Protein Kinase D2 drives chylomicron‐mediated lipid transport in the intestine and promotes obesity
2021; Springer Nature; Volume: 13; Issue: 5 Linguagem: Inglês
10.15252/emmm.202013548
ISSN1757-4684
AutoresJonathan Trujillo Viera, Rabih El‐Merahbi, Vanessa Schmidt, Till Karwen, Angel Loza‐Valdes, Akim Strohmeyer, Saskia Reuter, Minhee Noh, Magdalena Wit, Izabela Hawro, Sabine Mocek, Christina Fey, Alexander E. Mayer, Mona C. Löffler, Ilka Wilhelmi, Marco Metzger, Eri Ishikawa, Sho Yamasaki, Monika Rau, Andreas Geier, Mohammed K. Hankir, Florian Seyfried, Martin Klingenspor, Grzegorz Sumara,
Tópico(s)Diet, Metabolism, and Disease
ResumoArticle5 May 2021Open Access Source DataTransparent process Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity Jonathan Trujillo-Viera orcid.org/0000-0003-0566-4589 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany These authors contributed equally to this work. Search for more papers by this author Rabih El-Merahbi orcid.org/0000-0001-8133-8297 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany These authors contributed equally to this work. Search for more papers by this author Vanessa Schmidt Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Till Karwen Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Angel Loza-Valdes orcid.org/0000-0003-1957-4883 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Akim Strohmeyer Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany Search for more papers by this author Saskia Reuter Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Minhee Noh orcid.org/0000-0002-2028-6523 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Magdalena Wit orcid.org/0000-0002-0995-2732 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Izabela Hawro orcid.org/0000-0003-0987-5009 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Sabine Mocek Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany Search for more papers by this author Christina Fey Fraunhofer Institute for Silicate Research (ISC), Translational Center Regenerative Therapies (TLC-RT), Würzburg, Germany Search for more papers by this author Alexander E Mayer Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Mona C Löffler Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Ilka Wilhelmi Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany German Center for Diabetes Research (DZD), München-Neuherberg, Germany Search for more papers by this author Marco Metzger Fraunhofer Institute for Silicate Research (ISC), Translational Center Regenerative Therapies (TLC-RT), Würzburg, Germany Search for more papers by this author Eri Ishikawa Molecular Immunology, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Japan Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan Search for more papers by this author Sho Yamasaki Molecular Immunology, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Japan Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan Search for more papers by this author Monika Rau Division of Hepatology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Andreas Geier Division of Hepatology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Mohammed Hankir orcid.org/0000-0001-5218-9683 Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Florian Seyfried Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Martin Klingenspor orcid.org/0000-0002-4502-6664 Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany Search for more papers by this author Grzegorz Sumara Corresponding Author [email protected] [email protected] orcid.org/0000-0003-1502-6265 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Jonathan Trujillo-Viera orcid.org/0000-0003-0566-4589 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany These authors contributed equally to this work. Search for more papers by this author Rabih El-Merahbi orcid.org/0000-0001-8133-8297 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany These authors contributed equally to this work. Search for more papers by this author Vanessa Schmidt Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Till Karwen Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Angel Loza-Valdes orcid.org/0000-0003-1957-4883 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Akim Strohmeyer Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany Search for more papers by this author Saskia Reuter Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Minhee Noh orcid.org/0000-0002-2028-6523 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Magdalena Wit orcid.org/0000-0002-0995-2732 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Izabela Hawro orcid.org/0000-0003-0987-5009 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Sabine Mocek Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany Search for more papers by this author Christina Fey Fraunhofer Institute for Silicate Research (ISC), Translational Center Regenerative Therapies (TLC-RT), Würzburg, Germany Search for more papers by this