Defects in mitophagy promote redox‐driven metabolic syndrome in the absence of TP 53 INP 1
2015; Springer Nature; Volume: 7; Issue: 6 Linguagem: Inglês
10.15252/emmm.201404318
ISSN1757-4684
AutoresMarion Seillier, Laurent Pouyet, Prudence N’guessan, Marie Nollet, Florence Capo, Fabienne Guillaumond, Laure Peyta, Jean‐François Dumas, Annie Varrault, Gyslaine Bertrand, Stéphanie Bonnafous, Albert Tran, Gargi Meur, Piero Marchetti, Magalie A. Ravier, Stéphane Dalle, Philippe Gual, Dany Muller, Guy A. Rutter, Stéphane Servais, Juan Iovanna, Alice Carrier,
Tópico(s)Lipid metabolism and biosynthesis
ResumoResearch Article31 March 2015Open Access Source Data Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1 Marion Seillier Marion Seillier Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Laurent Pouyet Laurent Pouyet Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Prudence N'Guessan Prudence N'Guessan Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Marie Nollet Marie Nollet Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Florence Capo Florence Capo Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Fabienne Guillaumond Fabienne Guillaumond Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Laure Peyta Laure Peyta Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France Search for more papers by this author Jean-François Dumas Jean-François Dumas Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France Search for more papers by this author Annie Varrault Annie Varrault CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Gyslaine Bertrand Gyslaine Bertrand CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Stéphanie Bonnafous Stéphanie Bonnafous Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France Université de Nice-Sophia-Antipolis, Nice, France Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France Search for more papers by this author Albert Tran Albert Tran Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France Université de Nice-Sophia-Antipolis, Nice, France Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France Search for more papers by this author Gargi Meur Gargi Meur Cell Biology, Department of Medicine, Imperial College, London, UK Search for more papers by this author Piero Marchetti Piero Marchetti Islet Cell Laboratory, University of Pisa – Cisanello Hospital, Pisa, Italy Search for more papers by this author Magalie A Ravier Magalie A Ravier CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Stéphane Dalle Stéphane Dalle CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Philippe Gual Philippe Gual Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France Université de Nice-Sophia-Antipolis, Nice, France Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France Search for more papers by this author Dany Muller Dany Muller CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Guy A Rutter Guy A Rutter Cell Biology, Department of Medicine, Imperial College, London, UK Search for more papers by this author Stéphane Servais Stéphane Servais Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France Search for more papers by this author Juan L Iovanna Juan L Iovanna Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Alice Carrier Corresponding Author Alice Carrier Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Marion Seillier Marion Seillier Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Laurent Pouyet Laurent Pouyet Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Prudence N'Guessan Prudence N'Guessan Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Marie Nollet Marie Nollet Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Florence Capo Florence Capo Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Fabienne Guillaumond Fabienne Guillaumond Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Laure Peyta Laure Peyta Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France Search for more papers by this author Jean-François Dumas Jean-François