In this study, we found that Aldh2 knock-in mice mimicking the East-Asian-specific
ALDH2 Glu487Lys mutation are prone to develop obesity, fatty liver, insulin resistance
and glucose intolerance. The obesity phenotype is probably caused by reduced energy
expenditure and thermogenesis. Unexpectedly, we observe that the brown adipose tissue
is remarkably smaller in Aldh2 KI mice compared to wild-type mice, either on chow diet
or HFHSD. We further found the fatty acid oxidation capacity is reduced in BAT of Aldh2
KI mice, which may lead to the observed reduced thermogenesis.
Brown adipose tissue generates heat primarily within the mitochondria through
fatty acid oxidation and uncoupling of oxidative phosphorylation mediated by UCP1. The
fatty acid can be activators of the UCP1 for thermogenesis or be suppliers in the electron
transport chain for generating ATP. The oxidation of fatty acid in brown adipocyte
represents a major source of thermogenesis[57]. Suppression of fatty acid oxidation
compromise thermogenesis. For instance, adipose-specific knockout CPT2 mice
presented the hypothermic when they were exposed in a cold environment. And the
thermogenic genes in CPT2 mice brown adipose tissue didn't up-regulate under the
agonist-induced stimulation[45, 58]. When ACSL1, an enzyme catalyzing the formation
of acyl-CoA that are used for β-oxidation, are impaired in adipose tissue. The Acsl1A−/−
mice are also showed cold intolerance due to impaired fatty acid oxidation[44]. It
indicates that mitochondrial fatty acid β-oxidation is critical for the thermogenesis.
In this study, we did not observe altered UCP1 expression in Aldh2 knock-in mice.
However, either primary brown adipocytes or whole brown adipose tissue isolated from Aldh2 KI mice exhibited reduced fatty acid oxidation capacity. We hypothesize it may link to the higher aldehyde production in the BAT mitochondria of Aldh2 KI mice. Indeed, we observed that either 4-HNE-modified proteins or carbonylated proteins are enriched in the BAT isolated from Aldh2 KI mice.
Using LC-MS/MS, we further demonstrated that 4-HNE conjugated to the proteins
involved in mitochondrial electron transfer chain and fatty acid oxidation. And these
proteins demonstrated higher mount in Aldh2 KI mice. The conjugation of 4-HNE to these
proteins may interfere fatty acid oxidation. Consistent with our finding, mitochondrial
dysfunction caused by oxidative stress and the ROS-initiated lipid peroxidation
byproducts has been reported to be involved a range of disease in recent years[59, 60].
And it also indicates about 30% of 4-HNE-modified proteins formed in cell existing in
mitochondria and is considered as the major candidate to cause mitochondrial
dysfunction[61, 62]. Previous studies have demonstrated the 4-HNE modified proteins
plays important role in the pathogenesis of Alzheimer's disease[63, 64], rheumatological
diseases and other autoimmune disease[65], gastrointestinal diseases[66], myocardial
diseases[67], and cancer[68, 69].
Our study supports that 4-HNE or other toxic aldehydes may damper the function of
BAT, leading to obesity and related metabolic phenotypes. In addition to the reduced fatty
acid oxidation, which is probably caused by the adduction of aldehydes to protein
involved in fatty acid oxidation and mitochondrial electron transfer chain, we also observe
that the BAT of Aldh2 KI mice is much smaller in volume than wild-type controls. The
underlying mechanism is currently unknown. It may be related to the impaired embryonic
development of BAT. Therefore, we are currently investigating the embryonic
development of BAT in Aldh2 KI and WT mice.
FIGURE
A
B
C
Figure 2. Phenotype of Aldh2 *2/*2 knock-in mice and wild-type mice
(A) Body weight on HFHSD (n=21:24) and chow diet (n=19:15)
(B) Body composition on HFHSD (n=20:12) and chow diet(n=19:15)
(C) Distribution tissue weight of inguinal fat (subcutaneous), perigonadal fat
(intraabdominal fat), liver, and brown adipose (BAT) of Aldh2 *2/*2 knock-in mice (KI) and wild-type (WT) mice on HFHSD(n=78:67) or chow diet (n=12:10)
(D) Perigonadal fat adipocyte size and number from Aldh2 *2/*2 knock-in mice (KI)
and wild-type (WT) mice fed HFHSD (n=17:24)
D
E F
(E) Hepatic triglyceride content of HFHSD (n=23:20) and chow diet (n=8:10)
(F) Representative H&E stain of liver
* P < 0.05, ** P < 0.01, ***P<0.001. Data presented as mean ± s.e.m.
