased HIV-1 viral load by -0.921 (±0.858) log(10) copies/mL in HIV/HCV-coinfected patients. APOBEC3G/3F and BST-2 mRNA expression was significantly elevated during IFN-α/riba treatment in patient-derived CD4+ T cells (P < 0.04 and P < 0.008, paired Wilcoxon), and extent of BST-2 induction was correlated with reduction in HIV-1 viral load during treatment (P < 0.05, Pearson's r). APOBEC3 induction during treatment was correlated with degree of viral hypermutation (P < 0.03, Spearman's ρ), and evolution of the HIV-1 accessory protein viral protein U (Vpu) during IFN-α/riba treatment was suggestive of increased BST-2-mediated selection pressure. These data suggest that host restriction factors play a critical role in the antiretroviral capacity of IFN-α in vivo, and warrant investigation into therapeutic strategies that specifically enhance the expression of these intrinsic immune factors in HIV-1-infected individuals.

In vivo characterization of the circadian clock output gene usp2

Life on earth is subject to the repeated change between day and night periods. All organisms that undergo these alterations have to anticipate consequently the adaptation of their physiology and possess an endogenous periodicity of about 24 hours called circadian rhythm from the Latin circa (about) and diem (day). At the molecular level, virtually all cells of an organism possess a molecular clock which drives rhythmic gene expression and output functions. Besides altered rhythmicity in constant conditions, impaired clock function causes pathophysiological conditions such as diabetes or hypertension. These data unveil a part of the mechanisms underlying the well-described epidemiology of shift work and highlight the function of clock-driven regulatory mechanisms. The post-translational modification of proteins by the ubiquitin polypeptide is a central mechanism to regulate their stability and activity and is capital for clock function. Similarly to the majority of biological processes, it is reversible. Deubiquitylation is carried out by a wide variety of about ninety deubiquitylating enzymes and their function remains poorly understood, especially in vivo. This class of proteolytic enzymes is parted into five families including the Ubiquitin-Specific Proteases (USP), which is the most important with about sixty members. Among them, the Ubiquitin-Specific Protease 2 (Usp2) gene encodes two protein isoforms, USP2-45 and USP2-69. The first is ubiquitously expressed under the control of the circadian clock and displays all features of core clock genes or its closest outputs effectors. Additionally, Usp2-45 was also found to be induced by the mineralocorticoid hormone aldosterone and thought to participate in Na+ reabsorption and blood pressure regulation by Epithelial Na+ Channel ENaC in the kidneys. During my thesis, I aimed to characterize the role of Usp2 in vivo with respect to these two areas, by taking advantage of a total constitutive knockout mouse model. In the first project I aimed to validate the role of USP2-45 in Na+ homeostasis and blood pressure regulation by the kidneys. I found no significant alterations of diurnal Na+ homeostasis and blood pressure in these mice, indicating that Usp2 does not play a substantial role in this process. In urine analyses, we found that our Usp2-KO mice are actually hypercalciuric. In a second project, I aimed to understand the causes of this phenotype. I found that the observed hypercalciuria results essentially from intestinal hyperabsorption. These data reveal a new role for Usp2 as an output effector of the circadian clock in dietary Ca2+ metabolism in the intestine.

Mutations of the Serine Protease CAP1/Prss8 Lead to Reduced Embryonic Viability, Skin Defects, and Decreased ENaC Activity.

CAP1/Prss8 is a membrane-bound serine protease involved in the regulation of several different effectors, such as the epithelial sodium channel ENaC, the protease-activated receptor PAR2, the tight junction proteins, and the profilaggrin polypeptide. Recently, the V170D and the G54-P57 deletion mutations within the CAP1/Prss8 gene, identified in mouse frizzy (fr) and rat hairless (fr(CR)) animals, respectively, have been proposed to be responsible for their skin phenotypes. In the present study, we analyzed those mutations, revealing a change in the protein structure, a modification of the glycosylation state, and an overall reduction in the activation of ENaC of the two mutant proteins. In vivo analyses demonstrated that both fr and fr(CR) mutant animals present analogous reduction of embryonic viability, similar histologic aberrations at the level of the skin, and a significant decrease in the activity of ENaC in the distal colon compared with their control littermates. Hairless rats additionally had dehydration defects in skin and intestine and significant reduction in the body weight. In conclusion, we provided molecular and functional evidence that CAP1/Prss8 mutations are accountable for the defects in fr and fr(CR) animals, and we furthermore demonstrate a decreased function of the CAP1/Prss8 mutant proteins. Therefore, fr and fr(CR) animals are suitable models to investigate the consequences of CAP1/Prss8 action on its target proteins in the whole organism.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

Incrétines, sécrétion d'insuline et diabète

Nutrient ingestion triggers a complex hormonal response aimed at stimulating glucose utilization in liver, muscle and adipose tissue to minimize the raise in blood glucose levels. Insulin secretion by pancreatic beta cells plays a major role in this response. Although the beta cell secretory response is mainly controlled by blood glucose levels, gut hormones secreted in response to food intake have an important role in potentiating glucose-stimulated insulin secretion. These gluco-incretin hormones are GLP-1 (glucagon-like peptide-1) and GIP (gluco-dependent insulinotropic polypeptide). Their action on pancreatic beta cells depends on binding to specific G-coupled receptors linked to activation of the adenylyl cyclase pathway. In addition to their effect on insulin secretion both hormones also stimulate insulin production at the transcriptional and translational level and positively regulate beta cell mass. Because the glucose-dependent insulinotropic action of GLP-1 is preserved in type 2 diabetic patients, this peptide is now developed as a novel therapeutic drug for this disease.

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).