Acta Diabetol DOI 10.1007/s00592-015-0741-0
REVIEW ARTICLE
Understanding type 2 diabetes: from genetics to epigenetics Gregory Alexander Raciti1,2 • Michele Longo1,2 • Luca Parrillo1,2 • Marco Ciccarelli1,2 • Paola Mirra1,2 • Paola Ungaro1,2 • Pietro Formisano1,2 Claudia Miele1,2 • Francesco Be´guinot1,2
•
Received: 23 December 2014 / Accepted: 14 March 2015 Ó Springer-Verlag Italia 2015
Abstract The known genetic variability (common DNA polymorphisms) does not account either for the current epidemics of type 2 diabetes or for the family transmission of this disorder. However, clinical, epidemiological and, more recently, experimental evidence indicates that environmental factors have an extraordinary impact on the natural history of type 2 diabetes. Some of these environmental hits are often shared in family groups and proved to be capable to induce epigenetic changes which alter the function of genes affecting major diabetes traits. Thus, epigenetic mechanisms may explain the environmental origin as well as the familial aggregation of type 2 diabetes much easier than common polymorphisms. In the murine model, exposure of parents to environmental hits known to cause epigenetic changes reprograms insulin sensitivity as well as beta-cell function in the progeny, indicating that certain epigenetic changes can be transgenerationally transmitted. Studies from different laboratories revealed that, in humans, lifestyle intervention modulates the epigenome and reverts environmentally induced epigenetic modifications at specific target genes. Finally, specific human epigenotypes have been identified which predict adiposity and type 2 diabetes with much greater power than any polymorphism so far identified. These epigenotypes can be recognized in easily accessible white cells from Managed by Antonio Secchi. & Francesco Be´guinot [email protected] 1
Dipartimento di Scienze Mediche Traslazionali, ‘‘Federico II’’ University of Naples Medical School, Naples, Italy
2
Istituto per l’ Endocrinologia e l’ Oncologia Sperimentale del C.N.R, URT ‘‘Genomica Funzionale’’, Via Sergio Pansini, 5, 80131 Naples, Italy
peripheral blood, indicating that, in the future, epigenetic profiling may enable effective type 2 diabetes prediction. This review discusses recent evidence from the literature supporting the immediate need for further investigation to uncover the power of epigenetics in the prediction, prevention and treatment of type 2 diabetes. Keywords Type 2 diabetes Epigenetics Methylation Histone modifications MicroRNA Personalized medicine
Introduction It has become progressively clearer that environmental factors affect individual phenotypes by causing epigenetic modifications of the DNA [1]. In contrast to gene variants or SNPs, these modifications modulate gene function without affecting the genome sequence. Rather, the epigenetic changes often modify chromatin structure and gene accessibility to the transcription machinery [2]. Thus, environmental factors may also epigenetically impact on genes which play an important role in the physiological control of glucose tolerance [3–5], even when these genes have not been recognized in previous genome-wide efforts or their function in energy homeostasis is indirect [6, 7]. Similar to genetic polymorphisms [8], epigenetic modifications may alter the transcriptional activity of these genes and contribute to the insulin resistance, beta-cell and fat tissue dysfunction and type 2 diabetes phenotype, including response to anti-diabetic agents and occurrence of diabetes complications [9–11]. These effects may occur both during early development and, later in life, in the adulthood. At least in the mouse model, epigenetic marks can be transgenerationally transmitted [12]. In addition, current
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evidence now indicates that, in humans, they represent presymptomatic markers of the lifetime risk of diabetes [13]. As presented in this review, identification of these marks and their conclusive assignment to specific T2D subphenotypes offers unanticipated opportunities to elaborate novel diagnostic and therapeutic strategies tailored to meet individual T2D requirements. The global burden of diabetes The 2013 IDF report revealed that diabetes affects 382 million people or 8.3 % of the adult population [14]. Most of these individuals have type 2 diabetes. It is anticipated that, by 2035, some 592 million people, or one adult in 10, will have diabetes. This equates to approximately three new cases every 10 s or almost 10 million per year. The largest increases will take place in the regions where developing economies are predominant. In a cross-sectional survey dated 2010, the prevalence of type 2 diabetes was already 11.6 % while that of pre-diabetes was 50 % [15]. The causes of this epidemic remain a mystery. But unbiased considerations on type 2 diabetes genetics [16] and more recent evidence supporting the important role of epigenetic mechanisms in the natural history of this disorder [17, 18] strengthen our hope of a future without diabetes. Family transmission of type 2 diabetes Individuals with family history of diabetes have a risk of developing the disease up to tenfold higher, depending on the number of affected relatives and on whether maternal and paternal history is present [19–21]. Interestingly, this finding has long been adopted as an argument supporting the genetic origin of diabetes. In particular, it has often been underlined that (1) 39 % of affected individuals have at least one affected parent [22]; (2) among monozygotic twin pairs with one affected twin, approximately 90 % of unaffected twins eventually develop the disease through his or her life [23]; (3) the lifetime risk for a first-degree relative of a patient with type 2 diabetes is 5–10 times higher than that of age- and weight-matched subjects without a family history of diabetes [19]; and (4) first-degree relatives of patients with type 2 diabetes frequently have impaired nonoxidative glucose metabolism (indicative of insulin resistance) long before they develop type 2 diabetes. In addition, they may have beta-cell dysfunction, as evidenced by decreases in insulin and amylin release in response to glucose stimulation [24]. All of these findings have been considered to be determined by genetic variability and have prompted significant research efforts aiming at identifying risk genes.