A
B
C
D
Figure3. Glucose and insulin tolerance test of Aldh2 *2/*2 knock-in mice and
wild-type mice
(A) Intraperitoneal glucose tolerance test on HFHSD(n=44:49) or chow diet(n=19:15)
(B) Insulin sensitivity test on HFHSD(n=44:49) or chow diet(n=19:15)
(C) oral glucose tolerance on HFHSD(n=44:49) or chow diet(n=19:15)
(D) insulin response during OGTT on HFHSD(n=41:47)
* P < 0.05, ** P < 0.01, ***P<0.001. Data presented as mean ± s.e.m.
ipGTT: Intraperitoneal glucose tolerance ;OGTT: oral glucose tolerance; ITT: insulin
tolerance test
C D
A B
Figure4. Aldh2 *2/*2 knock-in mice decrease energy expenditure in response to
HFHSD
(A) Relative oxygen consumption (VO2) and energy expenditure over a 24-hr period
(n=12:12)
(B) Metabolic parameters of food intake and wheel meters (n=12:12)
(C) The temperature of cold-induced thermogenesis (n=7:8)
(D) The temperature of diet-induced thermogenesis (n=8:8)
* P < 0.05, ** P < 0.01, ***P<0.001. Data presented as mean ± s.e.m.
C D
E
B
A
Figure 5. The effect of impaired ALDH2 activity inhibits fatty acid oxidation in
brown adipose tissue
(A) Quantitative real-time polymerase chain reaction(qPCR) analysis of mRNA for fatty
acid oxidation and thermogenic genes in BAT (n=22:20)
(B) Western blot of UCP1 in BAT of Aldh2 *2/*2 knock-in mice and wild-type mice fed
HFHSD (n=4:4)
(C) Fatty acid oxidation was measured in primary brown adipocytes which treated
different 4HNE concentration
(D) Fatty acid oxidation was measured in primary brown adipocytes from Aldh2 *2/*2
knock-in and wild-type mice (n=15:15)
(E) Fatty acid oxidation was measured with BAT from Aldh2 *2/*2 knock-in and
wild-type mice fed 5week HFHSD. The body weight and BAT mass were measured(n=14:10)
* P < 0.05, ** P < 0.01, ***P<0.001. Data presented as mean ± s.e.m.
A
B
C
Figure 6. LC-MS/MS Analysis the BAT mitochondrial modified protein by 4HNE
(A) Detection of carbonylated proteins and 4-HNE modified proteins in BAT from Aldh2
*2/*2 knock-in mice and wild-type mice fed HFHSD (n=4:4)
(B) Experimental design and workflows used in the study
(C) Comparison of identified proteins from Aldh2 *2/*2 knock-in mice and wild-type
mice at the age of 30 weeks
(D) Venn diagram of identified proteins from Aldh2 *2/*2 knock-in mice and wild-type
mice
TABLE
Table1. 4HNE modified protein list of 30-week-old mice fed HFHSD
ene nameUniprot IDprotein namemodified sitebiological function a2Q8BWT13-ketoacyl-CoA thiolase, mitochondrial375Kfatty acid beta-oxidation 5f1aQ03265ATP synthase subunit alpha, mitochondrial175KATP biosynthetic process tQ8CAQ8MICOS complex subunit Mic60296Kmaintenance of mitochondrial architecture tE9Q800MICOS complex subunit MIC60(isoform)218Kmaintenance of mitochondrial architecture Q9CQB4Cytochrome b-c1 complex subunit 739Hmitochondrial electron transport s1Q9CR68Cytochrome b-c1 complex subunit Rieske, mitochondrial101Kmitochondrial electron transport a8P63017Heat shock cognate 71 kDa protein187KMAPK signaling pathway 1A0A0A6YVZ0Cytochrome b-c1 complex subunit 1, mitochondrial3Kmitochondrial electron transport 5a1D3Z6F5ATP synthase subunit alpha125KATP synthesis coupled proton transport d3D3Z0L4MICOS complex subunit24Kmaintenance of mitochondrial architecture ene nameUniprot IDprotein namemodified sitebiological function 5f1aQ03265ATP synthase subunit alpha, mitochondrial175Kmitochondrial electron transport tQ8CAQ8MICOS complex subunit Mic60296Kmaintenance of mitochondrial architecture tE9Q800MICOS complex subunit MIC60(isoform)218Kmaintenance of mitochondrial architecture Q9CQB4Cytochrome b-c1 complex subunit 739Hmitochondrial electron transport s1Q9CR68Cytochrome b-c1 complex subunit Rieske, mitochondrial101Kmitochondrial electron transport a8P63017Heat shock cognate 71 kDa protein187KMAPK signaling pathway 1A0A0A6YVZ0Cytochrome b-c1 complex subunit 