Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes
© Jufvas et al.; licensee BioMed Central Ltd. 2013
Received: 1 May 2013
Accepted: 12 July 2013
Published: 3 September 2013
Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies.
The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation compared with cells from normal-weight subjects. In contrast, trimethylation at lysine 4 was 40% higher in adipocytes from overweight diabetic subjects compared with normal-weight and overweight non-diabetic subjects. There was no association between level of modification and age of subjects.
The findings define genome-wide molecular modifications of histones in adipocytes that are directly associated with overweight and diabetes, and thus suggest a molecular basis for existing epidemiological evidence of epigenetic inheritance.
KeywordsEpigenetic Histone methylation Human Obesity Overweight Primary mature adipocytes Type 2 diabetes
The dynamics of chromatin regulate access to DNA and are therefore under tight control by the host cell and by external stimuli. Reversible covalent post-transcriptional modifications to histones are considered to form one of the major means by which gene transcription and DNA replication are controlled. Histone modifications have been associated with transcriptional control since the discovery of histone acetylation; hyperacetylated histones are linked to actively transcribed genes[2, 3].
Methylation of histone H3 at lysine 4 is associated with sites of active gene transcription[4, 5]. High levels of dimethylation and trimethylation (H3K4me2 and H3K4me3) are generally found near promoter regions of DNA. Trimethylation, particularly, is found at transcription start sites, while dimethylation flanks these sites of active genes[6, 7]. Enhancers appear to host higher levels of monomethylated lysine 4. Dimethylation of lysine 9 (H3K9me2), on the other hand, is a modification found in heterochromatin throughout silenced genes but is also found in actively transcribed genes. Methylation of histones is a reversible and dynamic process that is catalyzed by specific and general histone methyltransferases and demethylases, which are, in turn, dependent on metabolic coenzymes and thus responsive to changes in energy supply and metabolic status.
Obesity and type 2 diabetes (T2D) are characterized by strong hereditary components in addition to such lifestyle-related factors as overeating and physical inactivity; however, no simple relation to gene variants has been discovered. Conversely, genome-wide association studies have uncovered a number of genes that are associated with increased risks of developing the conditions, but the identified genes are each associated with a very low risk and are widely distributed in the population as a whole[10–13].
It is clear that lifestyle and environmental exposure can cause long-lasting susceptibility or resistance to disease, even in later generations, suggesting non-genetic memory and inheritance. Epidemiological data and clinical and experimental studies indicate that nutritional conditions during early life can strongly influence later susceptibility to T2D. Epigenetic mechanisms have been used to explain the discovery that the famine experienced by pregnant mothers in the Netherlands in World War II affected the birth weights of their children, and their children’s later development of obesity and impaired glucose tolerance[14–16]. In addition, it was found that different starvation or surfeit experiences by parents and grandparents in Överkalix in northern Sweden during the late nineteenth and early twentieth centuries was associated with different susceptibilities to death from cardiovascular disease or T2D in their offspring. Recently, a study of the whole population of Austria found a massively increased risk of diabetes in people born during or immediately after one of three different famines of the twentieth century. In experimental animal studies, the importance of the intrauterine environment has been demonstrated[19–21], as well as a paternal non-genetic transgenerational inheritance of propensity for obesity and diabetes[22, 23]. It has been suggested that methylation of DNA, modifications of histones, and noncoding RNA mediate epigenetic inheritance. Methylation of DNA and histone modifications have been shown to be affected by, for example, body mass index (BMI), age, intrauterine environment[26–28], glucose exposure[29, 30], and exercise.
In this study, we investigated whether there is a relation between overweight or obesity, T2D and genome-wide methylation of histone H3 at lysine 4 and at lysine 9 in isolated mature adipocytes.
We analyzed the global extent of H3K4me2, H3K4me3, and H3K9me2 in isolated primary mature adipocytes from subjects who were of normal weight, overweight, or overweight with type 2 diabetes. The extent of methylation was determined by SDS-PAGE and immunoblotting using site- and modification-specific antibodies. The extent of specific methylation was normalized for the total amount of histone H3 in each sample, and all values are the median value of three separate experiments. Hence, the extent of histone H3 methylation is determined as the fractional methylation of histone H3.