Dumas Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France Search for more papers by this author Annie Varrault Annie Varrault CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Gyslaine Bertrand Gyslaine Bertrand CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Stéphanie Bonnafous Stéphanie Bonnafous Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France Université de Nice-Sophia-Antipolis, Nice, France Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France Search for more papers by this author Albert Tran Albert Tran Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France Université de Nice-Sophia-Antipolis, Nice, France Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France Search for more papers by this author Gargi Meur Gargi Meur Cell Biology, Department of Medicine, Imperial College, London, UK Search for more papers by this author Piero Marchetti Piero Marchetti Islet Cell Laboratory, University of Pisa – Cisanello Hospital, Pisa, Italy Search for more papers by this author Magalie A Ravier Magalie A Ravier CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Stéphane Dalle Stéphane Dalle CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Philippe Gual Philippe Gual Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France Université de Nice-Sophia-Antipolis, Nice, France Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France Search for more papers by this author Dany Muller Dany Muller CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France Search for more papers by this author Guy A Rutter Guy A Rutter Cell Biology, Department of Medicine, Imperial College, London, UK Search for more papers by this author Stéphane Servais Stéphane Servais Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France Search for more papers by this author Juan L Iovanna Juan L Iovanna Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Alice Carrier Corresponding Author Alice Carrier Inserm, U1068, CRCM, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France CNRS, UMR7258, CRCM, Marseille, France Search for more papers by this author Author Information Marion Seillier1,2,3,4, Laurent Pouyet1,2,3,4, Prudence N'Guessan1,2,3,4, Marie Nollet1,2,3,4, Florence Capo1,2,3,4, Fabienne Guillaumond1,2,3,4, Laure Peyta5, Jean-François Dumas5, Annie Varrault6, Gyslaine Bertrand6, Stéphanie Bonnafous7,8,9, Albert Tran7,8,9, Gargi Meur10, Piero Marchetti11, Magalie A Ravier6, Stéphane Dalle6, Philippe Gual7,8,9, Dany Muller6, Guy A Rutter10, Stéphane Servais5, Juan L Iovanna1,2,3,4 and Alice Carrier 1,2,3,4 1Inserm, U1068, CRCM, Marseille, France 2Institut Paoli-Calmettes, Marseille, France 3Aix-Marseille Université, Marseille, France 4CNRS, UMR7258, CRCM, Marseille, France 5Inserm, U1069, Nutrition, Croissance et Cancer (N2C), Tours, France 6CNRS, UMR5203, Inserm, U661, Universités de Montpellier 1 & 2, IGF, Montpellier, France 7Inserm, U1065, C3M, Team 8 "Hepatic Complications in Obesity", Nice, France 8Université de Nice-Sophia-Antipolis, Nice, France 9Centre Hospitalier Universitaire de Nice, Pôle Digestif, Hôpital L'Archet, Nice, France 10Cell Biology, Department of Medicine, Imperial College, London, UK 11Islet Cell Laboratory, University of Pisa – Cisanello Hospital, Pisa, Italy *Corresponding author. Tel: +33 4 91 82 88 29; Fax: +33 4 91 82 60 83; E-mail: [email protected] EMBO Mol Med (2015)7:802-818https://doi.org/10.15252/emmm.201404318 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research. Synopsis TP53INP1, a p53-regulated protein with antioxidant and tumor suppressive functions, is shown to prevent redox-driven obesity, which leads to insulin resistance and type 2 diabetes (T2D), likely by impacting on mitochondria homeostasis and mitophagy. TP53INP1 is known for its tumor suppressive activity due to its implication in redox control. TP53INP1 also plays a role in T2D prevention by regulating redox-associated lipid metabolism. Excess of ROS in TP53INP1-deficient mice stems from accumulation of defective mitochondria producing ROS. Accumulation