author Alexander E Mayer Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Mona C Löffler Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Search for more papers by this author Ilka Wilhelmi Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany German Center for Diabetes Research (DZD), München-Neuherberg, Germany Search for more papers by this author Marco Metzger Fraunhofer Institute for Silicate Research (ISC), Translational Center Regenerative Therapies (TLC-RT), Würzburg, Germany Search for more papers by this author Eri Ishikawa Molecular Immunology, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Japan Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan Search for more papers by this author Sho Yamasaki Molecular Immunology, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Japan Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan Search for more papers by this author Monika Rau Division of Hepatology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Andreas Geier Division of Hepatology, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Mohammed Hankir orcid.org/0000-0001-5218-9683 Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Florian Seyfried Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Würzburg, Würzburg, Germany Search for more papers by this author Martin Klingenspor orcid.org/0000-0002-4502-6664 Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany Search for more papers by this author Grzegorz Sumara Corresponding Author [email protected] [email protected] orcid.org/0000-0003-1502-6265 Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Search for more papers by this author Author Information Jonathan Trujillo-Viera1, Rabih El-Merahbi1, Vanessa Schmidt1, Till Karwen1, Angel Loza-Valdes2, Akim Strohmeyer3,4,5, Saskia Reuter1, Minhee Noh1, Magdalena Wit2, Izabela Hawro2, Sabine Mocek3,4,5, Christina Fey6, Alexander E Mayer1, Mona C Löffler1, Ilka Wilhelmi7,8, Marco Metzger6, Eri Ishikawa9,10, Sho Yamasaki9,10, Monika Rau11, Andreas Geier11, Mohammed Hankir12, Florian Seyfried12, Martin Klingenspor3,4,5 and Grzegorz Sumara *,*,1,2 1Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany 2Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland 3Chair for Molecular Nutritional Medicine, Technical University of Munich, TUM School of Life Sciences Weihenstephan, Freising, Germany 4EKFZ - Else Kröner-Fresenius-Center for Nutritional Medicine, Technical University of Munich, Munich, Germany 5ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany 6Fraunhofer Institute for Silicate Research (ISC), Translational Center Regenerative Therapies (TLC-RT), Würzburg, Germany 7Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany 8German Center for Diabetes Research (DZD), München-Neuherberg, Germany 9Molecular Immunology, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Japan 10Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan 11Division of Hepatology, University Hospital Würzburg, Würzburg, Germany 12Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Würzburg, Würzburg, Germany *Corresponding author. Tel: +49 931 31 89263 or +48 22 5892 190; E-mails: [email protected] or [email protected] EMBO Mol Med (2021)13:e13548https://doi.org/10.15252/emmm.202013548 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 Lipids are the most energy-dense components of the diet, and their overconsumption promotes obesity and diabetes. Dietary fat content has been linked to the lipid processing activity by the intestine and its overall capacity to absorb triglycerides (TG). However, the signaling cascades driving intestinal lipid absorption in response to elevated dietary fat are largely unknown. Here, we describe an unexpected role of the protein kinase D2 (PKD2) in lipid homeostasis. We demonstrate that PKD2 activity promotes chylomicron-mediated TG transfer in enterocytes. PKD2 increases chylomicron size to enhance the TG secretion on the basolateral side of the mouse and human enterocytes, which is associated with decreased abundance of APOA4. PKD2 activation in intestine also correlates positively with circulating TG in obese human patients. Importantly, deletion, inactivation, or inhibition of PKD2 ameliorates high-fat diet-induced obesity and diabetes and improves gut microbiota profile in mice. Taken together, our findings suggest that PKD2 represents a key signaling node promoting dietary fat absorption and may serve as an attractive target for the treatment of obesity. Synopsis We show that upon fat ingestion, Protein Kinase D2 stimulates chylomicron-mediated triglyceride absorption in the intestine. Targeting PKD2, genetically or with small molecule inhibitors, reduces triglycerides absorption and prevents the development of obesity in mice and presumably in humans. PKD2 enhances chylomicron size and therefore chylomicron-mediated triglycerides absorption. PKD2 phosphorylates chylomicron-associated lipoprotein, APOA4. Inhibition of PKD2 diminishes