Myogenic differentiation and lipid-raft composition of L6 skeletal muscle cells are modulated by PUFAs.

Lipid composition and fatty acid analysis of the major classes of membrane phospholipids were determined during myogenic differentiation of L6 skeletal muscle cells. The cholesterol to glycerophospholipids ratio decreased during differentiation, both in total (TM) and detergent-resistant membranes (DRM). Analyses of the membrane lipids showed that differentiation had a major impact on the molecular composition of glycerophospholipids. A significant decrease in the concentration of saturated fatty acids was detected in glycerophospholipid classes, and to a lesser extent in sphingolipids, while the concentration of 16:1n-7, 18:1n-7 and 18:1n-9 increased. At the same time, the concentration of long polyunsaturated fatty acid chains decreased in TM and DRM glycerophospholipids, resulting in a lower saturated to unsaturated fatty acid ratio in myotubes as compared to myoblasts. Interestingly, the observed n-3/n-6 ratio was lower in differentiated cell membranes. PUFA supplementation of L6 cells led to an increase in myogenic differentiation correlated to an incorporation of added PUFAs in TM and DRM glycerophospholipids. As expected after n-3 PUFA supplementation, the n-3/n-6 ratio was clearly increased in TM and, surprisingly, this was also the case in isolated DRM. n-3 and n-6 PUFAs significantly and time-dependently increased the phosphorylation of kinase p70S6K1 during myogenic differentiation, revealing the activation of the upstream kinase mTORC1, a major regulator of cell cycle and protein translation. In contrast, PUFAs did not affect the phosphorylation of the kinase Akt, another pivotal regulator of cell metabolism. These results suggest that PUFA supplementation modified the membrane lipid composition and affected the differentiation of L6 cells.

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues.

Alk4 is a type I receptor that belongs to the transforming growth factor-beta (TGF-ß) family. It takes part in the signaling of TGF-ß ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult-tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed-allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre-mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen-inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue-specific contexts.

Phospholipase D regulates the size of skeletal muscle cells through the activation of mTOR signaling.

mTOR is a major actor of skeletal muscle mass regulation in situations of atrophy or hypertrophy. It is established that Phospholipase D (PLD) activates mTOR signaling, through the binding of its product phosphatidic acid (PA) to mTOR protein. An influence of PLD on muscle cell size could thus be suspected. We explored the consequences of altered expression and activity of PLD isoforms in differentiated L6 myotubes. Inhibition or down-regulation of the PLD1 isoform markedly decreased myotube size and muscle specific protein content. Conversely, PLD1 overexpression induced muscle cell hypertrophy, both in vitro in myotubes and in vivo in mouse gastrocnemius. In the presence of atrophy-promoting dexamethasone, PLD1 overexpression or addition of exogenous PA protected myotubes against atrophy. Similarly, exogenous PA protected myotubes against TNFα-induced atrophy. Moreover, the modulation of PLD expression or activity in myotubes showed that PLD1 negatively regulates the expression of factors involved in muscle protein degradation, such as the E3-ubiquitin ligases Murf1 and Atrogin-1, and the Foxo3 transcription factor. Inhibition of mTOR by PP242 abolished the positive effects of PLD1 on myotubes, whereas modulating PLD influenced the phosphorylation of both S6K1 and Akt, which are respectively substrates of mTORC1 and mTORC2 complexes. These observations suggest that PLD1 acts through the activation of both mTORC1 and mTORC2 to induce positive trophic effects on muscle cells. This pathway may offer interesting therapeutic potentialities in the treatment of muscle wasting.

Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes.

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.

The Transcription Factor YY1 Is Essential for Normal DNA Repair and Cell Cycle in Human and Mouse ß-Cells.

Identifying the mechanisms behind the ß-cell adaptation to failure is important to develop strategies to manage type 2 diabetes (T2D). Using db/db mice at early stages of the disease process, we took advantage of unbiased RNA sequencing to identify genes/pathways regulated by insulin resistance in ß-cells. We demonstrate herein that islets from 4-week-old nonobese and nondiabetic leptin receptor-deficient db/db mice exhibited downregulation of several genes involved in cell cycle regulation and DNA repair. We identified the transcription factor Yin Yang 1 (YY1) as a common gene between both pathways. The expression of YY1 and its targeted genes was decreased in the db/db islets. We confirmed the reduction in YY1 expression in ß-cells from diabetic db/db mice, mice fed a high-fat diet (HFD), and individuals with T2D. Chromatin immunoprecipitation sequencing profiling in EndoC-ßH1 cells, a human pancreatic ß-cell line, indicated that YY1 binding regions regulate cell cycle control and DNA damage recognition and repair. We then generated mouse models with constitutive and inducible YY1 deficiency in ß-cells. YY1-deficient mice developed diabetes early in life due to ß-cell loss. ß-Cells from these mice exhibited higher DNA damage, cell cycle arrest, and cell death as well as decreased maturation markers. Tamoxifen-induced YY1 deficiency in mature ß-cells impaired ß-cell function and induced DNA damage. In summary, we identified YY1 as a critical factor for ß-cell DNA repair and cell cycle progression.