Our findings reveal large genome-wide differences in the level of specific histone methylation in adipocytes from subjects with overweight or T2D compared with normal-weight and non-diabetic subjects. These differences were not related to the age of the subjects donating the adipocytes. The effects were restricted to H3K4 methylation, which is associated with actively transcribed genes, with no corresponding effects in the heterochromatin-defining H3K9 methylation. It is particularly interesting that overweight and T2D are associated with changes involving nearly half of the dimethylation and trimethylation levels at H3K4 in the adipocytes. This indicates that a large number of genes might be affected by the changed levels of modifications. The underlying cause of these differences probably originates from differences in activities of one or more of the involved methylases or demethylases, or their control. Histone methylation is a reversible process and we cannot exclude changes during surgical procedures and isolation or incubation of the cells, but our findings nevertheless demonstrate large genome-wide changes in overweight and T2D that are directly related to these specific histone modifications. Since most genetic variants associated with T2D appear to be linked to β-cell function and insulin release[10, 11] our findings indicate a potential importance of the adipose tissue in hereditability of T2D. An epigenetic link to overweight and T2D is in line with the epidemiological studies discussed previously[14, 15, 17, 18, 26].
H3K4me2 is demethylated by LSD1, a FAD-dependent demethylase[33–35]. Interestingly, it has been shown that LSD1 has an increased expression in adipocytes from high-fat diet-fed mice and that adipose energy-expenditure genes are direct targets of repression by LSD1. Inhibition of LSD1 increases global H3K4 methylation in P19 embryonal carcinoma cells and lowers the body weight of mice fed a high-fat diet. Histone methyltransferase MLL3 catalyzes methylation of H3K4. Mice with mutations in the catalytic SET-domain of MLL3 show altered gene expression of a number of metabolic genes in adipose tissue, such as Rbp4, which is associated with insulin resistance in human beings[39, 40]. The mutant mice also exhibit an altered phenotype, with less adipose tissue and improved insulin sensitivity compared with control mice. Collectively, these reports demonstrate that modifying the global levels of H3K4 methylation experimentally affects adiposity and sensitivity to insulin. This is further supported by experiments showing that the levels of H3K4me3 in PPARγ promoters correlate with expression levels of PPARγ during adipogenesis. Interestingly, H3K9me2 was selectively enriched in the entire PPARγ locus in 3T3-L1 preadipocytes, and the level of H3K9me2 correlated inversely with induction of PPARγ in both murine and human adipogenesis. However, globally we found no correspondence between levels of H3K9 and H3K4 methylation in the mature adipocytes of normal-weight, overweight, or diabetic individuals.
It may be that histone modifications do not determine sites of active transcription, but rather reinforce the effects of nucleosome binding during transcription, for example, in response to the targeting actions of noncoding RNAs. As such, our findings are indicators of large genome-wide changes in transcriptional activities associated with overweight and diabetes, which may be involved in an epigenetically affected propensity for these common disorders. In the future, it will be interesting to analyze to what extent particular sets of genes are affected in different individuals, who may be of normal weight, overweight, or diabetic.
Our findings define extensive genome-wide molecular modifications of histones in adipocytes that are directly associated with overweight and diabetes. Effects were restricted to H3K4 methylation, which is associated with actively transcribed genes, with no corresponding effects in the heterochromatin-defining H3K9 methylation. Changes involved 30% to 40% of the dimethylation and trimethylation levels at H3K4 in the adipocytes, indicating that a large number of genes might be affected by the changed levels of modifications. The findings suggest a molecular basis for existing epidemiological evidence of epigenetic inheritance.
Characteristics of participating subjects
(BMI < 25 kg/m2)
(BMI > 25 kg/m2)
64.4 ± 8.7
60.2 ± 11.4
55.2 ± 15.2
22.4 ± 1.5
34.5 ± 8.3
41.4 ± 10.8
Fasting glucose (mmol/l)
5.8 ± 1.0
6.2 ± 8.9
8.0 ± 0.5
Fasting insulin (pmol/l)
73.0 ± 64.0
54.5 ± 34.4
112.0 ± 114.2
Isolation and incubation of adipocytes
Adipocytes were isolated from adipose tissue samples by collagenase digestion (type 1, Worthington, NJ, USA) in modified Krebs-Ringer solution. Following overnight incubation, cells were washed with the modified Krebs-Ringer solution and incubated with 0.1 μM N6-phenylisopropyl adenosine and 2.5 μg/ml adenosine deaminase for 10 min, to control the intracellular concentration of cyclic AMP and establish a standardized level of basal lipolysis. Cells were separated from the medium by centrifugation through dinonyl phthalate and were then immediately dissolved in SDS and β-mercaptoethanol with protease and phosphatase inhibitors, frozen within 10 seconds and thawed in boiling water for further analysis.