1, mitochondrial3Kmitochondrial electron transport a2Q8BWT13-ketoacyl-CoA thiolase, mitochondrial25Kfatty acid beta-oxidation 375Kfatty acid beta-oxidation 2Q99KI0Aconitate hydratase, mitochondrial144KTricarboxylic acid cycle d2A2AQR0Glycerol-3-phosphate dehydrogenase668Kglycerol-3-phosphate metabolic process 652Kglycerol-3-phosphate metabolic process bQ9CQA3Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial246Hmitochondrial electron transport chain ,tricarboxylic acid cycle fc2Q9CQ54NADH dehydrogenase [ubiquinone] 1 subunit C28Hmitochondrial electron transport G5E8R3Pyruvate carboxylase574Hpyruvate metabolic process Q91ZA3Propionyl-CoA carboxylase alpha chain, mitochondrial275Kpropanoyl-CoA degradation fb4Q9CQC7NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 459Hmitochondrial electron transport a1P35486Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial121HTricarboxylic acid cycle 4Q5SX39Myosin-41525KMuscle contraction a8Q504P4Heat shock cognate 71 kDa protein168KATP binding
WT KI
REFERENCE
1. Yin, S.J., Alcohol dehydrogenase: enzymology and metabolism. Alcohol Alcohol Suppl, 1994. 2: p. 113-9.
2. Eriksson, C.J., M. Marselos, and T. Koivula, Role of cytosolic rat liver aldehyde dehydrogenase in the oxidation of acetaldehyde during ethanol metabolism in vivo. Biochem J, 1975. 152(3): p. 709-12.
3. Klyosov, A.A., et al., Possible role of liver cytosolic and mitochondrial aldehyde dehydrogenases in acetaldehyde metabolism. Biochemistry, 1996. 35(14): p.
4445-56.
4. Yoval-Sanchez, B. and J.S. Rodriguez-Zavala, Differences in susceptibility to inactivation of human aldehyde dehydrogenases by lipid peroxidation byproducts.
Chem Res Toxicol, 2012. 25(3): p. 722-9.
5. Chen, C.H., et al., Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev, 2014. 94(1): p. 1-34.
6. Enomoto, N., et al., Acetaldehyde metabolism in different aldehyde dehydrogenase-2 genotypes. Alcohol Clin Exp Res, 1991. 15(1): p. 141-4.
7. Edenberg, H.J., The genetics of alcohol metabolism: role of alcohol dehydrogenase and aldehyde dehydrogenase variants. Alcohol Res Health, 2007.
30(1): p. 5-13.
8. Larson, H.N., H. Weiner, and T.D. Hurley, Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant. The Journal of biological chemistry, 2005.
280(34): p. 30550-30556.
9. Perez-Miller, S., et al., Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant. Nature Structural &Amp;
Molecular Biology, 2010. 17: p. 159.
10. Eriksson, C.J., The role of acetaldehyde in the actions of alcohol (update 2000).
Alcohol Clin Exp Res, 2001. 25(5 Suppl ISBRA): p. 15S-32S.
11. Brooks, P.J., et al., The alcohol flushing response: an unrecognized risk factor for esophageal cancer from alcohol consumption. PLoS Med, 2009. 6(3): p. e50.
12. Li, H., et al., Refined geographic distribution of the oriental ALDH2*504Lys (nee 487Lys) variant. Ann Hum Genet, 2009. 73(Pt 3): p. 335-45.
13. Vasiliou, V., A. Pappa, and T. Estey, Role of Human Aldehyde Dehydrogenases in Endobiotic and Xenobiotic Metabolism. Drug Metabolism Reviews, 2004. 36(2):
p. 279-299.
14. Salaspuro, M., Acetaldehyde as a common denominator and cumulative carcinogen in digestive tract cancers. Scandinavian Journal of Gastroenterology, 2009. 44(8): p. 912-925.
15. O'Brien, P.J., A.G. Siraki, and N. Shangari, Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit Rev Toxicol, 2005. 35(7): p. 609-62.
16. LoPachin, R.M. and T. Gavin, Molecular mechanisms of aldehyde toxicity: a chemical perspective. Chemical research in toxicology, 2014. 27(7): p. 1081-1091.
17. Nelson, M.-A.M., S.P. Baba, and E.J. Anderson, Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease. Current opinion in pharmacology, 2017. 33:
p. 56-63.
18. Esterbauer, H., R.J. Schaur, and H. Zollner, Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med, 1991. 11(1): p. 81-128.