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The search of diabetes risk genes Several common polymorphisms (at least 50 to date) weakly contribute to the risk of type 2 diabetes. Some of these genes encode proteins that cause alterations in several pathways leading to diabetes, including pancreatic development, insulin synthesis, processing and secretion; amyloid deposition in beta-cells; cellular insulin resistance; and impaired regulation of gluconeogenesis. Importantly, however, this work revealed that most of the genes exerting their function in the control of glucose tolerance do not show naturally occurring variants associated with risk of diabetes. Whether and how these genes determine the natural history of diabetes contributes to the mystery of the ‘‘missing inheritance’’ [25]. The investigation of type 2 diabetes genetics performed with different approaches over the past 25 years has significantly contributed to the present understanding of betacell biology, mechanisms controlling glucose tolerance and responsivity to anti-diabetes drugs and has also generated evidence supporting the concept that insulin resistance, alone, is not sufficient to cause type 2 diabetes in humans. These same studies also led to the conclusion that genetic polymorphisms only marginally contribute to type 2 diabetes transmission within families [16]. Indeed, known polymorphisms have little impact on current strategies to predict type 2 diabetes, even when multiple genes are simultaneously taken into account through risk algorithms. Thus, genetic studies have so far left the issue of how type 2 diabetes is transmitted unsolved. From genetics to epigenetics Changes in DNA sequence are not the only family features that can be transmitted across generations. For example, habits, including eating habits, lifestyles and exposure to specific environmental factors, are commonly shared within families. All of these factors are potentially capable to physically impact upon the genome and affect the expression of genes [17, 18] (Fig. 1). Some of these genes play a major role in controlling glucose tolerance [17]. Epigenetic modifications amend the genotype without changing the DNA sequence and represent a common mechanism mediating environmental modulation of glucose tolerance genes. Different epigenetic modifications have been identified and described [26]. Quite frequently, DNA undergoes methylation at specific sites, and DNAassociated proteins (histones) are post-translationally modified through acetylation, methylation or sumoylation processes. These changes commonly affect chromatin state and DNA binding of transcription factors, thus modifying gene transcription. Thus, epigenetic changes may modulate gene function or turn it on and off, very much like SNPs
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Fig. 1 Health is a function of the interactions of genes and environment. A number of factors, such as eating habits, exercise, microbiome, aging or intrauterine environment, can regulate gene function through epigenetic modifications, thereby affecting (or shaping) health status
and other mutations. However, epigenetic changes may be determined by environmental hits much easier than mutations. In addition, some epigenetic modification may be stable enough to be transgenerationally transmitted [17]. To which extent epigenetic modifications determine the onset of type 2 diabetes in humans remains unclear at the moment. However, growing evidence indicates that epigenetics has a previously unanticipated role in the natural history as well as in transgenerational transmission of this disorder. In addition, epigenetic modifications potentially explain current type 2 diabetes epidemics and a number of unsolved issues related to diabetes pathogenesis and family transmission, such as the missing inheritance and identical twin discordance. In the future, this information may enable the generation of predicting tools more powerful than those offered by genetics and opportunities for intervention which are really tailored on individual needs, i.e., personalized. The role of epigenetics in the current type 2 diabetes epidemics: lesson from the study of the ped/pea-15 gene As mentioned earlier in this review, the number of people with diabetes has dramatically increased over the past
decades and is expected to double within the next 15 years [14]. This epidemic cannot be accounted for by the diffusion of genetic variants which are known to be associated with type 2 diabetes [16]. However, the analysis of clinical risk factors has taught that obesity, age and lifestyle determinants, including physical exercise, nutritional preferences and cigarette smoking, have an extraordinary impact on type 2 diabetes risk [27, 28]. We are currently very far from understanding how, mechanistically, these factors impact on the risk of type 2 diabetes. However, studies in mice have recently provided clues about this issue. In the mouse model, for instance, the overexpression of ped/pea15, a gene whose function is commonly increased in type 2 diabetic individuals, impairs glucose tolerance although it does not induce diabetes [29]. However, ped/pea-15 overexpressing mice fed a high-fat diet which render them obese also develop diabetes while their non-transgenic littermates only exhibit an impairment in glucose tolerance (Fig. 2a). At the molecular level, increased expression of ped/pea-15 in cultured skeletal muscle and adipose cells causes insulin resistance by impairing insulin-stimulated GLUT4 translocation and glucose uptake [30]. Our further studies have also demonstrated that ped/pea-15-induced resistance to insulin action on glucose disposal is accompanied by phospholipase D-dependent activation of the classical PKC isoform PKC-a [31]. In turn, the induction of PKC-a by ped/pea-15 prevents subsequent activation of the atypical PKC-f by insulin impairing thus GLUT4-containing vesicle translocation toward the plasma membrane [29, 31]. Phenotyping of transgenic mice overexpressing ped/pea-15 in the pancreatic b-cells revealed also that ped/pea-15 regulates glucose-induced insulin secretion by restraining potassium channel expression [32]. In b-cells, the overexpression of ped/pea-15, through the PKCa-dependent block of PKCf, impairs the phosphorylation of the transcription factor HNF-3b, thereby decreasing the expression of the Kir6.2 and the Sur1 potassium channel subunits [32]. This, in turn, prevents insulin exocytosis and secretion in response to increasing glucose concentration. Thus, as for other genes, ped/pea-15 could be defined a bona fide risk gene for type 2 diabetes, but association of polymorphic variants has never been identified in any subsequent study, leaving its role in diabetes a mystery. Several years later, however, studies in my laboratory revealed that diets causing obesity both in mice and in humans determine changes in the acetylation level of histone H3 at ped/pea-15 promoter (Fig. 2b). This epigenetic modification was demonstrated to be capable to enhance ped/pea-15 transcription and to contribute to the negative effect of these diets on glucose tolerance. Even more recently, we have further demonstrated that these effects on ped/pea-15 function are not reverted by weight reduction (PU and FB unpublished data).