SDS-PAGE and immunoblotting
Proteins were separated by SDS-PAGE (14.5% acrylamide) and transferred to a polyvinylidene difluoride blotting membrane (Immobilon-P, Millipore, MA, USA). The extent of H3K4 and H3K9 methylation was analyzed with antibodies against H3K4me2, H3K4me3, or H3K9me2 (Active Motif, Carlsbad, CA, USA). These antibodies are specific for dimethylation or trimethylation, such that the H3K4me2-specific antibodies do not cross-react with H3K4me3. Membranes were stripped of bound antibodies (2% SDS, 62.5 mM Tris, 100 mM β-mercaptoethanol, 60°C, 30 min) and the amount of histone H3 was determined in each sample with antibodies against histone H3 C-terminus (Active Motif), to calculate the ratio of histone H3 methylation to the amount of histone H3. To allow comparison between different gels, a standard sample (a mixture of aliquots from 23 subjects) was run in duplicate on every gel and all samples were normalized against the mean of the standard sample. Antibodies were detected using horseradish peroxidase conjugated IgG secondary antibody (Santa Cruz Biotechnical, Santa Cruz, CA, USA) and ECL-plus (Amersham Biosciences, Little Chalfont, Bucks, UK) using chemiluminescence imaging (LAS 1000; Image Gauge v.3.0, Fuji, Tokyo, Japan). Linearity of the antibodies’ responses was ascertained (Additional file1: Figure S1) and the amounts of each sample subjected to SDS-PAGE were adjusted to fall within this linear range. For the calculations, the median of three separate immunoblottings was used for each of the 43 subjects. Groups were compared with two-tailed Student’s t test, using GraphPad Prism v.5.00 (GraphPad software Inc., San Diego, CA, USA).
Body mass index
Histone H3 dimethylated at lysine 4
Histone H3 trimethylated at lysine 4
Histone H3 dimethylated at lysine 9
Standard error of the mean
Type 2 diabetes
This work was supported by Swedish Research Council grants to PS and AVV, and by Swedish Diabetes Fund and Novo Nordic Fund grants to PS.
- Zentner GE, Henikoff S: Regulation of nucleosome dynamics by histone modifications. Nat Struct Mol Biol. 2013, 20: 259-266. 10.1038/nsmb.2470.View ArticlePubMedGoogle Scholar
- Phillips DMP: The presence of acetyl groups in histones. Biochem J. 1961, 87: 258-263.View ArticleGoogle Scholar
- Pogo BG, Allfrey VG, Mirsky AE: RNA synthesis and histone acetylation during the course of gene activation in lymphocytes. Proc Natl Acad Sci USA. 1966, 55: 805-812. 10.1073/pnas.55.4.805.PubMed CentralView ArticlePubMedGoogle Scholar
- Orford K, Kharchenko P, Lai W, Dao MC, Worhunsky DJ, Ferro A, Janzen V, Park PJ, Scadden DT: Differential H3K4 methylation identifies developmentally poised hematopoietic genes. Devel Cell. 2008, 14: 798-809. 10.1016/j.devcel.2008.04.002.View ArticleGoogle Scholar
- Kouzarides T: Chromatin modifications and their function. Cell. 2007, 128: 693-705. 10.1016/j.cell.2007.02.005.View ArticlePubMedGoogle Scholar
- Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K: High-resolution profiling of histone methylations in the human genome. Cell. 2007, 129: 823-837. 10.1016/j.cell.2007.05.009.View ArticlePubMedGoogle Scholar
- Wang Z, Schones DE, Zhao K: Characterization of human epigenomes. Curr Opin Genet Dev. 2009, 19: 127-134. 10.1016/j.gde.2009.02.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Y, Reinberg D: Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Gen Devel. 2001, 15: 2343-2360. 10.1101/gad.927301.View ArticleGoogle Scholar
- Teperino R, Schoonjans K, Auwerx J: Histone methyl transferases and demethylases; can they link metabolism and transcription?. Cell Metab. 2010, 12: 321-327. 10.1016/j.cmet.2010.09.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Doria A, Patti ME, Kahn CR: The emerging genetic architecture of type 2 diabetes. Cell Metab. 2008, 8: 186-200. 10.1016/j.cmet.2008.08.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Billings LK, Florez JC: The genetics of type 2 diabetes: what have we learned from GWAS?. Ann New York Acad Sci. 2010, 1212: 59-77. 10.1111/j.1749-6632.2010.05838.x.View ArticleGoogle Scholar
