Postabsorptive and postprandial glucose and fat metabolism in postmenopausal women with breast cancer-Preliminary data after chemotherapy compared to healthy controls.

BACKGROUND: Breast cancer survivors are a growing population due to improved treatment. It is known that postmenopausal women treated for breast cancer may experience weight gain and increased insulin resistance, but detailed knowledge on how chemotherapy impact metabolic and endocrine mechanisms remain unknown. OBJECTIVES: We performed a thorough, preliminary study to elucidate the differing mechanisms of postprandial absorption and metabolism in postmenopausal early breast cancer (EBC) patients treated with adjuvant chemotherapy compared to healthy controls. We hypothesize that chemotherapy has a negative impact on metabolism in EBC patients. METHODS: We examined four postmenopausal women shortly after treatment with chemotherapy for EBC and four age-matched healthy women who served as controls using isotopic tracers during a mixed meal-test. Blood was sampled during the 240 min meal-test to examine postprandial absorption and endogenous synthesis of lipid and carbohydrate metabolites. RESULTS: We found that insulin concentrations were numerically higher before the meal-test in the EBC patients compared to controls (76.3 pmol/L vs 37.0 pmol/L; P = 0.06). Glucose kinetics was increased postprandial (most pronounced at 30 min, 9.46 mmol/L vs 7.33 mmol/L; P = 0.51), with no difference between the groups regarding liver glucose output. Fatty acid kinetics showed a numeric increase in oleic acid rate of appearance in BC patients, but only during the first hour after the mixed meal. There was no significant difference in VLDL-TAG synthesis between the two groups. CONCLUSIONS: This preliminary study is unique in using advanced tracer methods to investigate in vivo metabolism of EBC patients after chemotherapy although no statistical differences in glucose and fatty acid kinetics was seen compared to controls. However, during the first two postprandial hours, oral glucose and oleic acid appearance in the systematic circulation was elevated in the EBC patients. This could be due to changes in gastrointestinal uptake and further studies with altered set-up could provide valuable insights.

Loss of the KN Motif and AnKyrin Repeat Domain 1 (KANK1) Leads to Lymphoid Compartment Dysregulation in Murine Model.

The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.

Peripheral blood mononuclear cells exhibit increased mitochondrial respiration after adjuvant chemo- and radiotherapy for early breast cancer.

BACKGROUND: Adjuvant chemo- and radiotherapy cause cellular damage to tumorous and healthy dividing cells. Chemotherapy has been shown to cause mitochondrial respiratory dysfunction in non-tumorous tissues, but the effects on human peripheral blood mononuclear cells (PBMCs) remain unknown. AIM: We aimed to investigate mitochondrial respiration of PBMCs before and after adjuvant chemo- and radiotherapy in postmenopausal patients with early breast cancer (EBC) and relate these to metabolic parameters of the patients. METHODS: Twenty-three postmenopausal women diagnosed with EBC were examined before and shortly after chemotherapy with (n = 18) or without (n = 5) radiotherapy. Respiration (O2 flux per million PBMCs) was assessed by high-resolution respirometry of intact and permeabilized PBMCs. Clinical metabolic characteristics and mitochondrial DNA (mtDNA) content of PBMCs (mtDN relative to nuclear DNA) were furthermore assessed. RESULTS: Respiration of intact and permeabilized PBMCs from EBC patients significantly increased with adjuvant chemo- and radiotherapy (p = 6 × 10-5 and p = 1 × 10-7 , respectively). The oxygen flux attributed to specific mitochondrial complexes and respiratory states increased by 17-43% compared to before therapy initiation. Similarly, PBMC mtDNA content increased by 40% (p = 0.002). Leukocytes (p = 0.0001), hemoglobin (p = 0.0003), and HDL cholesterol (p = 0.003) concentrations decreased whereas triglyceride (p = 0.01) and LDL (p = 0.02) concentrations increased after treatment suggesting a worsened metabolic state. None of the metabolic parameters or the mtDNA content of PBMCs correlated significantly with PBMC respiration. CONCLUSION: This study shows that mitochondrial respiration and mtDNA content in circulating PBMCs increase after adjuvant chemo- and radiotherapy in postmenopausal patients with EBC. Besides the increased mtDNA content, a shift in PBMC subpopulation proportions towards cells relying on oxidative phosphorylation, who may be less sensitive to chemotherapy, might influence the increased mitochondrial respiration observed iafter chemotherapy.