19. Zarkovic, K., 4-hydroxynonenal and neurodegenerative diseases. Mol Aspects Med, 2003. 24(4-5): p. 293-303.
20. Zarkovic, N., 4-hydroxynonenal as a bioactive marker of pathophysiological processes. Mol Aspects Med, 2003. 24(4-5): p. 281-91.
21. Frohnert, B.I. and D.A. Bernlohr, Protein carbonylation, mitochondrial dysfunction, and insulin resistance. Adv Nutr, 2013. 4(2): p. 157-63.
22. Marnett, L.J., J.N. Riggins, and J.D. West, Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J Clin Invest, 2003. 111(5): p. 583-93.
23. Calingasan, N.Y., K. Uchida, and G.E. Gibson, Protein-bound acrolein: a novel marker of oxidative stress in Alzheimer's disease. J Neurochem, 1999. 72(2): p.
751-6.
24. LoPachin, R.M., D.S. Barber, and T. Gavin, Molecular mechanisms of the conjugated alpha,beta-unsaturated carbonyl derivatives: relevance to neurotoxicity and neurodegenerative diseases. Toxicol Sci, 2008. 104(2): p. 235-49.
25. Choi, K. and Y.B. Kim, Molecular mechanism of insulin resistance in obesity and type 2 diabetes. Korean J Intern Med, 2010. 25(2): p. 119-29.
26. Furukawa, S., et al., Increased oxidative stress in obesity and its impact on metabolic syndrome. The Journal of clinical investigation, 2004. 114(12): p. 1752-1761.
27. Li, C., et al., Aldehyde Dehydrogenase-2 Attenuates Myocardial Remodeling and Contractile Dysfunction Induced by a High-Fat Diet. Cellular Physiology and Biochemistry, 2018. 48(5): p. 1843-1853.
28. Pillon, N.J. and C.O. Soulage, Lipid peroxidation by-products and the metabolic syndrome, in Lipid Peroxidation. 2012, IntechOpen.
29. Uchida, K., et al., Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. J Biol Chem, 1999. 274(4): p. 2234-42.
30. Pillon, N.J., et al., The lipid peroxidation by-product 4-hydroxy-2-nonenal (4-HNE) induces insulin resistance in skeletal muscle through both carbonyl and oxidative stress. Endocrinology, 2012. 153(5): p. 2099-111.
31. Schauenstein, E., Autoxidation of polyunsaturated esters in water: chemical structure and biological activity of the products. J Lipid Res, 1967. 8(5): p. 417-28.
32. Singh, S.P., et al., Role of the electrophilic lipid peroxidation product 4-hydroxynonenal in the development and maintenance of obesity in mice.
Biochemistry, 2008. 47(12): p. 3900-11.
33. Frohnert, B.I., et al., Increased adipose protein carbonylation in human obesity.
2011. 19(9): p. 1735-1741.
34. Miwa, I., et al., Inhibition of glucose-induced insulin secretion by 4-hydroxy-2-nonenal and other lipid peroxidation products. Endocrinology, 2000. 141(8): p.
2767-72.
35. Ihara, Y., et al., Pancreatic (B-Cells of GK Rats, a Model of Type 2 Diabetes. 1999.
48.
36. Shearn, C.T., et al., Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells. Biochemistry, 2011. 50(19): p. 3984-96.
37. Demozay, D., et al., FALDH Reverses the Deleterious Action of Oxidative Stress Induced by Lipid Peroxidation Product 4-Hydroxynonenal on Insulin Signaling in 3T3-L1 Adipocytes. 2008. 57(5): p. 1216-1226.
38. Hill, J.O., H.R. Wyatt, and J.C. Peters, Energy balance and obesity. Circulation, 2012. 126(1): p. 126-132.
39. Galgani, J. and E. Ravussin, Energy metabolism, fuel selection and body weight regulation. International journal of obesity (2005), 2008. 32 Suppl 7(Suppl 7): p.
S109-S119.
40. Cannon, B. and J. Nedergaard, Brown adipose tissue: function and physiological significance. Physiol Rev, 2004. 84(1): p. 277-359.
41. Serra, D., et al., Mitochondrial fatty acid oxidation in obesity. Antioxidants &
redox signaling, 2013. 19(3): p. 269-284.
42. Calderon-Dominguez, M., et al., Fatty acid metabolism and the basis of brown adipose tissue function. Adipocyte, 2015. 5(2): p. 98-118.
43. Feldmann, H.M., et al., UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality.
Cell Metab, 2009. 9(2): p. 203-9.