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Fig. 2 Ped/pea-15 gene. a Under regular chow diet feeding (STD), transgenic mice overexpressing ped/pea-15 (Tg-ped) exhibit mildly elevated random-fed blood glucose levels and become hyperglycemic after glucose loading compared to their non-transgenic littermates. Moreover, Tg-ped mice become diabetic when body weight increases through administration of a high-fat diet (HFD). This finding suggests an important interaction of environmental modifiers with ped/pea-15 gene function, leading to a further derangement in glucose tolerance. b Epigenetic changes induced by HFD to ped/pea-15 gene expression. HFD feeding induces obesity and insulin resistance in C57BL/6 mice and enhances in the tibialis skeletal muscle of the obese mice ped/pea-15 gene transcription by causing chromatin remodeling of the ped/pea-15 promoter characterized by an increased acetylation at lysine 9 of histone H3 (AcH3K9), an hallmark of actively transcribed promoter
Ped/pea-15 is unlikely the only cause of the effect of diet-induced obesity on type 2 diabetes risk. However, these studies have taught that genes involved in the control of glucose tolerance, independent of DNA mutation, are at the interface with lifestyle factors. Indeed, lifestyle factors can induce stable epigenetic changes which modify their function. Epigenetic changes and transgenerational transmission We still do not know precisely how stable these diet-induced changes in ped/pea-15 promoter epigenetic profile are. However, work from different laboratories revealed that certain DNA methylation changes are stable enough to survive meiosis and be transgenerationally transmitted. Studies in rats demonstrated that the exposure of the father to an high-fat diet regimen determines metabolic sequelae in the progeny. Indeed, the high-fat diet was shown to generate an early onset of impaired insulin secretion and glucose tolerance that worsened with time in the progeny
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Fig. 3 Non-genetic, intergenerational transmission of metabolic sequelae of a high-fat diet (HFD) from parents to offspring. a Paternal HFD exposure programs b-cell dysfunction in rat F1 female offspring. Chronic HFD consumption in Sprague–Dawley fathers induced increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity. Relative to controls, their female offspring had an early onset of impaired insulin secretion and glucose tolerance, and normal adiposity. b Maternal HFD affects body size via the paternal lineage in F3 female mice. Maternal HFD exposure in mice resulted in an increase in body size and reduced insulin sensitivity that persisted across two generations (F1 and F2) via both maternal and paternal lineages, but only increased body weight, as transmitted through the paternal lineage to F3 female offspring
by modifying the methylation profile of genes responsible for maintaining beta-cell mass [12] (Fig. 3a). This finding has been confirmed and extended by an independent investigation published 1 year later [33]. In this second paper, it has been reported that the exposure of female mice to a high-fat diet is accompanied by reduced insulin sensitivity in the offspring. Importantly, it was shown that insulin resistance persists in the following two generations, indicating that an environmental hit, i.e., the exposure to the high-fat diet stably reprograms the epigenome through subsequent generations (Fig. 3b). Thus, rodent studies taught that parent nutrition affects metabolic control in the offspring through epigenetic mechanisms. Lifestyle effect upon epigenome plasticity: clinical significance Animal studies have further taught that the epigenome represents an interface through which gene transcription undergoes rapid adaptation to the environment, and recent work now indicates that these mechanisms also occur in humans [34]. In humans, epigenetic profile analysis revealed important differences between fat and lean individuals as well as in type 2 diabetics and non-diabetic controls [35]. In addition, some of the genes affected by
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these changes play an important role in metabolism. In the future, these studies may help to understand why obesity develops in certain individuals and not in other and why some individuals develop diabetes. More immediate, interventions for targeting epigenome plasticity through lifestyle changes proved feasible, offering innovative opportunities for prevention and treatment. Physical exercise, for instance, induces methylation as well as functional changes at several genes, both in muscle and in fat. In a recent study in human adipose tissue, 39 such genes were shown to play a potentially important role both in obesity and in type 2 diabetes development, despite lack of known polymorphisms associated with these disorders [36]. Weight loss following bariatric surgery also induced similar epigenetic changes [35]. These further studies strengthen the evidence that obesity and weight reduction exert a dynamic effect on the epigenome and support the concept that the epigenome represents an innovative target for pharmacological and environmental interventions aimed at preventing and treating type 2 diabetes. Epigenotypes as markers of risk of obesity and type 2 diabetes The possibility that certain epigenotypes may contribute to the assessment of risk of obesity and type 2 diabetes represents an even closer application. Godfrey et al. [37] have recently published a paper where it is demonstrated that the epigenetic profile at birth predicts both the adiposity level
