RAPTOR Controls Developmental Growth Transitions by Altering the Hormonal and Metabolic Balance

Vegetative growth requires the systemic coordination of numerous cellular processes, which are controlled by regulatory proteins that monitor extracellular and intracellular cues and translate them into growth decisions. In eukaryotes, one of the central factors regulating growth is the serine/threonine protein kinase Target of Rapamycin (TOR), which forms complexes with regulatory proteins. To understand the function of one such regulatory protein, Regulatory-Associated Protein of TOR 1B (RAPTOR1B), in plants, we analyzed the effect of raptor1b mutations on growth and physiology in Arabidopsis (Arabidopsis thaliana) by detailed phenotyping, metabolomic, lipidomic, and proteomic analyses. Mutation of RAPTOR1B resulted in a strong reduction of TOR kinase activity, leading to massive changes in central carbon and nitrogen metabolism, accumulation of excess starch, and induction of autophagy. These shifts led to a significant reduction of plant growth that occurred nonlinearly during developmental stage transitions. This phenotype was accompanied by changes in cell morphology and tissue anatomy. In contrast to previous studies in rice (Oryza sativa), we found that the Arabidopsis raptor1b mutation did not affect chloroplast development or photosynthetic electron transport efficiency; however, it resulted in decreased CO2 assimilation rate and increased stomatal conductance. The raptor1b mutants also had reduced abscisic acid levels. Surprisingly, abscisic acid feeding experiments resulted in partial complementation of the growth phenotypes, indicating the tight interaction between TOR function and hormone synthesis and signaling in plants.journal articl

mRNA expression levels, Mitochondrial DNA content, Complex I, II and III activities, COX-IV protein content, Cytochrome C Oxidase and Citrate Synthase Activities and mRNA expression levels of Myosin Heavy Chain types in Gastrocnemius of F1 mice.

<p>A) mRNA expression levels of genes encoding proteins involved in the mitochondrial function in Gastrocnemius from 7-months-old F1-CD and F1-LPD males mice. mRNA relative levels are normalized relatively to HPRT1 expression level and expressed as fold change relative to the control value measured in F1-CD. Values are means ± sem for at least 8 mice/group. *p≤0.05. B) Mitochondrial density is estimated by measuring the mitochondria DNA (mtDNA) content relatively to the nuclear DNA (nuclDNA) content in Gastrocnemius from 7-months-old F1-CD and F1-LPD males mice. mtDNA/nuclDNA ratio was calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real-time PCR, in DNA extracted from gastrocnemius of 7-months-old F1-CD and F1-LPD males mice. Results were normalized by the mean value of the control condition set to 1 unit. *p≤0.05. C) Complex I, II and III and Citrate Synthase (CS) activities are measured in gastrocnemius from 7-months-old F1-CD and F1-LPD males mice and expressed as a percentage of the Cplx/CS ratio of F1-CD mice. Values are means ± sem for at least 8 mice/group. D) Left panel: Cytochrome C Oxidase Sub-Unit IV (COX-IV) protein level was assessed by western blotting in gastrocnemius from 7-months-old F1-CD and F1-LPD males mice. Histograms shown represent a densitometrical quantification after Porin normalization. Values are means ± sem for at least 4 mice/group. *p≤0.05. Right panel: Cytochrome C Oxidase (COX) and Citrate Synthase (CS) activities are measured in gastrocnemius from 7-months-old F1-CD and F1-LPD males mice and expressed as a percentage of the COX/CS ratio of F1-CD mice. Values are means ± sem for at least 8 mice/group. *p≤0.05. E) mRNA expression levels of the 4 different types of Myosin Heavy Chain (MHC-I, MHC-IIa, MHC-IIb, MHC-IIx) in Gastrocnemius from 7-months-old F1-CD and F1-LPD males mice. mRNA relative levels are normalized relatively to HPRT1 expression level and expressed as fold change relative to the control value measured in F1-CD. Values are means ± sem for at least 8 mice/group. *p≤0.05.</p

Calorimetric parameters of F1 mice.

