Juliana de Almeida-Faria1,2, Daniella E Duque-Guimarães3, Thomas P Ong3,4, Lucas C Pantaleão3, Asha A Carpenter3, Elena Loche3, Laura C Kusinski3, Thomas J Ashmore3, Robin Antrobus5, Martin Bushell6, Denise S Fernandez-Twinn3, Susan E Ozanne7. 1. University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK. ju.almeidafaria@gmail.com. 2. Obesity and Comorbidities Research Center, Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil. ju.almeidafaria@gmail.com. 3. University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK. 4. Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, Food Research Center, São Paulo, Brazil. 5. Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, UK. 6. Cancer Research UK (CRUK), Beatson Institute, Glasgow, UK. 7. University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK. seo10@cam.ac.uk.
Abstract
AIMS/HYPOTHESIS: Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. METHODS: miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic-hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. RESULTS: The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. CONCLUSIONS/ INTERPRETATION: Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target.
AIMS/HYPOTHESIS: Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. METHODS: miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic-hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. RESULTS: The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. CONCLUSIONS/ INTERPRETATION: Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target.
Authors: R O Benatti; A M Melo; F O Borges; L M Ignacio-Souza; L A P Simino; M Milanski; L A Velloso; M A Torsoni; A S Torsoni Journal: Br J Nutr Date: 2014-03-25 Impact factor: 3.718
Authors: Katherine J Strissel; Zlatina Stancheva; Hideaki Miyoshi; James W Perfield; Jason DeFuria; Zoe Jick; Andrew S Greenberg; Martin S Obin Journal: Diabetes Date: 2007-09-11 Impact factor: 9.461
Authors: Siobhán E McQuaid; Leanne Hodson; Matthew J Neville; A Louise Dennis; Jane Cheeseman; Sandy M Humphreys; Toralph Ruge; Marjorie Gilbert; Barbara A Fielding; Keith N Frayn; Fredrik Karpe Journal: Diabetes Date: 2010-10-13 Impact factor: 9.461
Authors: Elena Loche; Heather L Blackmore; Asha A Carpenter; Jessica H Beeson; Adele Pinnock; Thomas J Ashmore; Catherine E Aiken; Juliana de Almeida-Faria; Josca M Schoonejans; Dino A Giussani; Denise S Fernandez-Twinn; Susan E Ozanne Journal: Cardiovasc Res Date: 2018-08-01 Impact factor: 10.787
Authors: Amir Ajoolabady; Simin Liu; Daniel J Klionsky; Gregory Y H Lip; Jaakko Tuomilehto; Sina Kavalakatt; David M Pereira; Afshin Samali; Jun Ren Journal: Trends Pharmacol Sci Date: 2021-12-08 Impact factor: 14.819
Authors: Patrycja A Jazwiec; Violet S Patterson; Tatiane A Ribeiro; Erica Yeo; Katherine M Kennedy; Paulo C F Mathias; Jim J Petrik; Deborah M Sloboda Journal: Biol Reprod Date: 2022-08-09 Impact factor: 4.161