in infancy and specific features of nutritional exposure during prenatal life. This is a prospective study where the methylation state of the retinoid receptor promoter has been analyzed in two independent cohorts. The retinoid receptor plays a major role in controlling adipose tissue metabolism and insulin sensitivity and, in both cohorts, the methylation of its promoter at specific sites correlates with the level of adiposity at 6 and 9 years of age. In this same paper, it has been further demonstrated that the amount of carbohydrate in the mother diet through her pregnancy first trimester correlates with the retinoid receptor promoter methylation. Even more surprisingly, the paper by Godfrey et al. reports that this single genotype, alone, accounts for at least 25 % of obesity variance in the two cohorts. This latter finding was totally unanticipated if one considers the modest effect of the common polymorphisms associated with obesity and type 2 diabetes. While the molecular mechanisms responsible for the differential methylation of the retinoid receptor promoter remain undefined, this work supports the concept that epigenetic analysis at the perinatal stage may identify the risk of obesity and type 2 diabetes with unprecedented accuracy [37]. More recently, this concept has been further strengthened by further studies which focused on the methylation state of a wellknown risk gene for obesity and type 2 diabetes termed FTO [13]. These authors showed that low methylation level of FTO represents an early marker of type 2 diabetes. The predictive power of FTO methylation, independent of any known polymorphism, is significantly greater than that of
Table 1 Methylation state of risk genes for T2D and obesity Risk genes
Tissue (s)
Disease phenotype (s)
References
PPARGC1a
Pancreatic islets, skeletal muscle
T2D
[38, 39]
FTO
Peripheral blood
T2D
[12]
INS PDX1
Pancreatic islets Pancreatic islets
T2D T2D
[40] [41]
CCL2
Peripheral blood mononuclear cells
T2D
[42]
MCHR1
Whole blood
Obesity, BMI
[43]
KCNQ1OT1, H19, IGF2, GRB10, MEST, SNRPN, GNAS
Saliva
Twins discordant for BMI
[44]
IL8, NOS3, PIK3CD, RXRA, SOD1
Umbilical cord tissue
Fat mass
[37]
POMC
Whole blood
Obesity
[45]
SLC6A4
Peripheral blood leukocytes
BMI, weight and waist circumference
[46]
TACSTD2
Whole blood
Fat mass
[47]
ALOX12, ALPL, BCL2A1, CASP10, CAV1, CCL3,
Umbilical cord blood
BMI, fat mass and lean mass
[48]
CD9, CDKN1C, DSC2, EPHA1, EVI2A, HLA, IRF5, KRT1, LCN2, MLLT4, MMP9, MPL, NID1, NKX31, PMP22, S100A12, TAL1, VIM BMI body mass index, T2D type 2 diabetes
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all genetic variants described so far. It further needs to be underlined that these studies have been conducted on peripheral blood cells, which are easily accessible. To date, few more studies have explored in humans the DNA methylation of selected candidate risk genes for T2D and/ or obesity in addition to FTO (Table 1) [49]. Despite some limitations in these studies, these findings may generate, in the future, innovative tools for predicting the risk of obesity and diabetes as well as their response to lifestyle and, possibly, pharmacological interventions.
4.
5.
6.
Conclusion and perspectives The work discussed in the present review has underlined important advancement derived from current understanding of type 2 diabetes genetics. In particular, it is now clear that genomic variability caused by mutations only marginally contributes to the etiology, pathogenesis and family risk of type 2 diabetes. These are all areas deserving further investigation. However, epigenetic variability proved to be able to contribute unanticipated power in solving these mysteries. Whether and how this advancement will be translated into clinical practice represents an exciting challenge for both the clinician and the molecular diabetologist
7.
8. 9.
10.
11. Acknowledgments This work has been supported, in part, by the European Foundation for the Study of Diabetes (EFSD), the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the Ministero dell’Universita` e della Ricerca Scientifica (Grants PRIN and FIRB-MERIT, and PON 01_02460). This work was also supported by the P.O.R. Campania FSE 2007-2013, Project CREMe. Disclosure of potential conflicts of interest that they have no conflict of interest.
13.
The authors declare
Statement of human rights This article does not contain any studies with human participants performed by any of the authors. Statement on the welfare of animals All animal procedures in studies conducted by the authors and cited in this review were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85-23, revised 1996), and experiments were approved by the ethics committee of the Federico II University.
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36. Ro¨nn T, Volkov P, Davega˚rdh C, Dayeh T, Hall E, Olsson AH, Nilsson E, Tornberg A, Dekker Nitert M, Eriksson KF, Jones HA, Groop L, Ling C (2013) A six months exercise intervention influences the genome-wide DNA methylation pattern in human adipose tissue. PLoS Genet 9(6):e1003572 37. Godfrey KM, Sheppard A, Gluckman PD, Lillycrop KA, Burdge GC, McLean C, Rodford J, Slater-Jefferies JL, Garratt E, Crozier SR, Emerald BS, Gale CR, Inskip HM, Cooper C, Hanson MA (2011) Epigenetic gene promoter methylation at birth is associated with child’s later adiposity. Diabetes 60(5):1528–1534 38. Ling C, Del Guerra S, Lupi R, Ro¨nn T, Granhall C, Luthman H, Masiello P, Marchetti P, Groop L, Del Prato S (2008) Epigenetic regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin secretion. Diabetologia 51(4):615–622 39. Barre`s R, Osler ME, Yan J, Rune A, Fritz T, Caidahl K, Krook A, Zierath JR (2009) Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density. Cell Metab 10(3):189–198 40. Yang BT, Dayeh TA, Kirkpatrick CL, Taneera J, Kumar R, Groop L, Wollheim CB, Nitert MD, Ling C (2011) Insulin promoter DNA methylation correlates negatively with insulin gene expression and positively with HbA(1c) levels in human pancreatic islets. Diabetologia 54(2):360–367 41. Yang BT, Dayeh TA, Volkov PA, Kirkpatrick CL, Malmgren S, Jing X, Renstro¨m E, Wollheim CB, Nitert MD, Ling C (2012) Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes. Mol Endocrinol 26(7):1203–1212 42. Liu ZH, Chen LL, Deng XL, Song HJ, Liao YF, Zeng TS, Zheng J, Li HQ (2012) Methylation status of CpG sites in the MCP-1 promoter is correlated to serum MCP-1 in type 2 diabetes. J Endocrinol Invest 35(6):585–589 43. Stepanow S, Reichwald K, Huse K, Gausmann U, Nebel A, Rosenstiel P, Wabitsch M, Fischer-Posovszky P, Platzer M (2011) Allele-specific, age-dependent and BMI-associated DNA methylation of human MCHR1. PLoS One 6(5):e17711 44. Souren NY, Tierling S, Fryns JP, Derom C, Walter J, Zeegers MP (2011) DNA methylation variability at growth-related imprints does not contribute to overweight in monozygotic twins discordant for BMI. Obesity 19(7):1519–1522 45. Kuehnen P, Mischke M, Wiegand S, Sers