obesity and associated diabetes. PKD2 activity correlates with triglycerides levels in obese patients. The Paper Explained Problem Consumption of highly caloric diets rich in triglycerides is one of the major causes for the development of obesity and associated disorders. However, up to date, limited pharmacological strategies exist to inhibit lipid absorption in the intestine. We have investigated the function of the diacylglycerol-activated protein kinase D2 in the regulation of triglyceride absorption in the intestine and its impact on the development of obesity. Results We have shown that PKD2 is activated upon triglyceride ingestion in the intestine. Moreover, we have demonstrated that PKD2 phosphorylates APOA4 to promote chylomicron size, and therefore, triglycerides transport in the intestine. Importantly, deletion, inactivation, or inhibition of PKD2 in mice suppresses triglyceride absorption in the intestine and ameliorates obesity as well as associated diabetes. Additionally, deletion of PKD2 is associated with improved microbiota in the intestine. Finally, our results indicate that PKD2 activity correlates with triglyceride levels in patients, and silencing of PKD2 in human enterocytes reduces chylomicron-mediated triglyceride transport. Impact Our findings indicate that PKD2 is an attractive target for pharmacological intervention to limit lipid absorption in the intestine and therefore ameliorate obesity and prevent the development of diabetes. Introduction The type of diet plays a major role in modulating organismal metabolism. Diets containing elevated fat content are generally more energy dense, which promotes a positive energy balance and, consequently, obesity. The digestive system is the first site to be challenged by elevated levels of fat in the diet. After emulsification of ingested fat by bile acids, triglycerides are broken down into glycerol, monoglyceride, and fatty acids (FAs) by pancreatic lipases in the small intestine lumen (Lowe, 2002; Hussain, 2014). Monoglycerides and FAs are then taken up by enterocytes either by passive diffusion or by an active mechanism involving FA transporters such as cluster of differentiation 36 (CD36) (Xu et al, 2013; Hussain, 2014). In enterocytes, FAs and monoglycerides or glycerol are re-esterified at the endoplasmic reticulum (ER). These monoglyceride and glycerol 3-phosphate pathways are responsible for the majority of TG synthesis in enterocytes (Yang & Nickels, 2015). Following re-esterification, TG are packed into pre-chylomicrons together with lipoproteins such as apolipoprotein B48 (APOB48) and apolipoprotein A4 (APOA4) by the microsomal transfer protein (MTTP) (Mansbach & Siddiqi, 2016). APOB48 is absolutely required for pre-chylomicron formation at the ER (Mansbach & Siddiqi, 2016), while APOA4 is likely responsible for determining final chylomicron size (Lu et al, 2006; Kohan et al, 2012; Weinberg et al, 2012; Kohan et al, 2015). Following their assembly, pre-chylomicrons are then transported to the Golgi apparatus to undergo further chemical modifications (possibly including lipidation) and, finally, are designated for secretion (Hesse et al, 2013). Increased dietary fat content leads to the elevation in expression and activity of enzymes critical for lipid uptake, FA re-esterification, TG packing, and lipoproteins required for assembly of pre-chylomicrons (Petit et al, 2007; Hernández Vallejo et al, 2009; Clara et al, 2017). Interestingly, an increase in chylomicron size might be a major factor determining the elevated capacity of enterocytes to process excessive dietary fat (Uchida et al, 2012). However, the signaling cascades driving the adaptation of enterocytes to increased lipid loads in the intestinal lumen remain largely unknown. Protein kinase D (PKD) family members are diacylglycerol (DAG) and protein kinase C (PKC) effectors, which recently emerged as central regulators of nutrient homeostasis (Sumara et al, 2009; Löffler et al, 2018; Mayer et al, 2019; Kolczynska et al, 2020). The PKD family includes three members (PKD1, PKD2, and PKD3), which regulate several aspects of cellular metabolism and pathophysiology (Fielitz et al, 2008; Kim et al, 2008; Sumara et al, 2009; Kleger et al, 2011; Konopatskaya et al, 2011; Rozengurt, 2011; Ittner et al, 2012; Löffler et al, 2018; Mayer et al, 2019; Kolczynska et al, 2020; preprint: Mayer et al, 2020). Our recent study suggested that PKDs might be activated in response to free FAs (FFAs) or DAG (Mayer et al, 2019). Moreover, high-fat diet (HFD) feeding activated PKDs in the liver (Mayer et al, 2019). However, the impact of PKDs activity on lipid metabolism in the intestine has not been investigated so far. Here, we show that PKD2 (also known as PRKD2) is activated upon lipids loading in intestine and promotes chylomicron growth and lipidation and consequently TG secretion by human and mouse enterocytes. Interestingly, PKD2 directly phosphorylates one of the apolipoproteins associated