Our carefully phenotyped MZ twins most discordant for obesity represent an ideal model to explore the effects of acquired human obesity independent of genetic factors. By constructing networks of global transcript profiles in subcutaneous fat biopsies, we identified several fundamental pathways with high relevance for obesity and its related insulin resistance (). Our data suggest that obesity that is already in its early stages in healthy young adults is characterized by marked inflammation of adipose tissue, significantly reduced mitochondrial DNA copy number, and disturbed mitochondrial energy metabolism—statistically most significantly, the decreased catabolism of insulin secretion–enhancing BCAAs. These impairments correlated with the critical clinical measures of obesity: liver fat accumulation, reduced whole-body insulin sensitivity, hyperinsulinemia, and hypoadiponectinemia.
Summary of Findings and Postulated Mechanisms and How They Associate with the Clinical Progression of Obesity-Related Pathologies
The observed impairments in adipose tissue function could be associated with insulin resistance by at least the following mechanisms: (1) Inflammation in fat and a direct action of cytokines or other mediators on the insulin signaling cascade [42
], (2) decline in mitochondrial DNA content or function [43
], and (3) diminished adipocyte differentiation within subcutaneous adipose tissue and subsequent increase in ectopic fat deposition [11
A wide range of inflammatory cascades, both humoral and cellular mediators, seemed to be activated in the obese co-twins' fat. In line with previous studies [6
], the major over-expressed innate inflammatory component was related to macrophages. These cells act as scavengers phagocytosing adipose debris [8
] and as antigen-presenting cells activating the adaptive immune system. Another up-regulated pathway in obese co-twins involved antigen presentation via MHC class II molecules. This result, together with signs of activation of lymphocytes and the complement system, suggest a widespread induction of previously unrecognized dimensions of the inflammatory response in obesity. In addition, complement component 3a receptor 1
), up-regulated in the obese co-twins, is among the three new genes with causal relationships for obesity in an elegant study in rodents integrating gene expression and DNA variation [44
A whole range of local and humoral mediators are known to inhibit insulin signaling [42
]. Additionally, recent studies show that specific proinflammatory lipid molecules, the lysophosphatidylcholines (LPCs), may impair insulin signaling at the level of insulin receptor substrate-1 (IRS1) and Akt/protein kinase C [45
]. LPCs, major components of oxidized low-density lipoprotein (LDL), are phospholipase A2-generated hydrolysis products of phosphatidylcholines, which are connected with the up-regulation of LDL-associated phospholipase A2 (PLA2G7
) observed in the fat of the obese twins in this study. Furthermore, LPCs activate RhoA GTPases [46
], MCP1, and other chemokines [47
], which were also up-regulated in this study's obese co-twins. In our previous study, LPCs were over-represented in the serum lipidomic assays in the obese co-twins, and were associated with lower insulin sensitivity [48
]. This observation supports the view that LPCs and possibly other lysophospholipids may act as triggers of inflammation in adipose tissue and play a role in obesity-related insulin resistance.
Our finding that the most over-expressed (5.9-fold) gene in the adipose tissue of the obese co-twins was the inflammatory cytokine osteopontin (SPP1
, also termed OPN
), involved in the recruitment of macrophages, is of great interest. During the review process of this article, Nomiyama and colleagues published their findings (in a mouse model of diet-induced obesity) that for the first time linked SPP1 to obesity and insulin resistance [49
]. Their results suggest that SPP1 plays a key role in the recruitment of macrophages into the adipose tissue in obese mice and thus in the development of insulin resistance. Secretion of SPP1 was increased during obesity and, unlike in wild-type mice, animals lacking SPP1 were protected from developing insulin resistance despite diet-induced obesity. The authors also showed that SPP1
expression decreased during the differentiation of 3T3-L1 preadipocytes, suggesting that alongside the decrease in PPARγ
expression observed here, the dramatic increase in SPP1
expression is yet another marker for poor differentiation of adipocytes in obesity. The over-expression of SPP1
in obese adipose tissue—here to our knowledge reported for the first time in humans—is an important observation in understanding the development of insulin resistance associated with obesity.