LEP promoter methylation in the initiation and progression of clonal cytopenia of undetermined significance and myelodysplastic syndrome.

BACKGROUND: Idiopathic non-clonal cytopenia (ICUS) and clonal cytopenia (CCUS) are common in the elderly population. While these entities have similar clinical presentations with peripheral blood cytopenia and less than 10% bone marrow dysplasia, their malignant potential is different and the biological relationship between these disorders and myeloid neoplasms such as myelodysplastic syndrome (MDS) is not fully understood. Aberrant DNA methylation has previously been described to play a vital role in MDS and acute myeloid leukemia (AML) pathogenesis. In addition, obesity confers a poorer prognosis in MDS with inferior overall survival and a higher rate of AML transformation. In this study, we measured DNA methylation of the promoter for the obesity-regulated gene LEP, encoding leptin, in hematopoietic cells from ICUS, CCUS and MDS patients and healthy controls. We investigated whether LEP promoter methylation is an early event in the development of myeloid neoplasms and whether it is associated with clinical outcome. RESULTS: We found that blood cells of patients with ICUS, CCUS and MDS all have a significantly hypermethylated LEP promoter compared to healthy controls and that LEP hypermethylation is associated with anemia, increased bone marrow blast percentage, and lower plasma leptin levels. MDS patients with a high LEP promoter methylation have a higher risk of progression, shorter progression-free survival, and inferior overall survival. Furthermore, LEP promoter methylation was an independent risk factor for the progression of MDS in a multivariate Cox regression analysis. CONCLUSION: In conclusion, hypermethylation of the LEP promoter is an early and frequent event in myeloid neoplasms and is associated with a worse prognosis.

Influence of the anti-oestrogens tamoxifen and letrozole on thyroid function in women with early and advanced breast cancer: A systematic review.

INTRODUCTION: Breast cancer (BC) is a common type of cancer in women. Advances in therapy options have resulted in higher overall survival rates but side effects of cancer treatment are increasingly in the spotlight. The beneficial effects of anti-oestrogen therapy with tamoxifen and letrozole in the prevention of BC recurrence are well documented. While the most common side-effects of this therapy are well-defined, less is known about its effects on thyroid function. In women treated for early BC, an average of 1-5 kg weight gain has been observed after treatment with chemotherapy/anti-oestrogens. We aim to evaluate the current knowledge on the side effects of tamoxifen and letrozole treatments on thyroid function, followed by its potential influence on the observed weight gain. METHODS: We searched PubMed and found 16 publications on thyroid function and tamoxifen treatment in pre- and post-menopausal women with early- and advanced BC, whereas five publications on letrozole treatment in post-menopausal women with advanced BC. RESULTS: According to the current literature, there is an overall tendency towards a mild and transient thyroid dysfunction, that is, subclinical hypothyroidism in tamoxifen-treated patients. Only one publication reported further significant changes in thyroid hormones beyond one year of tamoxifen treatment. No significant changes in thyroid function have been observed among letrozole-treated patients. CONCLUSION: Tamoxifen-treated patients can develop mild and transient thyroid dysfunction within the first 12 months, yet further significant changes in thyroid function beyond one year of tamoxifen treatment have been reported in a single study. There is no evidence of thyroid dysfunction in letrozole-treated patients. Current literature does not focus on subclinical hypothyroidism as a possible cause of weight gain in patients with BC. Subgrouping of BC patients and studies with a longer observation of thyroid hormones and weight changes during and after anti-oestrogen treatment are needed to further elucidate how anti-oestrogens affect thyroid function.

Independent phenotypic plasticity axes define distinct obesity sub-types.

Studies in genetically 'identical' individuals indicate that as much as 50% of complex trait variation cannot be traced to genetics or to the environment. The mechanisms that generate this 'unexplained' phenotypic variation (UPV) remain largely unknown. Here, we identify neuronatin (NNAT) as a conserved factor that buffers against UPV. We find that Nnat deficiency in isogenic mice triggers the emergence of a bi-stable polyphenism, where littermates emerge into adulthood either 'normal' or 'overgrown'. Mechanistically, this is mediated by an insulin-dependent overgrowth that arises from histone deacetylase (HDAC)-dependent ß-cell hyperproliferation. A multi-dimensional analysis of monozygotic twin discordance reveals the existence of two patterns of human UPV, one of which (Type B) phenocopies the NNAT-buffered polyphenism identified in mice. Specifically, Type-B monozygotic co-twins exhibit coordinated increases in fat and lean mass across the body; decreased NNAT expression; increased HDAC-responsive gene signatures; and clinical outcomes linked to insulinemia. Critically, the Type-B UPV signature stratifies both childhood and adult cohorts into four metabolic states, including two phenotypically and molecularly distinct types of obesity.

TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis.

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.

The role of vitamin C in epigenetic cancer therapy.

The role of vitamin C in the treatment of cancer has been subject to controversy for decades. Within the past 10 years, mechanistic insight into the importance of vitamin C in epigenetic regulation has provided a new rationale for its potential anti-cancer effects. At physiological concentrations, vitamin C is a potent antioxidant and thereby co-factor for a range of enzymes including the Fe(II)- and α-ketoglutarate-dependent dioxygenases that represent some of the most important epigenetic regulators; the ten-eleven translocation (TET) methylcytosine dioxygenases and the Jumonji-C domain-containing histone demethylases. Epigenetic deregulation is a hallmark of many cancers and reduced activity of these enzymes or somatic loss-of-function mutations in the genes encoding them, are observed in many cancer types. The present review outlines the growing literature on the role of vitamin C in epigenetic therapy of cancer. In the vast majority of in vitro, animal and clinical studies included in this review, vitamin C showed ability across cancer types to increase the hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine catalyzed by the TET enzymes - the first step in DNA demethylation. Most consistently, vitamin C in combination with the class of epigenetic drugs, DNA methyltransferase inhibitors, has demonstrated efficacy in the treatment of hematological malignancies in both preclinical and the limited number of available clinical studies. Yet, the pertinent question of what is the optimal dose of vitamin C in cancer studies remains to be answered. High-quality randomized placebo-controlled trials are needed to determine whether supplementation with vitamin C may benefit subgroups of patients with (pre-)cancer.

Activation of a Subset of Evolutionarily Young Transposable Elements and Innate Immunity Are Linked to Clinical Responses to 5-Azacytidine.

The DNA methyltransferase inhibitors (DNMTi) 5-azacytidine and 5-aza-2-deoxycytidine have been approved for the treatment of different types of hematologic malignancies. However, only about 50% of patients respond to treatment. Therefore, a more comprehensive understanding of the molecular changes in patients treated with DNMTi is needed. Here, we examined gene expression profiles in a total of 150 RNA samples from two adult cohorts and one pediatric cohort with hematologic cancers taken before, during, and after treatment with 5-azacytidine (40 patients; 15 nonresponders, 25 responders). Using each patient as their own control, malignant cells showed preferential activation of a subset of evolutionarily young transposable elements (TE), including endogenous retroviral long terminal repeats (LTR), short and long interspersed nuclear elements (SINE and LINE), and the type I IFN pathway in responders, all independent of disease classification. Transfection of eight upregulated LTRs into recipient human cells in culture showed robust and heterogenous activation of six genes in the type I IFN pathway. These results, obtained in diverse hematologic disease entities, show that common targets (TE) activated by the same drug (5-azacytidine) elicit an immune response, which may be important for patient's responses to DNMTi. SIGNIFICANCE: Activation of specific classes of evolutionarily young transposable elements can lead to activation of the innate immune system.

Angiotensinogen promoter methylation predicts bevacizumab treatment response of patients with recurrent glioblastoma.

Patients with recurrent glioblastoma achieving response to bevacizumab combined with chemotherapy have clinical improvement and prolonged survival. High gene expression of angiotensinogen (AGT) is associated with a poor bevacizumab response. Because AGT expression is epigenetically regulated, we aimed to investigate whether AGT promoter methylation in tumor tissue predicts response to bevacizumab combination therapy in patients with recurrent glioblastoma. The study included 159 patients with recurrent glioblastoma, treated with bevacizumab combination treatment (training cohort, n = 77; validation cohort, n = 82). All patients could be evaluated for treatment response and biomarkers. DNA methylation of 4 CpG sites in the AGT promoter was measured using pyrosequencing. A model for nonresponse was established using logistic regression analysis. In the training cohort, lower methylation of each of the four CpG sites in the AGT promoter was significantly associated with nonresponse (all P < 0.05). Moreover, the mean methylation level of all four CpG sites was associated with an increased likelihood of not achieving response to bevacizumab combination therapy (twofold decrease: odds ratio = 3.01; 95% confidence interval: 1.41-6.44; P = 0.004). We developed a model for nonresponse in the training cohort, where a threshold of mean AGT promoter methylation levels was set to below 12%. The model could predict bevacizumab nonresponse with 96% specificity. Importantly, this predictor was also significantly associated with nonresponse in the validation cohort (P = 0.037). Taken together, our findings suggest that low AGT promoter methylation in tumor tissue predicts nonresponse to bevacizumab combination treatment in patients with recurrent glioblastoma. We have, thus, established and successfully validated a predictor for nonresponse that can be used to identify patients who will not benefit from bevacizumab combination therapy.