of mitochondria in TP53INP1-deficient mice is due in part from defective autophagy, and in particular mitophagy. Introduction Metabolic syndrome (MS) describes a cluster of metabolic abnormalities including obesity, insulin resistance, hypertension and dyslipidemia (Pothiwala et al, 2009). The prevalence of MS has been increasing exponentially in the last few decades, paralleling the obesity epidemic. Obesity, which is defined as a body mass index ≥ 30 kg/m2, results from accumulation of white adipose tissue. It depends on both genetic and environmental factors, in particular lifestyles featuring increased nutrient caloric intake but decreased calorie consumption. Diet may have a major role in the pathogenesis and prevalence of obesity (Calder et al, 2011), but other causes have to be considered, such as gut microbiota which affect host nutritional metabolism (Musso et al, 2010; Greiner & Backhed, 2011), and lack of physical exercise (Calder et al, 2011). Excess body weight is a major public health concern since it is associated with increased risk of cardiovascular disease, type 2 diabetes (T2D), Alzheimer's disease and cancer (van Kruijsdijk et al, 2009; Siegel & Zhu, 2009; Forte et al, 2012; Leboucher et al, 2013). One major link between obesity and associated diseases is the chronic low-grade inflammatory state observed in obese patients (Calder et al, 2011; Gregor & Hotamisligil, 2011). Inflammation is induced by excessive accumulation of lipids in adipose tissue leading to adipocyte stress and release of inflammatory cytokines and adipokines. The resulting recruitment of immune cells to key metabolic organs further contributes to chronic inflammation. Obesity-associated immune signals concern all types of immune cells that prompt inflammation, as well as adipocytes themselves (Chawla et al, 2011; Deng et al, 2013). Importantly, the obesity-associated chronic low-grade inflammatory state impacts all organs in the body. Hence, obese patients are at increased risk of developing cancer in any localization even if pancreatic and liver cancers show the highest increase in risk (Siegel & Zhu, 2009). Inflammation is associated with oxidative stress which is one obesity-related feature participating in the development of MS (Bondia-Pons et al, 2012; Khoo et al, 2012; Rolo et al, 2012; Crujeiras et al, 2013). Oxidative stress results from excess of reactive oxygen species (ROS) production overwhelming antioxidant defenses (Pouyet & Carrier, 2010). ROS are mainly produced as by-products of the mitochondrial electron transport chain involved in ATP production (oxidative phosphorylation). Excess fatty acids and glucose are known to be deleterious for mitochondrial function, thus increasing ROS production. ROS can oxidize cell macromolecules, leading to impaired cellular homeostasis and associated pathologies such as cancer (Gupta et al, 2012; Crujeiras et al, 2013). In the recent years, we have provided evidence that tumor protein 53-induced nuclear protein 1 (TP53INP1) is a key stress protein with antioxidant-associated tumor suppressive function (Gironella et al, 2007; Gommeaux et al, 2007; Cano et al, 2009; N'Guessan et al, 2011; Seux et al, 2011). The TP53INP1 gene (a transcriptional target of p53 and other transcription factors) is highly conserved between human and rodents and over-expressed during stress response including inflammation (Tomasini et al, 2001; Jiang et al, 2004). TP53INP1-deficient mice, which lack participation of TP53INP1 in stress resolution, are prone to stress-induced dysfunctions including cancer (Gironella et al, 2007; Gommeaux et al, 2007; Cano et al, 2009; N'Guessan et al, 2011). Moreover, these mutant mice show a chronic oxidative stress characterized by an increase in the cell ROS level as well as a decrease of antioxidant defenses (Gommeaux et al, 2007; Cano et al, 2009; N'Guessan et al, 2011). Restoration of TP53INP1 expression in TP53INP1-deficient cells rescues the phenotype by alleviating ROS burden (Cano et al, 