We found considerable depletion of mtDNA in adipose tissue of obese co-twins; to our knowledge this is the first finding of its kind in humans. Such a decline in the mtDNA content of fat cells may decrease the maximal capacity for oxidative phosphorylation, i.e., utilization of fat for energy production. Our study does not yet answer the question of whether mtDNA depletion is a primary event in acquired obesity or whether it is secondary and reactive to, for example, inflammation. However, such a remarkable decrease in mtDNA copy number would have a considerable impact on energy metabolism of fat tissue. Supporting our findings, patients with mutations in polymerase gamma, the mtDNA replicative polymerase, show partial mtDNA depletion and obesity [50
]. Furthermore, in our weight-concordant control MZ pairs, the mtDNA copy number levels were nearly identical. Taken together, these results strongly suggest that mtDNA depletion in our study's obese co-twins is weight-dependent. Longitudinal data accessible to us for one obese male further supported this suggestion: after this twin's weight increased by 10 kg within 3 y, mtDNA copy number in his fat tissue declined by 44%.
MtDNA copy number is under nuclear control and can be regulated by proteins of mtDNA maintenance and by cytoplasmic nucleoside pools. Since MZ twins have identical nuclear genes, the depletion of mtDNA we saw in obese co-twins suggests that mtDNA levels in the adipose tissue respond to environmental factors, such as nutrition and exercise. Such an effect has been documented in human skeletal muscle [51
]. Genetic factors [18
] and age [52
] may also influence mtDNA levels and capacity for oxidative energy production. However, controversy remains over whether low mitochondrial oxidative capacity can be accounted for by reduced overall mitochondrial mass or by mitochondrial dysfunction [53
]. The role of mtDNA levels and mitochondrial dysfunction in obesity has not, to our knowledge, been shown in human adipose tissue before. Here we demonstrate that acquired obesity, at an early stage and independent of genetic variation or age, is associated with significant decreases in mtDNA copy number in fat, together with low expression of transcripts involved in mitochondrial oxidative energy metabolism and BCAA catabolism.
Observations in animal models of obesity (ob/ob
mouse) and type 2 diabetes (db/db
mouse) have recently shown a reduction in the mtDNA content of white adipocytes concomitant with smaller mitochondrial size, disturbed mitochondrial morphology, and reduced respiration rates [55
]. This pattern was observed only in adipose tissue, not in skeletal muscle or liver. In another study, treatment with the insulin sensitivity-enhancing drug rosiglitazone reversed the dysmorphologic features of ob/ob
mouse mitochondria and mitochondrial protein profile [19
]. These observations emphasize the role of adipose tissue, and especially of the associated mitochondria, in the development of insulin resistance and other obesity-associated pathologies.
Down-regulation of BCAA catabolism in subcutaneous adipose tissue was associated with liver fat accumulation, the mechanisms of which may involve not only reduced mitochondrial respiration in the existing adipocytes, but also reduced differentiation and formation of new adipocytes within the subcutaneous sites, which shifts lipid storage into peripheral tissues. BCAA catabolism is normally activated during adipocyte differentiation, together with the key transcription factor PPARγ and molecules of oxidative energy production (acetyl-CoA utilization and NAD(P) biosynthesis) [41
], all of which were coordinately down-regulated in our study's obese co-twins. Impaired adipogenesis, together with impaired mitochondrial biogenesis in subcutaneous adipose tissue, could connect obesity to liver fat accumulation and to the development of type 2 diabetes [11
Following the clue provided by the transcript profiles in fat, we searched for signs of the systemic effects of low adipose tissue mitochondrial BCAA catabolism. The decrease in BCAA catabolism activity was associated with low serum concentrations of leucine ketoacids. High serum BCAA (especially leucine) concentrations were associated with obesity and hyperinsulinemia in the males in our study, which is in line with earlier studies suggesting that BCAAs may augment insulin secretion from the pancreas in insulin-resistant states [38
]. Recent studies in rat adipocytes have identified that BCAAs act both as triggers of insulin secretion and also as important players in the augmentation of insulin signaling through the mTOR (mammalian target of rapamycin) and phosphatidylinositol 3-kinase signaling pathways [56