Oral vitamin C supplementation to patients with myeloid cancer on azacitidine treatment: Normalization of plasma vitamin C induces epigenetic changes.

BACKGROUND: Patients with haematological malignancies are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), the first step in active DNA demethylation. Here, we investigate whether oral vitamin C supplementation can correct vitamin C deficiency and affect the 5hmC/5mC ratio in patients with myeloid cancers treated with DNA methyltransferase inhibitors (DNMTis). RESULTS: We conducted a randomized, double-blinded, placebo-controlled pilot trial (NCT02877277) in Danish patients with myeloid cancers performed during 3 cycles of DNMTi-treatment (5-azacytidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n = 10) or placebo (n = 10) daily during the last 2 cycles. Fourteen patients (70%) were deficient in plasma vitamin C (< 23 µM) and four of the remaining six patients were taking vitamin supplements at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (< 11.4 µM; 4.997 vs 4.656% 5mC relative to deoxyguanosine, 95% CI [0.126, 0.556], P = 0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (mean increase 34.85 ± 7.94 µM, P = 0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in mononuclear myeloid cells from patients receiving oral vitamin C compared to placebo (0.037% vs - 0.029%, 95% CI [- 0.129, - 0.003], P = 0.041). CONCLUSIONS: Normalization of plasma vitamin C by oral supplementation leads to an increase in the 5hmC/5mC ratio compared to placebo-treated patients and may enhance the biological effects of DNMTis. The clinical efficacy of oral vitamin C supplementation to DNMTis should be investigated in a large randomized, placebo-controlled clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02877277 . Registered on 9 August 2016, retrospectively registered.

Vitamin C - A new player in regulation of the cancer epigenome.

Over the past few years it has become clear that vitamin C, as a provider of reduced iron, is an essential factor for the function of epigenetic regulators that initiate the demethylation of DNA and histones. Vitamin C deficiency is rare in the general population, but is frequently observed in patients with cancer. Genes encoding epigenetic regulators are often mutated in cancer, underscoring their central roles in carcinogenesis. In hematological cancers, such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), drugs that reverse epigenetic aberrations are now the standard of care. Recent in vitro studies suggest that vitamin C at physiological concentrations, combined with hypomethylating agents may act synergistically to cause DNA demethylation through active and passive mechanisms, respectively. Additionally, several recent studies have renewed interest in the use of pharmacological doses of vitamin C injected intravenously to selectively kill tumor cells. This review will focus on the potential of vitamin C to optimize the outcome of epigenetic therapy in cancer patients and alternatively to act as a therapeutic at high doses.

Impact of polyunsaturated and saturated fat overfeeding on the DNA-methylation pattern in human adipose tissue: a randomized controlled trial.

Background: Dietary fat composition can affect ectopic lipid accumulation and, thereby, insulin resistance. Diets that are high in saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) have different metabolic responses.Objective: We investigated whether the epigenome of human adipose tissue is affected differently by dietary fat composition and general overfeeding in a randomized trial.Design: We studied the effects of 7 wk of excessive SFA (n = 17) or PUFA (n = 14) intake (+750 kcal/d) on the DNA methylation of ∼450,000 sites in human subcutaneous adipose tissue. Both diets resulted in similar body weight increases. We also combined the data from the 2 groups to examine the overall effect of overfeeding on the DNA methylation in adipose tissue.Results: The DNA methylation of 4875 Cytosine-phosphate-guanine (CpG) sites was affected differently between the 2 diets. Furthermore, both the SFA and PUFA diets increased the mean degree of DNA methylation in adipose tissue, particularly in promoter regions. However, although the mean methylation was changed in 1797 genes [e.g., alpha-ketoglutarate dependent dioxygenase (FTO), interleukin 6 (IL6), insulin receptor (INSR), neuronal growth regulator 1 (NEGR1), and proopiomelanocortin (POMC)] by PUFAs, only 125 genes [e.g., adiponectin, C1Q and collagen domain containing (ADIPOQ)] were changed by SFA overfeeding. In addition, the SFA diet significantly altered the expression of 28 transcripts [e.g., acyl-CoA oxidase 1 (ACOX1) and FAT atypical cadherin 1 (FAT1)], whereas the PUFA diet did not significantly affect gene expression. When the data from the 2 diet groups were combined, the mean methylation of 1444 genes, including fatty acid binding protein 1 (FABP1), fatty acid binding protein 2 (FABP2), melanocortin 2 receptor (MC2R), MC3R, PPARG coactivator 1 α (PPARGC1A), and tumor necrosis factor (TNF), was changed in adipose tissue by overfeeding. Moreover, the baseline DNA methylation of 12 CpG sites that was annotated to 9 genes [e.g., mitogen-activated protein kinase 7 (MAPK7), melanin concentrating hormone receptor 1 (MCHR1), and splicing factor SWAP homolog (SFRS8)] was associated with the degree of weight increase in response to extra energy intake.Conclusions: SFA overfeeding and PUFA overfeeding induce distinct epigenetic changes in human adipose tissue. In addition, we present data that suggest that baseline DNA methylation can predict weight increase in response to overfeeding in humans. This trial was registered at clinicaltrials.gov as NCT01427140.