C, Horsthemke B, Lau S, Keil T, Lee YA, Grueters A, Krude H (2012) An Alu elementassociated hypermethylation variant of the POMC gene is associated with childhood obesity. PLoS Genet 8(3):e1002543 46. Zhao J, Goldberg J, Vaccarino V (2013) Promoter methylation of serotonin transporter gene is associated with obesity measures: a monozygotic twin study. Int J Obes 37(1):140–145 47. Groom A, Potter C, Swan DC, Fatemifar G, Evans DM, Ring SM, Turcot V, Pearce MS, Embleton ND, Smith GD, Mathers JC, Relton CL (2012) Postnatal growth and DNA methylation are associated with differential gene expression of the TACSTD2 gene and childhood fat mass. Diabetes 61(2):391–400 48. Relton CL, Groom A, St Pourcain B, Sayers AE, Swan DC, Embleton ND, Pearce MS, Ring SM, Northstone K, Tobias JH, Trakalo J, Ness AR, Shaheen SO, Smith GD (2012) DNA methylation patterns in cord blood DNA and body size in childhood. PLoS One 7(3):e31821 49. Drong AW, Lindgren CM, McCarthy MI (2012) The genetic and epigenetic basis of type 2 diabetes and obesity. Clin Pharmacol Ther 92(6):707–715
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REVIEW ARTICLE
Understanding type 2 diabetes: from genetics to epigenetics Gregory Alexander Raciti1,2 • Michele Longo1,2 • Luca Parrillo1,2 • Marco Ciccarelli1,2 • Paola Mirra1,2 • Paola Ungaro1,2 • Pietro Formisano1,2 Claudia Miele1,2 • Francesco Be´guinot1,2
•
Received: 23 December 2014 / Accepted: 14 March 2015 Ó Springer-Verlag Italia 2015
Abstract The known genetic variability (common DNA polymorphisms) does not account either for the current epidemics of type 2 diabetes or for the family transmission of this disorder. However, clinical, epidemiological and, more recently, experimental evidence indicates that environmental factors have an extraordinary impact on the natural history of type 2 diabetes. Some of these environmental hits are often shared in family groups and proved to be capable to induce epigenetic changes which alter the function of genes affecting major diabetes traits. Thus, epigenetic mechanisms may explain the environmental origin as well as the familial aggregation of type 2 diabetes much easier than common polymorphisms. In the murine model, exposure of parents to environmental hits known to cause epigenetic changes reprograms insulin sensitivity as well as beta-cell function in the progeny, indicating that certain epigenetic changes can be transgenerationally transmitted. Studies from different laboratories revealed that, in humans, lifestyle intervention modulates the epigenome and reverts environmentally induced epigenetic modifications at specific target genes. Finally, specific human epigenotypes have been identified which predict adiposity and type 2 diabetes with much greater power than any polymorphism so far identified. These epigenotypes can be recognized in easily accessible white cells from Managed by Antonio Secchi. & Francesco Be´guinot [email protected] 1
Dipartimento di Scienze Mediche Traslazionali, ‘‘Federico II’’ University of Naples Medical School, Naples, Italy
2
Istituto per l’ Endocrinologia e l’ Oncologia Sperimentale del C.N.R, URT ‘‘Genomica Funzionale’’, Via Sergio Pansini, 5, 80131 Naples, Italy
peripheral blood, indicating that, in the future, epigenetic profiling may enable effective type 2 diabetes prediction. This review discusses recent evidence from the literature supporting the immediate need for further investigation to uncover the power of epigenetics in the prediction, prevention and treatment of type 2 diabetes. Keywords Type 2 diabetes Epigenetics Methylation Histone modifications MicroRNA Personalized medicine
Introduction It has become progressively clearer that environmental factors affect individual phenotypes by causing epigenetic modifications of the DNA [1]. In contrast to gene variants or SNPs, these modifications modulate gene function without affecting the genome sequence. Rather, the epigenetic changes often modify chromatin structure and gene accessibility to the transcription machinery [2]. Thus, environmental factors may also epigenetically impact on genes which play an important role in the physiological control of glucose tolerance [3–5], even when these genes have not been recognized in previous genome-wide efforts or their function in energy homeostasis is indirect [6, 7]. Similar to genetic polymorphisms [8], epigenetic modifications may alter the transcriptional activity of these genes and contribute to the insulin resistance, beta-cell and fat tissue dysfunction and type 2 diabetes phenotype, including response to anti-diabetic agents and occurrence of diabetes complications [9–11]. These effects may occur both during early development and, later in life, in the adulthood. At least in the mouse model, epigenetic marks can be transgenerationally transmitted [12]. In addition, current
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evidence now indicates that, in humans, they represent presymptomatic markers of the lifetime risk of diabetes [13]. As presented in this review, identification of these marks and their conclusive assignment to specific T2D subphenotypes offers unanticipated opportunities to elaborate novel diagnostic and therapeutic strategies tailored to meet individual T2D requirements. The global burden of diabetes The 2013 IDF report revealed that diabetes affects 382 million people or 8.3 % of the adult population [14]. Most of these individuals have type 2 diabetes. It is anticipated that, by 2035, some 592 million people, or one adult in 10, will have diabetes. This equates to approximately three new cases every 10 s or almost 10 million per year. The largest increases will take place in the regions where developing economies are predominant. In a cross-sectional survey dated 2010, the prevalence of type 2 diabetes was already 11.6 % while that of pre-diabetes was 50 % [15]. The causes of this epidemic remain a mystery. But unbiased considerations on type 2 diabetes genetics [16] and more recent evidence supporting the important role of epigenetic mechanisms in the natural history of this disorder [17, 18] strengthen our hope of a future without diabetes. Family transmission of type 2 diabetes Individuals with family history of diabetes have a risk of developing the disease up to tenfold higher, depending on the