with chylomicrons, namely APOA4. Deletion of PKD2 in intestine of mice or in human enterocytes results in increased abundance of intracellular and secreted APOA4. Consistently with these results, the ablation of PKD2 activity or the specific deletion of this kinase in the intestine resulted in reduced absorption of fat, increased excretion of energy in the feces and resistance to high-fat diet-induced obesity. Moreover, deletion of PKD2 resulted in resistance to high-fat diet-induced diabetes and pathological changes in the gut microbiota. Additionally, we demonstrate that a PKD-specific inhibitor decreases fat absorption and is effective in the treatment of obesity and associated diseases in animal models. Finally, our data indicate that human subject activity of PKD2 in the intestine correlates with TG levels in blood. Therefore, we establish PKD2 as a key component of the intestinal fat absorption and an attractive target for future anti-obesity therapies. Results PKD2 inactivation protects from diet-induced obesity Our previous studies revealed that PKD1 promotes obesity by blocking energy dissipation in adipocytes (Löffler et al, 2018), while PKD3 promotes hepatic insulin resistance (Mayer et al, 2019). However, the role of PKD2 in regulating glucose and lipid metabolism and in the development of obesity-induced diabetes is not known. We addressed this by utilizing mice with global defective PKD2 enzymatic activity, because of point mutations in serines 707 and 711 to alanines (Pkd2ki/ki mice) (Matthews et al, 2010a). We maintained Pkd2ki/ki mice and corresponding control animals (Pkd2wt/wt) on a normal chow diet (ND) or high-fat diet (HFD) for 22 weeks after weaning. Remarkably, while Pkd2ki/ki and Pkd2wt/wt mice on ND gained similar weight, Pkd2ki/ki mice maintained on HFD gained significantly less weight than corresponding control animals (Fig 1A). Weight reduction in Pkd2ki/ki mice fed HFD was associated with decreased adiposity and reduced adipocyte size (Fig 1B–E). However, the weight of other organs was not affected by PKD2 inactivation (Fig 1C). As previous studies indicated that deletion of PKD1 kinase (closely related to PKD2), in adipose tissue, promotes the expression of beige adipocyte-specific markers (Löffler et al, 2018), we have tested the expression of Ucp1, Cidea, Bmp7, Prdm16, Ppara, Pgc1a, Adrb3, Cidec, Myh2, Ckm, Mck, Slc6a8, Slc27a2, Ucp3, and Myh1 subcutaneous adipose tissue of Pkd2ki/ki and control Pkd2wt/wt mice fed HFD and except Slc6a8, which was downregulated in the mice without active PKD2; there were no significant changes in the expression of these genes (Fig EV1A). Similarly, liver histology and markers of hepatic function, aspartate transaminase (AST), and alanine transaminase (ALT) did not differ between genotypes, while hepatic content of TG was decreased in Pkd2ki/ki mice (Figs 1F and EV1B–D). Of note, mice deficient for PKD2 enzymatic activity were protected from diet-induced glucose intolerance and displayed better insulin sensitivity when fed HFD (Fig 1G and H). Moreover, in the Pkd2ki/ki mice, we found a small but not significant increase in insulin levels during the glucose-stimulated insulin secretion test (Fig EV1E). Furthermore, the islet area (relative to pancreas area) is increased in Pkd2ki/ki mice (Fig EV1F–H). Inactivation of PKD2 in mice fed HFD also reduced circulating triglyceride and free FA levels (Fig 1I and J). Altogether, these findings indicate that inactivation of PKD2 protects from diet-induced obesity as well as associated hyperglycemia and hyperlipidemia. Figure 1. PKD2 inactivation protects from diet-induced obesity A. Body weight gain of male mice with the specified genotypes under normal (ND) or high-fat diet (HFD). B. Quantification of fat, free fluid, and lean mass by nuclear magnetic resonance (NMR) of mice in HFD in panel (A). C. Organ weight (percentage of total weight) of different fat depots, liver, and quadriceps of Pkd2wt/wt and Pkd2ki/ki male mice after 22 weeks in HFD. D. Quantification of the average adipocyte size in SubWAT and EpiWAT of male mice of the specified genotypes in normal and high-fat diet. E. Representative pictures of H&E staining of SubWAT and EpiWAT of indicated male mice fed HFD. F. Triglyceride content in liver of Pkd2wt/wt and Pkd2ki/ki male mice in panel (A). Relative to Pkd2wt/wt. G. Glucose tolerance test of the specified genotypes after 16 weeks in HFD. H. Insulin tolerance test of Pkd2wt/wt and Pkd2ki/ki male mice after 18 weeks in HFD. I, J. Triglycerides (I) and free fatty acids (J) in circulation of specified mice after 18 weeks in HFD. K, L. Energy expenditure (K) and energy intake (L) of mice after 20 weeks in HFD. Data information: Male mice were subjected to ND or HFD directly after weaning, and the specified metabolic parameters were measured. n = 7 for ND. In HFD, n = 7 for Pkd2wt/wt and n = 8 for Pkd2ki/ki. Data presented as average ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Significances were assessed by using a two-tailed Student's t-test for independent groups. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Inactivation of PKD2 does not affect adipose tissue or liver function but increases pancreatic islets size - Related to Fig 1 A. Relative expression of specified genes in SubWAT of Pkd2wt/wt and Pkd2ki/ki male mice after 22 weeks in HFD. n = 6. B. Representative pictures of H&E staining of liver of indicated male mice in panel (A). C, D. Aspartate aminotransferase (C) and alanine aminotransferase (D) levels in serum of male mice in panel (A). n = 5. E. Glucose-stimulated insulin secretion of Pkd2wt/wt and Pkd2ki/ki male mice after 8 weeks in HFD. n = 8. F. Representative pictures of immunofluorescent staining for insulin (red) and DAPI (blue) of Pkd2wt/wt and Pkd2ki/ki male mice. n = 3. For each subject, three different sections of the pancreas were taken and a distance of 50 μm was kept between the sections. G. Mean fluorescence intensity (MFI) for insulin of mice in panel (F). Relative to Pkd2wt/wt. n = 3. MFI was quantified from all the islets found in each of the three sections per experimental subject. H. Islet area compared to pancreas area of mice in panel (F). Relative to Pkd2wt/wt. All the islets found in each of the three sections per mouse were quantified. I. Activity of Pkd2wt/wt and Pkd2ki/ki male mice after 20 weeks in HFD. Expressed as counts per 12 h of intervals. n = 7 for Pkd2wt/wt and n = 8 for Pkd2ki/ki. Data information: Data presented as average ± SEM, *P ≤ 0.05, **P ≤ 0.01. Significances were assessed by using a two-tailed Student's t-test for independent groups. Download figure Download PowerPoint In order to investigate the mechanisms underlying the amelioration of obesity in mice deficient for PKD2 activity, we used integrated analyses of metabolic parameters which revealed that inactivation of PKD2 does not affect food intake, energy expenditure, or voluntary movements of mice fed HFD (Figs 1K and L, and EV1I), suggesting that reduced body weight gain of Pkd2ki/ki upon HFD feeding must be caused by misregulation of other processes. PKD2 promotes the absorption of lipids from food Since inactivation of PKD2 in mice did not affect food intake and energy expenditure, we hypothesized that absorption of nutrients might be reduced in the absence of PKD2 activity. Therefore, we collected feces from Pkd2ki/ki mice and control animals. We observed that feces collected from Pkd2ki/ki mice fed HFD were yellowish in contrast to the excrements derived from Pkd2wt/wt control mice which displayed a typical dark-brown color (Fig 2A). Moreover, in contrast to stool from control mice, feces from Pkd2ki/ki mice did not sink in water (Fig 2A). Additionally, Pkd2ki/ki mice fed HFD produced more stool than control animals per week also when extrapolated to food intake (Fig 2B and C). Moreover, HFD feeding was significantly less efficient in promoting body weight gain in mice expressing inactive PKD2 relative to control animals (Fig 2D). Of note, Pkd2ki/ki fed ND produced the same amount of feces as corresponding control animals and their color did not differ from feces from control animals (Fig EV2A and B). These data suggest that PKD2 inactivation dramatically modulates the physicochemical properties of feces of mice fed HFD but not of mice fed ND. Figure 2. Pkd2 knockin mice excrete more energy in feces and present lower absorption of lipids in the intestine A. Pictures of feces collected from male mice fed HFD and photo of them placed in water. B. Weight of feces collected in a week. C. Feces excreted per gram of food consumed. D. Body weight gained per gram of food consumed. E, F. Energy content (E) and lipid content (F) per gram of feces. G. Percentage of energy excreted in feces from the total energy ingested. H. Metabolizable energy calculated from intake minus excreted and assuming a urinary excretion of 2%. Data information: For panels (A–H), male mice after weaning were kept in individual cages during 2 weeks fed with HFD. For first 2 weeks, animals were acclimatized in the cages, and then during two more weeks, mice were monitored for food consumption and feces were analyzed for deposition of lipids and energy. n = 7 for Pkd2wt/wt and n = 8 for Pkd2ki/ki. Data presented as average ± SEM. **P ≤ 0.01, ***P ≤ 0.001. Significances were assessed by using a two-tailed Student's t-test for independent groups. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. PKD2 does not regulate the expression of genes implicated in alimentary lipid processing in the intestine or the liver - Related to Fig 2 Weight of feces collected from Pkd2wt/wt and Pkd2ki/ki male mice fed normal diet per week. n = 6. Pictures of feces from Pkd2wt/wt and Pkd2ki/ki male mice under normal diet. n = 6. Western blot for pancreatic lipase in duodenum of Pkd2wt/wt and Pkd2ki/ki male mice 1 h after olive oil gavage. n = 6. The animals were dissected at the ind
Referência(s)