44. Ellis, J.M., et al., Adipose acyl-CoA synthetase-1 directs fatty acids toward beta-oxidation and is required for cold thermogenesis. Cell metabolism, 2010. 12(1): p.
53-64.
45. Lee, J., J.M. Ellis, and M.J. Wolfgang, Adipose fatty acid oxidation is required for thermogenesis and potentiates oxidative stress-induced inflammation. Cell reports, 2015. 10(2): p. 266-279.
46. Cypess, A.M., et al., Identification and importance of brown adipose tissue in adult humans. The New England journal of medicine, 2009. 360(15): p. 1509-1517.
47. Luu, S.U., et al., Ethanol and acetaldehyde metabolism in chinese with different aldehyde dehydrogenase-2 genotypes. Proc Natl Sci Counc Repub China B, 1995.
19(3): p. 129-36.
48. Wen, W., et al., Meta-analysis of genome-wide association studies in East Asian-ancestry populations identifies four new loci for body mass index. Human molecular genetics, 2014. 23(20): p. 5492-5504.
49. Chang, Y.C., et al., Common ALDH2 genetic variants predict development of hypertension in the SAPPHIRe prospective cohort: gene-environmental interaction with alcohol consumption. BMC Cardiovasc Disord, 2012. 12: p. 58.
50. Fedorenko, A., P.V. Lishko, and Y. Kirichok, Mechanism of fatty-acid-dependent
UCP1 uncoupling in brown fat mitochondria. Cell, 2012. 151(2): p. 400-413.
51. Tolwani, R.J., et al., Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice. PLoS genetics, 2005. 1(2): p. e23-e23.
52. Chondronikola, M., et al., Brown Adipose Tissue Activation Is Linked to Distinct Systemic Effects on Lipid Metabolism in Humans. Cell Metab, 2016. 23(6): p. adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner. Free radical biology & medicine, 2013. 63: p. 390-398.
55. Castro, J.P., et al., 4-Hydroxynonenal (HNE) modified proteins in metabolic diseases. Free Radic Biol Med, 2017. 111: p. 309-315.
56. Ruskovska, T. and D.A. Bernlohr, Oxidative stress and protein carbonylation in adipose tissue - implications for insulin resistance and diabetes mellitus. Journal of proteomics, 2013. 92: p. 323-334.
57. Tseng, Y.-H., A.M. Cypess, and C.R. Kahn, Cellular bioenergetics as a target for obesity therapy. Nature Reviews Drug Discovery, 2010. 9: p. 465.
58. Gonzalez-Hurtado, E., et al., Fatty acid oxidation is required for active and quiescent brown adipose tissue maintenance and thermogenic programing.
Molecular metabolism, 2018. 7: p. 45-56.
59. Bhat, A.H., et al., Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight. Biomed Pharmacother, 2015.
74: p. 101-10.
60. Bhatti, J.S., G.K. Bhatti, and P.H. Reddy, Mitochondrial dysfunction and oxidative stress in metabolic disorders - A step towards mitochondria based therapeutic strategies. Biochim Biophys Acta Mol Basis Dis, 2017. 1863(5): p. 1066-1077.
61. Zhao, Y., et al., Redox proteomic identification of HNE-bound mitochondrial proteins in cardiac tissues reveals a systemic effect on energy metabolism after doxorubicin treatment. Free Radic Biol Med, 2014. 72: p. 55-65.
62. Poli, G., et al., 4-hydroxynonenal: a membrane lipid oxidation product of medicinal interest. Med Res Rev, 2008. 28(4): p. 569-631.
63. Shringarpure, R., et al., 4-Hydroxynonenal-modified amyloid-β peptide inhibits the proteasome: possible importance in Alzheimer’s disease. 2000. 57(12): p.
1802-1809.
64. Castegna, A., et al., Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J Neurochem, 2002. 82(6): p. 1524-32.
65. Grune, T., et al., Increased levels of 4-hydroxynonenal modified proteins in plasma of children with autoimmune diseases. 1997. 23(3): p. 357-360.
66. Naito, Y., et al., Lipid hydroperoxide-derived modification of proteins in gastrointestinal tract. Subcell Biochem, 2014. 77: p. 137-48.
67. Nakamura, K., et al., Carvedilol decreases elevated oxidative stress in human failing myocardium. 2002. 105(24): p. 2867-2871.
68. Biasi, F., et al., Associated changes of lipid peroxidation and transforming growth factor β1 levels in human colon cancer during tumour progression. 2002. 50(3):
p. 361-367.
69. Marquez-Quiñones, A., et al., HNE-protein adducts formation in different pre-carcinogenic stages of hepatitis in LEC rats. 2010. 44(2): p. 119-127.