Metabolic and Transcriptional Changes in Cultured Muscle Stem Cells from Low Birth Weight Subjects.

CONTEXT/OBJECTIVE: Developmental programming of human muscle stem cells could in part explain why individuals born with low birth weight (LBW) have an increased risk of developing type 2 diabetes (T2D) later in life. We hypothesized that immature muscle stem cell functions including abnormal differentiation potential and metabolic function could link LBW with the risk of developing T2D. Design/Settings/Participants: We recruited 23 young men with LBW and 16 age-matched control subjects with normal birth weight. Biopsies were obtained from vastus lateralis, and muscle stem cells were isolated and cultured into fully differentiated myotubes. MAIN OUTCOME MEASURES: We studied glucose uptake, glucose transporters, insulin signaling, key transcriptional markers of myotube maturity, selected site-specific DNA methylation, and mitochondrial gene expression. RESULTS: We found reduced glucose uptake as well as decreased levels of glucose transporter-1 and -4 mRNA and of the Akt substrate of 160-kDa mRNA and protein in myotubes from LBW individuals compared with normal birth weight individuals. The myogenic differentiation markers, myogenin and myosin heavy chain 1 and 2, were decreased during late differentiation in LBW myotubes. Additionally, mRNA levels of the peroxisome proliferator-activated receptor-γ coactivator-1α and cytochrome c oxidase polypeptide 7A were reduced in LBW myotubes. Decreased gene expression was not explained by changes in DNA methylation levels. CONCLUSION: We demonstrate transcriptional and metabolic alterations in cultured primary satellite cells isolated from LBW individuals after several cell divisions, pointing toward a retained intrinsic defect conserved in these myotubes.

Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood.

Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic biomarkers of aging in blood, including ELOVL2, FHL2, KLF14 and GLRA1, also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2. We identified 2825 genes (e.g. FTO, ITIH5, CCL18, MTCH2, IRS1 and SPP1) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28-46 mmol/mol) correlated significantly with the methylation of 711 sites, annotated to, for example, RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue.

Impaired leptin gene expression and release in cultured preadipocytes isolated from individuals born with low birth weight.

Low birth weight (LBW) is associated with increased risk of the development of type 2 diabetes (T2D). The appetite-regulating hormone leptin is released from mature adipocytes, and its production may be decreased in immature preadipocytes from LBW individuals. We recruited 14 men born with LBW and 13 controls born with normal birth weight (NBW). Biopsy samples were obtained from subcutaneous abdominal fat depots, and preadipocytes were isolated and cultured. Gene expression of leptin and selected differentiation markers were analyzed during preadipocyte differentiation, and cell culture media were collected to analyze leptin secretion. DNA methylation of CpG sites in the leptin promoter was measured using pyrosequencing. We found that differentiating preadipocytes from LBW individuals showed reduced leptin gene expression and a corresponding reduced leptin release compared with NBW individuals. Mean DNA methylation of the proximal LEP promoter was increased in LBW compared with NBW individuals. The notion of impaired adipocyte maturation in LBW individuals was supported by a lower mRNA expression of the differentiation markers; fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, and GLUT4. Our findings are consistent with impaired preadipocyte maturation, contributing to an increased risk of the development of T2D in LBW subjects.