number of affected relatives and on whether maternal and paternal history is present [19–21]. Interestingly, this finding has long been adopted as an argument supporting the genetic origin of diabetes. In particular, it has often been underlined that (1) 39 % of affected individuals have at least one affected parent [22]; (2) among monozygotic twin pairs with one affected twin, approximately 90 % of unaffected twins eventually develop the disease through his or her life [23]; (3) the lifetime risk for a first-degree relative of a patient with type 2 diabetes is 5–10 times higher than that of age- and weight-matched subjects without a family history of diabetes [19]; and (4) first-degree relatives of patients with type 2 diabetes frequently have impaired nonoxidative glucose metabolism (indicative of insulin resistance) long before they develop type 2 diabetes. In addition, they may have beta-cell dysfunction, as evidenced by decreases in insulin and amylin release in response to glucose stimulation [24]. All of these findings have been considered to be determined by genetic variability and have prompted significant research efforts aiming at identifying risk genes.
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The search of diabetes risk genes Several common polymorphisms (at least 50 to date) weakly contribute to the risk of type 2 diabetes. Some of these genes encode proteins that cause alterations in several pathways leading to diabetes, including pancreatic development, insulin synthesis, processing and secretion; amyloid deposition in beta-cells; cellular insulin resistance; and impaired regulation of gluconeogenesis. Importantly, however, this work revealed that most of the genes exerting their function in the control of glucose tolerance do not show naturally occurring variants associated with risk of diabetes. Whether and how these genes determine the natural history of diabetes contributes to the mystery of the ‘‘missing inheritance’’ [25]. The investigation of type 2 diabetes genetics performed with different approaches over the past 25 years has significantly contributed to the present understanding of betacell biology, mechanisms controlling glucose tolerance and responsivity to anti-diabetes drugs and has also generated evidence supporting the concept that insulin resistance, alone, is not sufficient to cause type 2 diabetes in humans. These same studies also led to the conclusion that genetic polymorphisms only marginally contribute to type 2 diabetes transmission within families [16]. Indeed, known polymorphisms have little impact on current strategies to predict type 2 diabetes, even when multiple genes are simultaneously taken into account through risk algorithms. Thus, genetic studies have so far left the issue of how type 2 diabetes is transmitted unsolved. From genetics to epigenetics Changes in DNA sequence are not the only family features that can be transmitted across generations. For example, habits, including eating habits, lifestyles and exposure to specific environmental factors, are commonly shared within families. All of these factors are potentially capable to physically impact upon the genome and affect the expression of genes [17, 18] (Fig. 1). Some of these genes play a major role in controlling glucose tolerance [17]. Epigenetic modifications amend the genotype without changing the DNA sequence and represent a common mechanism mediating environmental modulation of glucose tolerance genes. Different epigenetic modifications have been identified and described [26]. Quite frequently, DNA undergoes methylation at specific sites, and DNAassociated proteins (histones) are post-translationally modified through acetylation, methylation or sumoylation processes. These changes commonly affect chromatin state and DNA binding of transcription factors, thus modifying gene transcription. Thus, epigenetic changes may modulate gene function or turn it on and off, very much like SNPs
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Fig. 1 Health is a function of the interactions of genes and environment. A number of factors, such as eating habits, exercise, microbiome, aging or intrauterine environment, can regulate gene function through epigenetic modifications, thereby affecting (or shaping) health status
and other mutations. However, epigenetic changes may be determined by environmental hits much easier than mutations. In addition, some epigenetic modification may be stable enough to be transgenerationally transmitted [17]. To which extent epigenetic modifications determine the onset of type 2 diabetes in humans remains unclear at the moment. However, growing evidence indicates that epigenetics has a previously unanticipated role in the natural history as well as in transgenerational transmission of this disorder. In addition, epigenetic modifications potentially explain current type 2 diabetes epidemics and a number of unsolved issues related to diabetes pathogenesis and family transmission, such as the missing inheritance and identical twin discordance. In the future, this information may enable the generation of predicting tools more powerful than those offered by genetics and opportunities for intervention which are really tailored on individual needs, i.e., personalized. The role of epigenetics in the current type 2 diabetes epidemics: lesson from the study of the ped/pea-15 gene As mentioned earlier in this review, the number of people with diabetes has dramatically increased over the past