2009). We demonstrated that TP53INP1 impacts on p53 and p73 transcriptional activity by direct interaction and mediates the antioxidant activity of p53 (Tomasini et al, 2003, 2005; Cano et al, 2009). The tumor suppressor p53 is a fascinating protein endowed with multiple functions, including metabolic regulation, in common with two other members of this family: p63 and p73 (Maddocks & Vousden, 2011; Rufini et al, 2012; Su et al, 2012; Liang et al, 2013). We also provided evidence for a role of TP53INP1 in autophagy by direct interaction with mammalian Atg8 orthologs including LC3 (Seillier et al, 2012). Autophagy is a catabolic process involved in the cellular energetic balance and lipid homeostasis thus regulating obesity (Singh & Cuervo, 2011; Lavallard et al, 2012). Interestingly, a genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus (Voight et al, 2010). Collectively, those observations led us to address the role of TP53INP1 in metabolic regulation. We used TP53INP1-deficient mice to assess in vivo the effect of a high-fat diet which favors obesity, insulin resistance and T2D, and we investigated the cellular metabolic defects induced by TP53INP1 deficiency. In this work, we provide the demonstration that TP53INP1 is a primary molecular link between oxidative stress and MS. Results Absence of TP53INP1 favors obesity in a redox-dependent manner in vivo We initially observed that the body weight of 5-month-old TP53INP1-deficient (KO or −/−) mice was higher than WT (+/+) in both males and females (Supplementary Fig S1A) and that fat mass was more abundant in TP53INP1-deficient than in WT mice (Supplementary Fig S1B). We supplemented drinking water with the anti-oxidant N-acetylcysteine (NAC) which alleviates chronic oxidative stress associated with TP53INP1 deficiency through increase of intracellular glutathione level (Cano et al, 2009; N'Guessan et al, 2011). NAC supplementation completely abolished fat mass difference between TP53INP1-deficient and WT mice (Supplementary Fig S1A and B). We then fed 8-week-old mice with a high-fat diet (HFD, 60% fat) (control (CTRL) food is 10% fat) during 16 weeks. We observed a higher body weight gain in HFD-fed TP53INP1 KO than WT mice (Fig 1A and Supplementary Fig S2A), although food consumption did not differ between genotypes (Supplementary Fig S3). Epididymal and renal fat masses were higher in KO than in WT mice upon HFD (Fig 1B and Supplementary Fig S2A). HFD-induced liver weight increase was also higher in HFD-fed TP53INP1 KO mice than WT (Fig 1B and Supplementary Fig S2A). HFD-induced steatosis (accumulation of lipid droplets in hepatocytes), assessed by histological analysis, was greater in KO than in WT mice (Supplementary Fig S2B). Taken together, those data show that TP53INP1-deficient mice are prone to obesity and liver complications, suggesting a role of TP53INP1 in dampening fat storage. Interestingly, the gene encoding TP53INP1 was over-expressed in the liver of HFD-fed C57BL/6 mice (Supplementary Fig S2C). Furthermore, in human, morbidly obese patients with hepatic steatosis showed an increase in hepatic TP53INP1 expression, and TP53INP1 expression was correlated with the level of a marker of hepatocyte death (keratin 18), with the grade of steatosis and with the expression level of the stress marker NQO1 (Supplementary Fig S2D–H and Supplementary Table S2). This suggests that TP53INP1 expression is induced as part of an obesity-associated stress response and that this protective function is lacking in TP53INP1-deficient mice, thus impairing fat homeostasis. Figure 1. TP53INP1-deficient mice are highly susceptible to HFD-induced obesity owing to their chronic oxidative stressTP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%). Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013. At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047. Data information: Results are expressed as the mean ± SEM and are representative of two independent experiments. * −/− versus +/+; £ HFD versus CTRL; § NAC versus no NAC; 1 character: P < 0.05; 2 characters: P < 0.005; 4 characters: P < 0.00005. Download figure Download PowerPoint In order to evaluate the impact of chronic oxidative stress in obesity predisposition of TP53INP1 KO mice, we treated the mice with NAC at the starting of HFD. Whereas NAC treatment did not modify final weight gain in HFD-fed WT mice, it completely abolished all body weight, organ weight and hepatic steatosis differences between HFD-fed KO and WT mice, bringing the KO mice values to those of the WT (Fig 1 and Supplementary Fig S2B). These results illustrate that chronic oxidative stress affecting the TP53INP1-deficient mice predisposes them to increased weight gain and adiposity, further favoring obesity and hepatic steatosis when challenged with a lipid-rich diet. Insulin resistance establishment is elicited by chronic oxidative stress induced by TP53INP1 deficiency in vivo As obesity is generally associated with insulin resistance (IR), we investigated the susceptibility of HFD-fed TP53INP1-deficient mice to develop IR, glucose intolerance and hyperinsulinemia. We monitored glycemia and insulinemia at the beginning and end of HFD protocol and determined the HOMA-IR index (Fig 2A and B, respectively, and Supplementary Table S1). We also performed glucose tolerance (GTT) and insulin tolerance (ITT) tests at the end of HFD protocol (Fig 2C and D, respectively). In HFD-fed WT animals, glucose utilization (GTT, Fig 2C) and insulin sensitivity (ITT, Fig 2D) were both altered as expected. This was compensated by hyperinsulinemia (Fig 2B), while blood glucose remained unchanged (Fig 2A), indicating that WT mice under HFD have developed IR. Interestingly, TP53INP1 knockout mice fed a standard diet were also glucose intolerant and insulin resistant, but neither hyperglycemic nor hyperinsulinemic. Glucose intolerance and IR further developed when TP53INP1-deficient mice were fed a HFD, and hyperinsulinemia finally occurred in such experimental conditions with plasma insulin levels twice as high in HFD-fed TP53INP1-deficient as in WT animals. As a consequence, the combined effects of HFD-induced obesity and the absence of TP53INP1 led to hyperglycemia (Fig 2A and C), suggesting that these mice had developed T2D. In contrast, NAC-treated HFD-fed TP53INP1-deficient mice showed similar metabolic profiles to HFD-fed WT animals (Fig 2A and B) indicative of chronic oxidative stress predisposing those mice to systemic IR, hyperinsulinemia, glucose intolerance and therefore T2D. Figure 2. TP53INP1-deficient mice have moderate redox-related insulin resistance syndrome which is exacerbated by HFD protocolMale TP53INP1 KO and WT mice were fed a high-fat diet (HFD, 60% fat) or a control diet (CTRL) during 16 weeks. Mice drank tap water or NAC-supplemented tap water (1%). A, B. Histograms show blood glucose (A) or plasma insulin (B) levels of 6-h-fasted mice at the beginning (Week 0) and/or at the end of the protocol (Week 16). Fasting blood glucose week 16: P (−/− versus +/+; HFD) = 0.0052; P (CTRL versus HFD; −/−) = 0.000081; P (NAC versus no NAC; −/− HFD) = 0.0019; P (w16 versus w0; +/+ CTRL) = 0.012; P (w16 versus w0; −/− CTRL) = 0.050; P (w16 versus w0; −/− HFD) = 0.0023. Fasting plasma insulin: P (−/− versus +/+; HFD) = 0.043; P (CTRL versus HFD; +/+) = 0.028; P (CTRL versus HFD; −/−) = 0.013; P (CTRL versus HFD; +/+ NAC) = 0.011; P (CTRL versus HFD; −/− NAC) = 0.0015; P (NAC versus no NAC; −/− HFD) = 0.038. C. Glucose tolerance test (GTT) was performed on 6-h-fasted mice during 120 min after injection of 1 g glucose/kg of body weight. Curves on the left show blood glucose level monitored after injection of glucose. Histograms on the right show area under curve (AUC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 0 min) = 0.045; P (−/− versus +/+; CTRL; t = 15 min) = 0.013; P (−/− versus +/+; CTRL; t = 30 min) = 0.016; P (−/− versus +/+; CTRL; t = 60 min) = 0.030; P (−/− versus +/+; CTRL; t = 90 min) = 0.017; P (−/− versus +/+; HFD; t = 60 min) = 0.041; P (−/− versus +/+; HFD; t = 90 min) = 0.043; P (−/− versus +/+; HFD; t = 120 min) = 0.034; P (HFD versus CTRL; +/+; t = 15 min) = 0.0076; P (HFD versus CTRL; +/+; t = 30 min) = 0.0067; P (HFD versus CTRL; +/+; t = 60 min) = 0.00058; P (HFD versus CTRL; +/+; t = 90 min) = 0.0010; P (HFD versus CTRL; +/+; t = 120 min) = 0.023; P (HFD versus CTRL; −/−; t = 60 min) = 0.032. AUC: P (−/− versus +/+; CTRL) = 0.023; P (−/− versus +/+; HFD) = 0.035; P (HFD versus CTRL; +/+) = 0.042. D. Insulin tolerance test (ITT) was performed on 6-h-fasted mice during 150 min after injection of 0.70 U insulin/kg of body weight. Curves on the left show blood glucose level monitored after injection of insulin. Histograms on the right show area above curve (AAC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 15 min = 0.012; P (−/− versus +/+; CTRL; t = 30 min) = 0.022; P (−/− versus +/+; HFD; t = 0 min) = 0.027; P (−/− versus +/+; HFD; t = 15 min) = 0.011; P (−/− versus +/+; HFD; t = 30 min) = 0.0037; P (−/− versus +/+; HFD; t = 60 min) = 0.0028; P (−/− versus +/+; HFD; t = 90 min) = 0.041; P (−/− versus +/+; HFD; t = 120 min) = 0.0032; P (−/− versus +/+; HFD; t = 150 min) = 0.0025; P (HFD versus CTRL; +/+; t = 15 min) = 0.0082; P (HFD versus CTRL; +/+; t = 30 min) = 0.033; P (HFD versus CTRL; +/+; t = 90 min) = 0.047; P (HFD versus CTRL; +/+; t = 150 min) = 0.028; P (HFD versus CTRL; −/−; t = 15 min) = 0.026; P (HFD versus CTRL; −/−; t = 30 min) = 0.0095; P (HFD versus CTRL; −/−; t = 60 min) = 0.0031; P (HFD versus CTRL; −/−; t = 90 min) = 0.033; P (HFD versus CTRL; −/−; t = 120 min) = 0.0068; P (HFD versus CTRL; −/−; t = 150 min) = 0.0082. AAC: P (−/− versus +/+; HFD) = 0.030; P (HFD versus CTRL; −/−) = 0.037. Data information: Results are expressed as the mean ± SEM and are representative of two independent experiments. * TP53INP1 −/− versus TP53INP1 +/+; £ HFD versus CTRL; $ Week 16 versus Week 0; § NAC versus no NAC; 1 character: P < 0.05; 2 characters: P < 0.005; 4 characters: P < 0.00005. Download figure Download PowerPoint TP53INP1 mRNA has been reported to be present in human islets of Langerhans (~30th centile) (Eizirik et al, 2012). Using immunofluorescence to examine mouse pancreatic sections and human isolated β-cells (Fig 3A and B), and quantitative PCR analysis of rodent cells and endocrine tissues (Fig 3C and D), we found that TP53INP1 was expressed both by pancreatic exocrine cells and by the insulin-secreting β-cells which play a central role in the control of glucose homeostasis. Because TP53INP1-deficient mice were glucose intolerant, and since TP53INP1 transcripts were significantly increased in islets isolated from HFD-fed mice (Fig 3E), we next hypothesized that defects in β-cell function or plasticity could occur in TP53INP1 knockout mice. However, neither functional modifications (glucose-induced insulin secretion, NADP(H) or cytosolic free calcium concentration, [Ca2+]c) nor changes in islet mass were detected in the absence of TP53INP1 (Supplementary Fig S4). These results suggest that HFD-fed TP53INP1 KO mice developed diabetes due to severe IR, which resulted from whole-body redox deregulation rather than specific endocrine pancreatic alterations. Nonetheless, the observed failure of β-cell mass or function to increase in response to elevated insulin demand suggests that TP53INP1 may also be required in β-cells to mount a compensatory response to IR. Figure 3. The gene encoding TP53INP1 is expressed in pancreatic endocrine cells A, B. (A, B) Immunocytofluorescent staining of TP53INP1 (red) and insulin (green) in mouse pancreatic sections (A) and single human islet beta cell (B). Scale bars represent 50 μm (A) and 10 μm (B). C–E. Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat (C) and C57BL/6J mice fed with a normal diet (5% fat; ND) or an high-fat diet (45% fat; HFD) (D, E). Results are expressed as the mean ± SEM and are representative of two independen
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