]. Thus, normal BCAA metabolism may be critical for the maintenance of an appropriate insulin response. We propose that reduced mitochondrial BCAA catabolism (possibly as a consequence of low mtDNA levels), inhibition of adipose tissue differentiation, increased liver fat accumulation, and stimulation of insulin secretion by high serum BCAA levels may explain part of the well-documented association between fatty liver and hyperinsulinemia during the development of insulin resistance syndrome [57
An additional novel finding in our study was the down-regulation of several NAD+
-dependent histone deacetylases or their associated proteins (e.g., HDAC5, SIN3, SIRT1, SIRT3, and SIRT7
) in the fat tissue of the obese co-twins. Sirtuins (SIRTs) have been shown to mediate the response to caloric restriction, and their overexpression mimics this response in disease models (SIR2 in C. elegans
), leading to an extended life span. Furthermore, they also regulate insulin-signaling and energy metabolic pathways [40
]. In humans, SIRT1
gene expression in skeletal muscle has been shown to increase in caloric restriction with or without exercise and in vitro by adiponectin treatment [58
]. Our findings of the coordinated down-regulation of sirtuin expression in obese-human fat are, to our knowledge, first of its kind, and may be a consequence of decreased NAD synthesis and reduced energy utilization when abundant calories are available. Low NAD levels and increased NADH and nicotinamide levels could inhibit the sirtuins, thus initiating a cascade of events through numerous pathways that would (1) decrease transcription of genes for mitochondrial biogenesis and potentially reduce mtDNA copy number; and (2) decrease lipolysis through PPARs, thus promoting obesity [40
The MZ co-twin control study represents perhaps the best controlled study design available in humans because of the complete or close match for genes, age, gender, and intrauterine and childhood environment. However, when used retrospectively as done here, it does not solve the problem of direction of causation. For this reason, we include some results from one obese study participant who had gained weight upon a follow-up visit. These data, although they essentially amount to a case report, do show convincingly a further pathological progression in the same measures identified as differing between the obese and non-obese twins. Despite the limited sample size, this, in addition to the trend for increased effects in twins with greater obesity discordance, lend support to the notion that the changes identified in this limited study sample are indeed sequelae, not proximal causes of obesity.
This study provides evidence for the contribution of lifestyle to the metabolic disturbances in obesity. However, it does not devalue the effects of genes. The fact that it was extremely difficult to find young, healthy MZ twins with large weight differences (14 twin pairs out of 2,453) itself suggests a genetic basis for obesity. The large differences in BMI within these most-discordant pairs may have arisen because of the presence of “environmental sensitivity/variability” genes that make these individuals particularly susceptible to environmental variation (such as diet and exercise), whereas other pairs who were concordant for BMI may carry alleles that render them relatively impervious to dietary or physical activity patterns. While the contribution of possible epigenetic changes [59
] cannot be conclusively ruled out, they seem unlikely to explain this adult discordance, as all twins analyzed in the current study exhibited remarkably similar development of BMI until the onset of obesity in their early adulthood [23
In summary, this study identified several changes in transcript profiles of adipose tissue early in acquired obesity by using the obesity-discordant MZ co-twin control design that allows full control for genomic sequence variations, a significant confounder in most studies comparing obese and non-obese participants. Our results highlight the metabolic plasticity and alterations of human adipose tissue. Our most critical findings were that, in the fat of obese co-twins compared to their non-obese counterparts, (1) mtDNA content was reduced, (2) mitochondrial energy production pathways were down-regulated along with adipocyte differentiation within subcutaneous adipose tissue, and (3) increase in ectopic fat deposition was subsequently increased. These events probably represent important drivers of insulin resistance, hyperinsulinemia, and accumulation of liver fat, pathognomonic characteristics of obesity acquired already in the young. It is likely that proper management of obesity, primarily by lifestyle changes, and perhaps with a new generation of therapies directed at several targets in mitochondrial biogenesis pathways, will correct these abnormalities and favorably modify the risk, course, and outcome of diabetes and cardiovascular diseases.