decades and is expected to double within the next 15 years [14]. This epidemic cannot be accounted for by the diffusion of genetic variants which are known to be associated with type 2 diabetes [16]. However, the analysis of clinical risk factors has taught that obesity, age and lifestyle determinants, including physical exercise, nutritional preferences and cigarette smoking, have an extraordinary impact on type 2 diabetes risk [27, 28]. We are currently very far from understanding how, mechanistically, these factors impact on the risk of type 2 diabetes. However, studies in mice have recently provided clues about this issue. In the mouse model, for instance, the overexpression of ped/pea15, a gene whose function is commonly increased in type 2 diabetic individuals, impairs glucose tolerance although it does not induce diabetes [29]. However, ped/pea-15 overexpressing mice fed a high-fat diet which render them obese also develop diabetes while their non-transgenic littermates only exhibit an impairment in glucose tolerance (Fig. 2a). At the molecular level, increased expression of ped/pea-15 in cultured skeletal muscle and adipose cells causes insulin resistance by impairing insulin-stimulated GLUT4 translocation and glucose uptake [30]. Our further studies have also demonstrated that ped/pea-15-induced resistance to insulin action on glucose disposal is accompanied by phospholipase D-dependent activation of the classical PKC isoform PKC-a [31]. In turn, the induction of PKC-a by ped/pea-15 prevents subsequent activation of the atypical PKC-f by insulin impairing thus GLUT4-containing vesicle translocation toward the plasma membrane [29, 31]. Phenotyping of transgenic mice overexpressing ped/pea-15 in the pancreatic b-cells revealed also that ped/pea-15 regulates glucose-induced insulin secretion by restraining potassium channel expression [32]. In b-cells, the overexpression of ped/pea-15, through the PKCa-dependent block of PKCf, impairs the phosphorylation of the transcription factor HNF-3b, thereby decreasing the expression of the Kir6.2 and the Sur1 potassium channel subunits [32]. This, in turn, prevents insulin exocytosis and secretion in response to increasing glucose concentration. Thus, as for other genes, ped/pea-15 could be defined a bona fide risk gene for type 2 diabetes, but association of polymorphic variants has never been identified in any subsequent study, leaving its role in diabetes a mystery. Several years later, however, studies in my laboratory revealed that diets causing obesity both in mice and in humans determine changes in the acetylation level of histone H3 at ped/pea-15 promoter (Fig. 2b). This epigenetic modification was demonstrated to be capable to enhance ped/pea-15 transcription and to contribute to the negative effect of these diets on glucose tolerance. Even more recently, we have further demonstrated that these effects on ped/pea-15 function are not reverted by weight reduction (PU and FB unpublished data).
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Fig. 2 Ped/pea-15 gene. a Under regular chow diet feeding (STD), transgenic mice overexpressing ped/pea-15 (Tg-ped) exhibit mildly elevated random-fed blood glucose levels and become hyperglycemic after glucose loading compared to their non-transgenic littermates. Moreover, Tg-ped mice become diabetic when body weight increases through administration of a high-fat diet (HFD). This finding suggests an important interaction of environmental modifiers with ped/pea-15 gene function, leading to a further derangement in glucose tolerance. b Epigenetic changes induced by HFD to ped/pea-15 gene expression. HFD feeding induces obesity and insulin resistance in C57BL/6 mice and enhances in the tibialis skeletal muscle of the obese mice ped/pea-15 gene transcription by causing chromatin remodeling of the ped/pea-15 promoter characterized by an increased acetylation at lysine 9 of histone H3 (AcH3K9), an hallmark of actively transcribed promoter
Ped/pea-15 is unlikely the only cause of the effect of diet-induced obesity on type 2 diabetes risk. However, these studies have taught that genes involved in the control of glucose tolerance, independent of DNA mutation, are at the interface with lifestyle factors. Indeed, lifestyle factors can induce stable epigenetic changes which modify their function. Epigenetic changes and transgenerational transmission We still do not know precisely how stable these diet-induced changes in ped/pea-15 promoter epigenetic profile are. However, work from different laboratories revealed that certain DNA methylation changes are stable enough to survive meiosis and be transgenerationally transmitted. Studies in rats demonstrated that the exposure of the father to an high-fat diet regimen determines metabolic sequelae in the progeny. Indeed, the high-fat diet was shown to generate an early onset of impaired insulin secretion and glucose tolerance that worsened with time in the progeny
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Fig. 3 Non-genetic, intergenerational transmission of metabolic sequelae of a high-fat diet (HFD) from parents to offspring. a Paternal HFD exposure programs b-cell dysfunction in rat F1 female offspring. Chronic HFD consumption in Sprague–Dawley fathers induced increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity. Relative to controls, their female offspring had an early onset of impaired insulin secretion and glucose tolerance, and normal adiposity. b Maternal HFD affects body size via the paternal lineage in F3 female mice. Maternal HFD exposure in mice resulted in an increase in body size and reduced insulin sensitivity that persisted across two generations (F1 and F2) via both maternal and paternal lineages, but only increased body weight, as transmitted through the paternal lineage to F3 female offspring
by modifying the methylation profile of genes responsible for maintaining beta-cell mass [12] (Fig. 3a). This finding has been confirmed and extended by an independent investigation published 1 year later [33]. In this second paper, it has been reported that the exposure of female mice to a high-fat diet is accompanied by reduced insulin sensitivity in the offspring. Importantly, it was shown that insulin resistance persists in the following two generations, indicating that an environmental hit, i.e., the exposure to the high-fat diet stably reprograms the epigenome through subsequent generations (Fig. 3b). Thus, rodent studies taught that parent nutrition affects metabolic control in the offspring through epigenetic mechanisms. Lifestyle effect upon epigenome plasticity: clinical significance Animal studies have further taught that the epigenome represents an interface through which gene transcription undergoes rapid adaptation to the environment, and recent work now indicates that these mechanisms also occur in humans [34]. In humans, epigenetic profile analysis revealed important differences between fat and lean individuals as well as in type 2 diabetics and non-diabetic controls [35]. In addition, some of the genes affected by