<p>A) Minimum Energy Expenditure, Daily Energy Expenditure and Respiratory Quotient (RQ) of 7-mo-old male F1-CD and F1-LPD mice were measured by indirect calorimetry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104896#s2" target="_blank">Materials and Methods</a>. Energy Expenditure is expressed as kJ per day relatively to the total body weight (BW) or to the Lean Body Mass (LBM) of each mouse. Values are the mean ± sem for 6 mice/group. *p≤0.05. B) Body temperature (°C) of 7-mo-old male F1-CD and F1-LPD mice were measured using a rectal thermoprobe. Values are expressed as the mean temperature ± sem for at least 10 mice/group. *p≤0.05. C) Spontaneous locomotor activity of 7-mo-old male F1-CD and F1-LPD mice were measured in the home cage using infrared light-beam. Values are expressed as the mean distance (10⁺⁰³ cm) ± sem for 6 mice/group. *p≤0.05.</p

Phenotypic characterization of F1 mice.

<p>A) Body weight of 7-mo-old F1-CD and F1-LPD male mice. Results are expressed in grams as means ± sem for at least 10 mice/group. *p≤0.05. B) Body composition parameters of 7-mo-old F1-CD and F1-LPD male mice. Adiposity is expressed as a percentage of fat relative to total body weight. Lean mass is expressed in grams. Brown Adipose Tissue weight is expressed as weight in mg relatively to the weight of each mouse. All values are means ± sem for at least 10 mice/group. *p≤0.05. C) Food intake of 7-mo-old male F1-CD and F1-LPD mice was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104896#s2" target="_blank">Materials and Methods</a>. Values represent the weight of food consumed daily relatively to the total body weight (BW) or to the Lean Body Mass (LBM) of each mouse and are expressed as means ± sem for at least 10 mice/group. *p≤0.05.</p

神経・筋骨格系を有する3次元股義足歩行シミュレーションモデルの開発

本研究では,股義足を使用した歩行を再現することのできるシミュレーションモデルを開発した.まず,股義足歩行の動作分析を行い,股義足歩行の特徴を調べた.その結果から,妥当な股義足歩行をシミュレーションで再現するには3次元で全身のモデルが必要であることがわかった.そこで,股義足の機械的モデルを導入した股義足・筋骨格系モデルと股離断者の神経系モデルを統合した3次元股義足歩行シミュレーションモデルを構築した.数値シミュレーションにおいて,20,000試行の神経系のパラメータ探索計算を経て,3次元,多数歩,全身動作の股義足歩行を生成した.生成された歩行は,実際の股義足歩行の特徴を定性的に再現していた.This paper describes the development of a model which simulates the walking with a hip disarticulation prosthesis (HDP). In order to characterize walking with an HDP, the motion analyses were performed and incorporated into this study. Results of these analyses suggested that a three-dimensional model of the entire body was necessary to generate an accurate walking with an HDP. Therefore, a three-dimensional neuro-musculo-skeletal model of a human wearing an HDP was proposed including the mechanical model of an HDP and a neural model of a hip disarticulation amputee. After 20,000 iteration computation for the optimization process of neural parameters, simulated walking qualitatively represents the measured characteristics and dynamics of walking with an HDP.journal articl

Activation of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway triggers autophagy response to Crohn disease-associated adherent-invasive <i>Escherichia coli</i> infection