- Drong AW, Lindgren CM, McCarthy MI: The genetic and epigenetic basis of type 2 diabetes and obesity. Clin Pharmacol Ther. 2012, 92: 707-715. 10.1038/clpt.2012.149.View ArticlePubMedGoogle Scholar
- Sandholt CH, Hansen T, Pedersen O: Beyond the fourth wave of genome-wide obesity association studies. Nutr Diabetes. 2012, 2: e37-10.1038/nutd.2012.9.PubMed CentralView ArticlePubMedGoogle Scholar
- Ravelli AC, van der Meulen JH, Michels RP, Osmond C, Barker DJ, Hales CN, Bleker OP: Glucose tolerance in adults after prenatal exposure to famine. Lancet. 1998, 351: 173-177. 10.1016/S0140-6736(97)07244-9.View ArticlePubMedGoogle Scholar
- Ravelli AC, van Der Meulen JH, Osmond C, Barker DJ, Bleker OP: Obesity at the age of 50 y in men and women exposed to famine prenatally. Am J Clin Nutr. 1999, 70: 811-816.PubMedGoogle Scholar
- Heijmans BT, Tobi EW, Stein AD, Putter H, Blauw GJ, Susser ES, Slagboom PE, Lumey LH: Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci USA. 2008, 105: 17046-17049. 10.1073/pnas.0806560105.PubMed CentralView ArticlePubMedGoogle Scholar
- Kaati G, Bygren LO, Edvinsson S: Cardiovascular and diabetes mortality determined by nutrition during parents’ and grandparents’ slow growth period. Eur J Hum Gen. 2002, 10: 682-688. 10.1038/sj.ejhg.5200859.View ArticleGoogle Scholar
- Thurner S, Klimek P, Szell M, Duftschmid G, Endel G, Kautzky-Willer A, Kasper DC: Quantification of excess risk for diabetes for those born in times of hunger, in an entire population of a nation, across a century. Proc Natl Acad Sci USA. 2013, 110: 4703-4707. 10.1073/pnas.1215626110.PubMed CentralView ArticlePubMedGoogle Scholar
- Jousse C, Parry L, Lambert-Langlais S, Maurin AC, Averous J, Bruhat A, Carraro V, Tost J, Letteron P, Chen P, Jockers R, Launay JM, Mallet J, Fafournoux P: Perinatal undernutrition affects the methylation and expression of the leptin gene in adults: implication for the understanding of metabolic syndrome. FASEB J. 2011, 25: 3271-3278. 10.1096/fj.11-181792.View ArticlePubMedGoogle Scholar
- Raychaudhuri N, Raychaudhuri S, Thamotharan M, Devaskar SU: Histone code modifications repress glucose transporter 4 expression in the intrauterine growth-restricted offspring. J Biol Chem. 2008, 283: 13611-13626. 10.1074/jbc.M800128200.PubMed CentralView ArticlePubMedGoogle Scholar
- Seki Y, Williams L, Vuguin PM, Charron MJ: Minireview. Epigenetic programming of diabetes and obesity: animal models. Endocrinology. 2012, 153: 1031-1038.PubMedGoogle Scholar
- Ng S-F, Lin RCY, Laybutt DR, Barres R, Owens JA, Morris MJ: Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring. Nature. 2010, 467: 963-966. 10.1038/nature09491.View ArticlePubMedGoogle Scholar
- Carone BR, Fauquier L, Habib N, Shea JM, Hart CE, Li R, Bock C, Li C, Gu H, Zamore PD, Meissner A, Weng Z, Hofmann HA, Friedman N, Rando OJ: Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell. 2010, 143: 1084-1096. 10.1016/j.cell.2010.12.008.PubMed CentralView ArticlePubMedGoogle Scholar
- Feinberg AP, Irizarry RA, Fradin D, Aryee MJ, Murakami P, Aspelund T, Eiriksdottir G, Harris TB, Launer L, Gudnason V, Fallin MD: Personalized epigenomic signatures that are stable over time and covary with body mass index. Sci Transl Med. 2010, 2: 49ra-67ra.View ArticleGoogle Scholar
- Han S, Brunet A: Histone methylation makes its mark on longevity. Trends Cell Biol. 2012, 22: 42-49. 10.1016/j.tcb.2011.11.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Dabelea D, Hanson RL, Lindsay RS, Pettitt DJ, Imperatore G, Gabir MM, Roumain J, Bennett PH, Knowler WC: Intrauterine exposure to diabetes conveys risks for type 2 diabetes and obesity: a study of discordant sibships. Diabetes. 2000, 49: 2208-2211. 10.2337/diabetes.49.12.2208.View ArticlePubMedGoogle Scholar
- Oge A, Isganaitis E, Jimenez-Chillaron J, Reamer C, Faucette R, Barry K, Przybyla R, Patti ME: In utero undernutrition reduces diabetes incidence in non-obese diabetic mice. Diabetologia. 2007, 50: 1099-1108. 10.1007/s00125-007-0617-0.View ArticlePubMedGoogle Scholar
- Park JH, Stoffers DA, Nicholls RD, Simmons RA: Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1. J Clin Invest. 2008, 118: 2316-2324.PubMed CentralView ArticlePubMedGoogle Scholar