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these changes play an important role in metabolism. In the future, these studies may help to understand why obesity develops in certain individuals and not in other and why some individuals develop diabetes. More immediate, interventions for targeting epigenome plasticity through lifestyle changes proved feasible, offering innovative opportunities for prevention and treatment. Physical exercise, for instance, induces methylation as well as functional changes at several genes, both in muscle and in fat. In a recent study in human adipose tissue, 39 such genes were shown to play a potentially important role both in obesity and in type 2 diabetes development, despite lack of known polymorphisms associated with these disorders [36]. Weight loss following bariatric surgery also induced similar epigenetic changes [35]. These further studies strengthen the evidence that obesity and weight reduction exert a dynamic effect on the epigenome and support the concept that the epigenome represents an innovative target for pharmacological and environmental interventions aimed at preventing and treating type 2 diabetes. Epigenotypes as markers of risk of obesity and type 2 diabetes The possibility that certain epigenotypes may contribute to the assessment of risk of obesity and type 2 diabetes represents an even closer application. Godfrey et al. [37] have recently published a paper where it is demonstrated that the epigenetic profile at birth predicts both the adiposity level
in infancy and specific features of nutritional exposure during prenatal life. This is a prospective study where the methylation state of the retinoid receptor promoter has been analyzed in two independent cohorts. The retinoid receptor plays a major role in controlling adipose tissue metabolism and insulin sensitivity and, in both cohorts, the methylation of its promoter at specific sites correlates with the level of adiposity at 6 and 9 years of age. In this same paper, it has been further demonstrated that the amount of carbohydrate in the mother diet through her pregnancy first trimester correlates with the retinoid receptor promoter methylation. Even more surprisingly, the paper by Godfrey et al. reports that this single genotype, alone, accounts for at least 25 % of obesity variance in the two cohorts. This latter finding was totally unanticipated if one considers the modest effect of the common polymorphisms associated with obesity and type 2 diabetes. While the molecular mechanisms responsible for the differential methylation of the retinoid receptor promoter remain undefined, this work supports the concept that epigenetic analysis at the perinatal stage may identify the risk of obesity and type 2 diabetes with unprecedented accuracy [37]. More recently, this concept has been further strengthened by further studies which focused on the methylation state of a wellknown risk gene for obesity and type 2 diabetes termed FTO [13]. These authors showed that low methylation level of FTO represents an early marker of type 2 diabetes. The predictive power of FTO methylation, independent of any known polymorphism, is significantly greater than that of
Table 1 Methylation state of risk genes for T2D and obesity Risk genes
Tissue (s)
Disease phenotype (s)
References
PPARGC1a
Pancreatic islets, skeletal muscle
T2D
[38, 39]
FTO
Peripheral blood
T2D
[12]
INS PDX1
Pancreatic islets Pancreatic islets
T2D T2D
[40] [41]
CCL2
Peripheral blood mononuclear cells
T2D
[42]
MCHR1
Whole blood
Obesity, BMI
[43]
KCNQ1OT1, H19, IGF2, GRB10, MEST, SNRPN, GNAS
Saliva
Twins discordant for BMI
[44]
IL8, NOS3, PIK3CD, RXRA, SOD1
Umbilical cord tissue
Fat mass
[37]
POMC
Whole blood
Obesity
[45]
SLC6A4
Peripheral blood leukocytes
BMI, weight and waist circumference
[46]
TACSTD2
Whole blood
Fat mass
[47]
ALOX12, ALPL, BCL2A1, CASP10, CAV1, CCL3,
Umbilical cord blood
BMI, fat mass and lean mass
[48]
CD9, CDKN1C, DSC2, EPHA1, EVI2A, HLA, IRF5, KRT1, LCN2, MLLT4, MMP9, MPL, NID1, NKX31, PMP22, S100A12, TAL1, VIM BMI body mass index, T2D type 2 diabetes
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all genetic variants described so far. It further needs to be underlined that these studies have been conducted on peripheral blood cells, which are easily accessible. To date, few more studies have explored in humans the DNA methylation of selected candidate risk genes for T2D and/ or obesity in addition to FTO (Table 1) [49]. Despite some limitations in these studies, these findings may generate, in the future, innovative tools for predicting the risk of obesity and diabetes as well as their response to lifestyle and, possibly, pharmacological interventions.
4.
5.
6.
Conclusion and perspectives The work discussed in the present review has underlined important advancement derived from current understanding of type 2 diabetes genetics. In particular, it is now clear that genomic variability caused by mutations only marginally contributes to the etiology, pathogenesis and family risk of type 2 diabetes. These are all areas deserving further investigation. However, epigenetic variability proved to be able to contribute unanticipated power in solving these mysteries. Whether and how this advancement will be translated into clinical practice represents an exciting challenge for both the clinician and the molecular diabetologist
7.
8. 9.
10.
11. Acknowledgments This work has been supported, in part, by the European Foundation for the Study of Diabetes (EFSD), the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the Ministero dell’Universita` e della Ricerca Scientifica (Grants PRIN and FIRB-MERIT, and PON 01_02460). This work was also supported by the P.O.R. Campania FSE 2007-2013, Project CREMe. Disclosure of potential conflicts of interest that they have no conflict of interest.
13.
The authors declare
Statement of human rights This article does not contain any studies with human participants performed by any of the authors. Statement on the welfare of animals All animal procedures in studies conducted by the authors and cited in this review were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85-23, revised 1996), and experiments were approved by the ethics committee of the Federico II University.
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