Phospholipase D2 regulates endothelial permeability through cytoskeleton reorganization and occludin downregulation.

Endothelial permeability is controlled by adhesive strengths which connect cells to each other through interendothelial junctions and by contractile forces associated with cytoskeleton reorganization. Phospholipase D (PLD) activation resulting in the generation of phosphatidic acid (PA) is increasingly recognized as a key event in the initiation of various cell responses. In human umbilical vein endothelial cells (HUV-EC), enhancement of intracellular PA by a variety of approaches increased the permeability of endothelial cell monolayers and induced stress fibre formation. Using adenovirus-mediated overexpression and siRNA silencing, we showed that PLD2 but not PLD1 was involved in the enhancement of basal permeability through cytoskeleton reorganization. Furthermore, PLD2 overexpression induced ERK1/2 activation and downregulated the expression of occludin, a major component of tight junctions. A substantial part of PLD2 protein was associated with the low-density caveolin-rich fractions isolated on sucrose gradients. The Raf-1 specific inhibitor GW-5074 drastically reduced hyperpermeability induced by PLD2 overexpression, and inhibited PA-mediated increase of endothelial permeability and ERK1/2 activation. On the whole, the present results demonstrate the selective role of PLD2 isoform in the control of endothelial permeability through a mechanism involving both stress fibre formation and contraction, and occludin downregulation, possibly resulting from PA-mediated activation of Raf-1.

mTORC1 to AMPK switching underlies ß-cell metabolic plasticity during maturation and diabetes.

Pancreatic beta cells (ß-cells) differentiate during fetal life, but only postnatally acquire the capacity for glucose-stimulated insulin secretion (GSIS). How this happens is not clear. In exploring what molecular mechanisms drive the maturation of ß-cell function, we found that the control of cellular signaling in ß-cells fundamentally switched from the nutrient sensor target of rapamycin (mTORC1) to the energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK), and that this was critical for functional maturation. Moreover, AMPK was activated by the dietary transition taking place during weaning, and this in turn inhibited mTORC1 activity to drive the adult ß-cell phenotype. While forcing constitutive mTORC1 signaling in adult ß-cells relegated them to a functionally immature phenotype with characteristic transcriptional and metabolic profiles, engineering the switch from mTORC1 to AMPK signaling was sufficient to promote ß-cell mitochondrial biogenesis, a shift to oxidative metabolism, and functional maturation. We also found that type 2 diabetes, a condition marked by both mitochondrial degeneration and dysregulated GSIS, was associated with a remarkable reversion of the normal AMPK-dependent adult ß-cell signature to a more neonatal one characterized by mTORC1 activation. Manipulating the way in which cellular nutrient signaling pathways regulate ß-cell metabolism may thus offer new targets to improve ß-cell function in diabetes.

ActivinB Is Induced in Insulinoma To Promote Tumor Plasticity through a ß-Cell-Induced Dedifferentiation.

Loss of pancreatic ß-cell maturity occurs in diabetes and insulinomas. Although both physiological and pathological stresses are known to promote ß-cell dedifferentiation, little is known about the molecules involved in this process. Here we demonstrate that activinB, a transforming growth factor ß (TGF-ß)-related ligand, is upregulated during tumorigenesis and drives the loss of insulin expression and ß-cell maturity in a mouse insulinoma model. Our data further identify Pax4 as a previously unknown activinB target and potent contributor to the observed ß-cell dedifferentiation. More importantly, using compound mutant mice, we found that deleting activinB expression abolishes tumor ß-cell dedifferentiation and, surprisingly, increases survival without significantly affecting tumor growth. Hence, this work reveals an unexpected role for activinB in the loss of ß-cell maturity, islet plasticity, and progression of insulinoma through its participation in ß-cell dedifferentiation.

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors.

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a(+) and neurogenin 3-expressing (Ngn3(+)) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors.

Both PAX4 and MAFA are expressed in a substantial proportion of normal human pancreatic alpha cells and deregulated in patients with type 2 diabetes.

Pax4 and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A) are two transcription factors crucial for normal functions of islet beta cells in the mouse. Intriguingly, recent studies indicate the existence of notable difference between human and rodent islet in terms of gene expression and functions. To better understand the biological role of human PAX4 and MAFA, we investigated their expression in normal and diseased human islets, using validated antibodies. PAX4 was detected in 43.0±5.0% and 39.1±4.0% of normal human alpha and beta cells respectively. We found that MAFA, detected in 88.3±6.3% insulin(+)cells as in the mouse, turned out to be also expressed in 61.2±6.4% of human glucagons(+) cells with less intensity than in insulin(+) cells, whereas MAFB expression was found not only in the majority of glucagon(+) cells (67.2±7.6%), but also in 53.6±10.5% of human insulin(+) cells. Interestingly, MAFA nuclear expression in both alpha and beta cells, and the percentage of alpha cells expressing PAX4 were found altered in a substantial proportion of patients with type 2 diabetes. Both MAFA and PAX4 display, therefore, a distinct expression pattern in human islet cells, suggesting more potential plasticity of human islets as compared with rodent islets.