<p>The intestinal mucosa of Crohn disease (CD) patients is abnormally colonized by adherent-invasive <i>E. coli</i> (AIEC). Upon AIEC infection, autophagy is induced in host cells to restrain bacterial intracellular replication. The underlying mechanism, however, remains unknown. Here, we investigated the role of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway in the autophagic response to AIEC infection. We showed that infection of human intestinal epithelial T84 cells with the AIEC reference strain LF82 activated the EIF2AK4-EIF2A-ATF4 pathway, as evidenced by increased phospho-EIF2AK4, phospho-EIF2A and ATF4 levels. <i>EIF2AK4</i> depletion inhibited autophagy activation in response to LF82 infection, leading to increased LF82 intracellular replication and elevated pro-inflammatory cytokine production. Mechanistically, <i>EIF2AK4</i> depletion suppressed the LF82-induced ATF4 binding to promoters of several autophagy genes including <i>MAP1LC3B, BECN1, SQSTM1, ATG3</i> and <i>ATG7</i>, and this subsequently inhibited transcription of these genes. LF82 infection of wild-type (WT), but not <i>eif2ak4</i>^−/−, mice activated the EIF2AK4-EIF2A-ATF4 pathway, inducing autophagy gene transcription and autophagy response in enterocytes. Consequently, <i>eif2ak4</i>^−/− mice exhibited increased intestinal colonization by LF82 bacteria and aggravated inflammation compared to WT mice. Activation of the EIF2AK4-EIF2A-ATF4 pathway was observed in ileal biopsies from patients with noninflamed CD, and this was suppressed in inflamed CD, suggesting that a defect in the activation of this pathway could be one of the mechanisms contributing to active disease. In conclusion, we show that activation of the EIF2AK4-EIF2A-ATF4 pathway upon AIEC infection serves as a host defense mechanism to induce functional autophagy to control AIEC intracellular replication.</p

Physiological parameters.

<p>Plasma metabolites and hormones parameters of 7-mo-old male F1-CD and F1-LPD mice were measured in plasma of over-night starved animals and are expressed as means ± sem for n = 10 (Glucose) or n = 25 (Insulin, TG, Cholesterol, Cholesterol-HDL).</p

mRNA expression levels in Brown and White Adipose Tissue, Cytochrome C Oxidase and Citrate Synthase Activities and Mitochondrial DNA content in White Adipose Tissue of F1 mice.

<p>A, B) mRNA expression levels of genes encoding proteins involved in the mitochondrial function in (A) Brown Adipose Tissue (BAT) and (B) White Adipose Tissue (WAT) from 7-months-old F1-CD and F1-LPD males mice. mRNA relative levels are normalized relatively to Nono expression level and expressed as fold change relative to the control value measured in F1-CD. Values are means ± sem for at least 8 mice/group. **p≤0.05, *p≤0.08. C) Cytochrome C Oxidase (COX  =  Complex IV) and Citrate Synthase (CS) activities are measured in WAT from 7-months-old F1-CD and F1-LPD males mice and expressed as a percentage of the COX/CS ratio of F1-CD mice. Values are means ± sem for at least 8 mice/group. D) Mitochondrial density is estimated by measuring the mitochondria DNA (mtDNA) content relatively to the nuclear DNA (nuclDNA) content in White Adipose Tissue. mtDNA/nuclDNA ratio was calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real-time PCR, in DNA extracted from White Adipose Tissue of 7-months-old F1-CD and F1-LPD males mice. Results were normalized by the mean value of the control condition set to 1 unit. *p≤0.05.</p

Weight gain and food intake of F1 mice subjected to HFD.

<p>Five-mo-old F1-CD and F1-LPD male mice were fed for 7 months with either a Chow diet or a HFD. A) Body weight variation was determined at the end of the diet challenge as a percentage of the initial body weight and is expressed as a mean ± sem for at least 8 mice/group. *p≤0.05. B) Food intake of 12-mo-old Chow or HFD-fed F1-CD and F1-LPD male mice was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104896#s2" target="_blank">Materials and Methods</a>. Values represent the weight of food consumed daily (kCal) relatively to the weight of each mouse and are expressed as means ± sem for at least 8 mice/group. *p≤0.05.</p

GDF15 provides an endocrine signal of nutritional stress in mice and humans

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress