In this study we aimed to gain insight into the regulation of fat cell insulin sensitivity by miRNAs. So far there is only a handful of studies of miRNAs in relation to IR of human fat cell (103,104). We selected obese individuals based on insulin sensitivity that was determined
by insulin-stimulated minus basal lipogenesis of isolated fat cells. This way we could generate the cohort consisting of 21 OIS, 18 OIR and 9 lean subjects as controls. The obese groups were matched for BMI, waist circumference and fat cell size, all of which may influence insulin sensitivity independently of lipogenesis values. More clinical data could be found below in the manuscript of study IV.
A combination of global miRNA measurements and validations of miRNA expression with RT-qPCR identified eleven adipose miRNAs to be differently regulated in IOS compared to IOR. These candidate miRNAs were further pursued for functional studies in vitro. The miRNAs were overexpressed in human differentiated adipocytes followed by measurements of basal and insulin-stimulated glucose incorporation of lipids. Two miRNAs (miR-143-3p and miR-652-3p) increased glucose incorporation into lipids in response to insulin.
The possible clinical impact was evaluated by performing association analysis of the miRNAs expression determined by RT-qPCR in intact WAT with the lipogenesis values in isolated fat cells ex vivo. Indeed, expression of both miR-143-3p and miR-652-3p correlated to insulin-stimulated lipogenesis independently of BMI. To identify targets of miR-143-3p and miR-652-3p involved in lipogenic pathways and to delineate their possible regulatory pathways, we selected for known genes involved in insulin signaling, insulin sensitivity/resistance and lipogenesis. To identify if these genes could possibly be targeted by the miRNAs, they were overlapped with the list of predicted targets of the miR-143-3p and miR-652-3p. We identified 13 and 8 genes for miR-143-3p and miR-652-3p respectively thus, mRNA expression was measured after overexpression of the miRNAs in adipocytes.
Overexpression of miR-143-3p increased PRKAA1 mRNA levels by about 35 % while overexpression of miR-652-3p downregulated ENPP1 by 30 %. The findings were also confirmed at the protein levels for both genes. As miR-652-3p reduced its predicted target, one could suspect that it directly targeted ENPP1. Indeed, the direct binding of miRNA- 652-3p to the 3'-UTR of ENPP1 was confirmed by the luciferase reporter assay.
As mentioned in section 1.3.1.1, insulin signaling comprises of numerous phosphorylation events and the protein kinase AMPK is an important regulator for energy metabolism responsible for direct phosphorylation of metabolic enzymes. PRKAA2 is a catalytic subunit of AMPK. The fact that overexpression of miRNA-143-3p increased expression of PRKAA2 allowed us to hypothesize for indirect effects where posttranslational events could be involved. Obviously, miRNA-652-3p also targets multiple targets, many of them indirectly.
Thus we overexpressed both miRNAs in adipocytes, and measured total and phospho-proteins at the activating residues of the main insulin signaling players using ELISA (Akt2, phospho-Akt2, total IRS-1, phospho-IRS1, Insulin Receptor β and phospho-Insulin Receptor β). We found that phosphorylation of AKT2 at Ser474-site was induced by miR-143-3p and miR-652-3p increased phosphorylation of AMPK at activating residue Thr172 and total phosphorylation of IRS-1.
In summary, miR-143-3p and miR-652-3p were downregulated in OIR compared to OIS
women. We found that they increase insulin-stimulated lipogenesis directly by reducing ENPP1 and indirectly by inducing post-translational (PRKAA2) and phosphorylation’s events. The main findings are illustrated in a schematic picture (Figure 7).
Figure 7. A schematic summarization of miRNAs involved in glucose incorporation in human white adipocytes. Two miRNAs was found to be downregulated in obese insulin resistant adipose tissue and suggested to regulate lipid incorporation. The circuit of the experimentally validated miRNAs indicated that miR-143-3p indirectly affects PRKAA2 (indicated by dashed line), whereas miR-652-3p was found to bind and regulate ENPP1. Both miRNAs was moreover engaged in phosphorylating important insulin signaling factors such as AKT2, AMPK and IRS-1 through undefined mechanism.
Here, we included phosphorylation events that positively regulate insulin signaling due to the stimulatory effects of the miRNAs on glucose incorporation to the lipids. Phosphorylation of residues promoting inhibitory effects is also important but was not covered in here. It would be interesting to more thoroughly investigate and characterize miRNAs effect on insulin signaling post-translationally and especially the phosphorylation actions.
Table 3. An overview of the obesity dysregulated miRNAs in WAT and their effect on adipocyte function.
MicroRNA Expression in WAT Combinatorial effect Affect
Effect on Lipolysis
Obese/lean IS/IR Morphology CCL2 Adiponectin TNF- α
Let-7a-5p down - - - yes no Yes no
Let-7d-5p down - up in hypertrophy - yes no yes yes
miR-26a-5p down down up in hypertrophy - yes yes yes yes
miR -30c-5p down - - - no no no yes
miR -92a-3p down - - on CCL2
(miR-92a+193b/126) yes no yes no
miR -126-3p down - - on CCL2 (miR-126+92a) yes yes no no
miR -143-3p down down up in hypertrophy - yes no yes no
miR-145-5p down down - - yes no yes yes
miR-193a-5p down - - - yes no yes no
miR-193b-3p down - - on CCL2 (miR-92a+193b) yes yes yes yes
miR-652-3p down down - - yes no no yes
miR-361-5p - - up in hypertrophy on EBF1 (miR-361+574) - - - -
miR-574-5p - - up in hypertrophy on EBF1 (miR-361+574) - - - -
References Arner et al.
(2012) (73)
Dahlman et al. (MS)
Belarbi et al. (Manuscript 2016)
Kulyté et al. (2014) (105), Belarbi et al. (Manuscript 2016)
Kulyté et al.
(2014) (105)
Belarbi et al.
(2015)
Lorente-Cebrian et al.
(2014) (106)
Lorente-Cebrian et al. (2014) (106)
Abbreviations: IS, insulin sensitive; IR, insulin resistance; CCL2, chemokine (C-C Motif) Ligand 2; TNF- α, tumor necrosis factor α; EBF1, early B cell factor 1.
5 CONCLUSION AND FUTURE PERSPECTIVES
WAT has a remarkable ability to adapt and remodel in response to under- and over nutrition.
The flexibility of WAT includes alterations in the structure and composition that consequently affects adipocyte metabolism. The biology of the different conditions of WAT is complex and extensive research is performed in order to understand the molecular events and its clinical impact. The focus of this thesis was to identify miRNAs in WAT and map their regulatory pathways in relation to obesity and IR. Our results provide novel insights on the function of miRNAs in human adipocytes. Nevertheless, many questions remain to be addressed.
Future investigations are needed to evaluate the clinical impact of the dysregulated miRNAs and to elucidate the complete gene regulatory networks in WAT and their contribution to insulin sensitivity and inflammation. For all four studies, the experimental pipeline was to identify miRNAs by global expression analysis of the miRNA transcriptome.
Further finding and validating comprehensive pathways leading to clinical pathologies is even more important. Studies I-III are based on the same cohort (cohort 1) consisting of lean and obese individuals, where expression analysis of miRNA and genes were assessed. Data revealed that 11 miRNAs were dysregulated in obesity (73). We further studied miRNAs and miRNA-gene regulatory networks to get insights into different aspects of WAT function, e.g.
inflammation (studies on CCL2 and adiponectin) and WAT morphology (EBF1 study). In study IV, we evaluated the impact of miRNAs on insulin sensitivity in obese subjects.
Altogether, these studies demonstrate the impact of miRNAs in regulating WAT function.
A further apparent and very relevant investigation is to create networks that include additional levels of regulation including chromatin marks (DNA methylation, histone modifications) and post-translational modifications such as phosphorylation and methylations events. In addition, it would be of interest to map the obesity-regulated miRNAs which were excluded from more detailed analyses due to various reasons. By including these miRNAs, the regulatory network could be extended. For example in study III it would be interesting to study the remaining 9 miRNAs, and how they may be involved in regulating WAT morphology through e.g. EBF1, PPARG, and C/EBPα. These three genes encode TFs that cross talk and regulate each other and all are downregulated in hypertrophic non-obese WAT (42,101). In general, studies on the regulation of miRNAs themselves are lacking and it would be highly relevant to consider this extra layer. Other aspects of interest are the ability of miRNAs to act in a combinatorial manner and to characterize if/how miRNAs crosstalk with other tissues e.g. through exosome secretion.
6 ACKNOWLEDGEMENT
I am truly happy for the journey of this experience and all that it has taught me about science and myself. I am grateful for my colleagues, friends and my family who have all supported throughout this thesis.
Firstly, I would like to thank my main supervisor, Agné Kulyté, for teaching me all about science and molecular techniques. Your great skills in the lab make everything look easy. I have tried to keep up with your structured way of working and quick pace. I have been spoiled with your unlimited help and I don’t know if I am ready to let go yet. Thank you for having patience and for taking time to support me at any time, for letting me grow and encouraging me towards new challenges. Lastly, I want to thank you for all the fun moments we have shared in the lab and on conferences, especially in Japan. In our next life I will teach you all about FOCUS and STRUCUTURE.
Mikael Rydén, an excellent scientist and a great teacher. Thank you for giving me this opportunity. It has been a true inspiration to observe your work and without your input this thesis would have been unreadable. Your fun personality adds an extra layer to the scientific world.
Peter Arner for being the most kind and happy professor I have ever met. Your optimism and genuine interest will always be an inspiration. Thank you for your encouragement and for taking time to answer my questions. You are a true genius, great boss and a brilliant character contributing with lots of fun.
Niklas Mejhert, I don’t know where to start. I am forever thankful for all the hidden doors you have presented and opened for me, even outside of science. You have made me laugh my pants off and contributed with great times. It has been a true pleasure working with you. You took me under your wing and introduced me to the scientific world. Thanks for always believing in me and for being a great support!
Thank you Gaby Åström, Eva Sjölin, Kerstin Wåhlén and Elisabeth Dungner, the most valuable players (MVP) in the lab. You are always helpful and your skills in the lab are truly impressive. Thanks for contributing to a vibrant atmosphere and for the fun talks in the lab.
Thank you for teaching me molecular techniques and for covering-up my mistakes. Gaby Åström, thank you for all the deep talks and your warm heart, for all the music sessions and discussion about society. Eva Sjölin, thanks for introducing me to the pipette and all the fun exchanges of experience in life. The lab is not the same without you. Kerstin Wåhlén, thank you for all the support and nice talks in the lab. Your way of handling problems and finding solutions is inspiring. Elisabeth Dungner, thanks for always helping out and always solving problems when everyone else have given up. I have enjoyed all the laughs and interesting
talks while pipetting.
Hui Gao, Thank you for taking time and teaching me every detail about western blot “the old school way”. It has always been a pleasure working with you. You have offered different perspectives perspective on things that I never thought about. I wish I could have your mind set for only one day.
Paul Petrus, not only a colleague but also a friend. It has been nice to share all my weaknesses with you, from coffee and chewing gum addiction to not understanding proper Swedish. Thanks for explaining and making molecular mechanisms understandable, I will miss your summarizing drawings while you chewing on candy, a perfect fat researcher.
My mentor Maja Ullberg, thanks for listening to me and giving me good advice. I have enjoyed those moments with you, always contributing with bright ideas and solutions to problems. The way you put things in perspective is inspiring.
Ingrid Dahlman, Jurga Laurencikiene, Anna Ehrlund, Juan Acosta, Christel Björk for being great inspiring scientists. Thanks for all your comments and help at work. I have enjoyed your company in the lab and on conferences. Special thanks to Christel for always being able to help, giving advice for coping with an IBS stomach and also all the nice talks about life. Juan.for doing odd things and contributing to a comic environment.
Amitha, Ana and Patricia, I have not known you for too long but it has been a blast this period, GO GIRLS.
Britt-Marie Leijonhufvud, Daniel Eriksson Hogling, Daniel P Andersson, Jesper
Bäckdahl, Katarina Hertel, Lena Lindberg, Patrik Löfgren, Yvonne Widlund , (current and former lab members) – for all discussions and inputs.
Former colleagues, Silvia Lorente-Cebrián, Annie Pettersson, Britta Stenson and Clara Bambace thank you for taking care of me in the lab my first years, teaching me everything about pranks, HOT chocolate and contributing to a fun environment.
Prince Joel, for always being happy and spreading good energy with your laugh. I loved our Thursday sessions.
All the nice people I have meet in the kitchen sharing nice conversation. Muhammad Al Mustafa Ismail for being a great friend and for motivating me to stay positive. Thanks for all the nice coffee sessions and for bringing cookies from all over the world. Heela Sarius, I have appreciated all our interesting discussion and your warm heart. You have broadened my view on life, giving me new perspectives.
My friend and dance teacher, Marita Halldén. For showing and carefully guiding me into a new exiting world where I have met wonderful people (Alma, Agnes but also others).
Without you I would never have made it, I will never forget what you have given to me.
My family, I owe all my gratitude for the endless love and support even though times have been tough. There is not enough space here to explain my appreciation. To my lovely parents, Satu and Sadek, for always being true heroes in my life. To my wonderful siblings, Sabria, Merieme, Yacine and Mohamed for making me understand what life really is about. Sabria, for not only being my sister but also friend. Thank you for your endless support, motivating talks and for inputs in my work. I have not only been blessed with a double set of sisters and brothers but also parents, love you Abbassia and Jerry. Lots of love to my relatives spread out in the world, some extra emphasis on my family in Algeria and US.
Last but definitely not least, all my great friends. For all fun, laugh and crazy moments we have shared, I know I have been busy, rude and unavailable but times are changing. I hope you understand how much you mean to me and thank you for always being there. We have shared so many memories and I wish for many more. Thank you for supporting me for throughout this period. Love attack to my closest friends; Alice, Susie, Marieme, Fanny Anna, Tyson, Sara, Julia, Winta, Sebastian, Ida and Madjiguene. Susie and Winta, for being the best study friends ever, I would never have passed a single course without you. I am so glad that we found each other the first day. Other people who are equally important are Diane, Marko, Josefin, Siri, Evelin, Sofie, Tomas, Meshach, Amelie, Kendis, Manzoura, Mohammed, Love, Lindsay, John, Kathy, Larry, Lina, Lea, Yasmine and all the others that I may have forgot. <3<3<3
7 REFERENCES
1. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, et al. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study 2013.
Lancet. 2014;384:766–81.
2. World Health Organization. WHO | Obesity and overweight [Internet]. 2013 [cited
2016 Jan 1].
Available from: http://www.who.int/mediacentre/factsheets/fs311/en/index.html 3. Cornier MA, Dabelea D, Hernandez TL, Lindstrom RC, Steig AJ, Stob NR, et al. The
metabolic syndrome. Endocr Rev. 2008;29:777–822.
4. NCD Risk Factor Collaboration (NCD-RisC). Worldwide trends in diabetes since 1980: a pooled analysis of 751 population-based studies with 4.4 million participants.
Lancet. 2016;387:1513–30.
5. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic burden of the projected obesity trends in the USA and the UK. Lancet. 2011;378:815–
25.
6. Bish CL, Blanck HM, Serdula MK, Marcus M, Kohl HW, Khan LK. Diet and physical activity behaviors among Americans trying to lose weight: 2000 Behavioral Risk Factor Surveillance System. Obes Res. 2005;13:596–607.
7. Hales CN, Barker DJP. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. 1992. Int J Epidemiol. 2013;42:1215–22.
8. Vaag AA, Grunnet LG, Arora GP, Brøns C. The thrifty phenotype hypothesis revisited. Diabetologia. 2012;55:2085–8.
9. Pampel FC, Denney JT, Krueger PM. Obesity, SES, and economic development: A test of the reversal hypothesis. Soc Sci Med. 2012;74:1073–81.
10. Walley AJ, Asher JE, Froguel P. The genetic contribution to non-syndromic human obesity. Nat Rev Genet. 2009;10:431–42.
11. Nishida C, Ko G, Kumanyika S. Body fat distribution and noncommunicable diseases in populations: overview of the 2008 WHO Expert Consultation on Waist Circumference and Waist–Hip Ratio. Eur J Clin Nutr. 2010;64:2–5.
12. World Health Organization. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet. 2004;363:157–63.
13. Sjöström L. Review of the key results from the Swedish Obese Subjects (SOS) trial - a prospective controlled intervention study of bariatric surgery. J Intern Med.
2013;273:219–34.
14. Bueter M, le Roux CW. Gastrointestinal hormones, energy balance and bariatric surgery. Int J Obes (Lond). 2011;35:S35–9.
15. Spalding KL, Arner E, Westermark PO, Bernard S, Buchholz BA, Bergmann O, et al.
Dynamics of fat cell turnover in humans. Nature. 2008;453:783–7.
16. Arner P. Human fat cell lipolysis: Biochemistry, regulation and clinical role. Best Pr
Res Clin Endocrinol Metab. 2005;19:471–82.
17. Frayn KN, Arner P, Yki-Jarvinen H. Fatty acid metabolism in adipose tissue, muscle and liver in health and disease. Essays Biochem. 2006;42:89–103.
18. Samuel VT, Shulman GI. The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest. 2016;126:12–22.
19. Leto D, Saltiel AR. Regulation of glucose transport by insulin: traffic control of GLUT4. Nat Rev Mol Cell Biol. 2012;13:383–96.
20. Wong RH, Sul HS. Insulin signaling in fatty acid and fat synthesis: A transcriptional perspective. Curr Opin Pharmacol. 2010;10:684–91.
21. Arner P. Insulin resistance in type 2 diabetes: role of fatty acids. Diabetes Metab Res Rev. 2002;18:S5–9.
22. Ouchi N, Parker JL, Lugus JJ, Walsh K. Adipokines in inflammation and metabolic disease. Nat Rev Immunol. 2011;11:85–97.
23. Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem. 1995;270:26746–9.
24. Gil-campos M, Gil A, Cañetea R. Adiponectin, the missing link in insulin resistance and obesity. Clin Nutr. 2004;23:963–74.
25. Kaser S, Tatarczyk T, Stadlmayr A, Ciardi C, Ress C, Tschoner A, et al. Effect of obesity and insulin sensitivity on adiponectin isoform distribution. Eur J Clin Invest.
2008;38:827–34.
26. Lara-Castro C, Luo N, Wallace P, Klein RL, Garvey WT. Adiponectin Multimeric Complexes and the Metabolic Syndrome Trait Cluster. Diabetes. 2006;55:249–59.
27. Shehzad A, Iqbal W, Shehzad O, Lee YS. Adiponectin: regulation of its production and its role in human diseases. Hormones (Athens). 2012;11:8–20.
28. Tomas E, Tsao T, Saha AK, Murrey HE, Zhang Cc Cc, Itani SI, et al. Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain : Acetyl – CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci U S A. 2002;99:16309–13.
29. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med. 2002;8:1288–95.
30. Mao X, Kikani CK, Riojas RA, Langlais P, Wang L, Ramos FJ, et al. APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function. Nat Cell Biol.
2006;8:516–23.
31. Berg AH, Combs TP, Du X, Brownlee M, Scherer PE. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med. 2001;7:947–53.
32. Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, et al.
Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med.
2002;8:731–7.
contributes to macrophage infiltration into adipose tissue , insulin resistance , and hepatic steatosis in obesity. J Clin Invest. 2006;116:1494-1505.
34. Weisberg SP, Hunter D, Huber R, Lemieux J, Slaymaker S, Vaddi K, et al. CCR2 modulates inflammatory and metabolic effects of high-fat feeding. J Clin Invest.
2006;116:115–24.
35. Sartipy P, Loskutoff DJ. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A. 2003;100:7265–70.
36. Bruun JM, Lihn AS, Pedersen SB, Richelsen B. Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): Implication of macrophages resident in the AT. J Clin Endocrinol Metab. 2005;90:2282–9.
37. Sun K, Kusminski CM, Scherer PE. Adipose tissue remodeling and obesity. J Clin Invest. 2011;121:2094–101.
38. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6:13.
39. Arner E, Westermark PO, Spalding KL, Britton T, Rydén M, Frisén J, et al.
Adipocyte Turnover : Relevance to Human Adipose Tissue. Diabetes. 2010;59:105–9.
40. Hoffstedt J, Arner E, Wahrenberg H, Andersson DP, Qvisth V, Löfgren P, et al.
Regional impact of adipose tissue morphology on the metabolic profile in morbid obesity. Diabetologia. 2010;53:2496–503.
41. Weyer C, Foley JE, Bogardus C, Tataranni PA, Pratley RE. Enlarged subcutaneous abdominal adipocyte size, but not obesity itself, predicts type II diabetes independent of insulin resistance. Diabetologia. 2000;43:1498–506.
42. Gao H, Mejhert N, Fretz J a, Arner E, Lorente-Cebrián S, Ehrlund A, et al. Early B cell factor 1 regulates adipocyte morphology and lipolysis in white adipose tissue. Cell Metab. 2014;19:981–92.
43. Lin H, Grosschedl R. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature. 1995;376:263–7.
44. Liberg D, Sigvardsson M, Åkerblad P, Peter A. The EBF/Olf/Collier family of transcription factors: regulators of differentiation in cells originating from all three embryonal germ layers. Mol Cell Biol. 2002;22:8389–97.
45. Jimenez MA, Akerblad P, Sigvardsson M, Rosen ED. Critical role for Ebf1 and Ebf2 in the adipogenic transcriptional cascade. Mol Cell Biol. 2007;27:743–57.
46. Hesslein DG, Fretz JA, Xi Y, Nelson T, Zhou S, Lorenzo JA, et al. Ebf1-dependent control of the osteoblast and adipocyte lineages. Bone. 2009;44:537–46.
47. Petrus P, Mejhert N, Gao H, Bäckdahl J, Arner E, Arner P, et al. Low early B-cell factor 1 (EBF1) activity in human subcutaneous adipose tissue is linked to a pernicious metabolic profile. Diabetes Metab. 2015;1:2–5.
48. Reggio S, Pellegrinelli V, Clément K, Tordjman J. Fibrosis as a Cause or a Consequence of White Adipose Tissue Inflammation in Obesity. Curr Obes Rep.
2012;2:1–9.
49. Divoux a, Clément K. Architecture and the extracellular matrix: the still