- Brasacchio D, Okabe J, Tikellis C, Balcerczyk A, George P, Baker EK, Calkin AC, Brownlee M, Cooper ME, El-Osta A: Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with gene-activating epigenetic marks that co-exist on the lysine tail. Diabetes. 2009, 58: 1229-1236. 10.2337/db08-1666.PubMed CentralView ArticlePubMedGoogle Scholar
- El-Osta A, Brasacchio D, Yao D, Pocai A, Jones PL, Roeder RG, Cooper ME, Brownlee M: Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. J Exp Med. 2008, 205: 2409-2417. 10.1084/jem.20081188.PubMed CentralView ArticlePubMedGoogle Scholar
- Barrès R, Yan J, Egan B: Treebak Jonas T, Rasmussen M, Fritz T, Caidahl K, Krook A, O’Gorman Donal J, Zierath Juleen R: Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab. 2012, 15: 405-411. 10.1016/j.cmet.2012.01.001.View ArticlePubMedGoogle Scholar
- Feinberg AP: Epigenetics at the epicenter of modern medicine. J Am Med Assoc. 2008, 299: 1345-1350. 10.1001/jama.299.11.1345.View ArticleGoogle Scholar
- Fang R, Barbera AJ, Xu Y, Rutenberg M, Leonor T, Bi Q, Lan F, Mei P, Yuan GC, Lian C, Peng J, Cheng D, Sui G, Kaiser UB, Shi Y, Shi YG: Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation. Mol Cell. 2010, 39: 222-233. 10.1016/j.molcel.2010.07.008.PubMed CentralView ArticlePubMedGoogle Scholar
- Hino S, Sakamoto A, Nagaoka K, Anan K, Wang Y, Mimasu S, Umehara T, Yokoyama S, Kosai K, Nakao M: FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure. Nat Commun. 2012, 3: 758-PubMed CentralView ArticlePubMedGoogle Scholar
- Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y: Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell. 2004, 119: 941-953. 10.1016/j.cell.2004.12.012.View ArticlePubMedGoogle Scholar
- Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R: Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol. 2006, 13: 563-567. 10.1016/j.chembiol.2006.05.004.View ArticlePubMedGoogle Scholar
- Shilatifard A: Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. Curr Opin Cell Biol. 2008, 20: 341-348. 10.1016/j.ceb.2008.03.019.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee J, Saha PK, Yang QH, Lee S, Park JY, Suh Y, Lee SK, Chan L, Roeder RG, Lee JW: Targeted inactivation of MLL3 histone H3-Lys-4 methyltransferase activity in the mouse reveals vital roles for MLL3 in adipogenesis. Proc Natl Acad Sci USA. 2008, 105: 19229-19234. 10.1073/pnas.0810100105.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang Q, Graham TE, Mody N, Preitner F, Peroni OD, Zabolotny JM, Kotani K, Quadro L, Kahn BB: Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature. 2005, 436: 356-362. 10.1038/nature03711.View ArticlePubMedGoogle Scholar
- Öst A, Danielsson A, Lidén M, Eriksson U, Nystrom FH, Strålfors P: Retinol-binding protein-4 attenuates insulin-induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes. FASEB J. 2007, 21: 3696-3704. 10.1096/fj.07-8173com.View ArticlePubMedGoogle Scholar
- Mikkelsen TS, Xu Z, Zhang X, Wang L, Gimble JM, Lander ES, Rosen ED: Comparative epigenomic analysis of murine and human adipogenesis. Cell. 2010, 143: 156-169. 10.1016/j.cell.2010.09.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang L, Xu S, Lee J-E, Baldridge A, Grullon S, Peng W, Ge K: Histone H3K9 methyltransferase G9a represses PPARgamma expression and adipogenesis. EMBO J. 2013, 32: 45-59.PubMed CentralView ArticlePubMedGoogle Scholar
- Strålfors P, Honnor RC: Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity. Eur J Biochem. 1989, 182: 379-385. 10.1111/j.1432-1033.1989.tb14842.x.View ArticlePubMedGoogle Scholar
- Danielsson A, Öst A, Lystedt E, Kjolhede P, Gustavsson J, Nystrom FH, Strålfors P: Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes. FEBS J. 2005, 272: 141-151.View ArticlePubMedGoogle Scholar
- Honnor RC, Dhillon GS, Londos C: cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior. J Biol Chem. 1985, 260: 15122-15129.PubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.View ArticlePubMedGoogle Scholar
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