Objective: Several studies have shown that mitochondrial metabolism may be disrupted if the rate of the specific 4,977 bp deletion of mitochondrial DNA (mtDNA) reaches a threshold. This study aimed to investigate the possible associations between the mtDNA4977 deletion load and obesity-related metabolic abnormalities in the adipose tissue. Methods: The study included thirty obese individuals, who underwent bariatric surgery, and twelve control subjects. mtDNA4977 deletion, adenine nucleotides, and lactate levels, which show the bioenergetic status were evaluated in visceral adipose tissues. Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural variations and composition of adipose tissues in the context of deletion load. Results: There were no differences between the two groups in terms of mtDNA4977 deletion, adenine nucleotides, and lactate levels. The FTIR spectra indicated a few obesity-related alterations in adipose tissues that were not related to the mtDNA deletion load. Also, statistical analysis showed a correlation between the deletion load and a band shift of 1,744 cm−1, which assigns C = O stretching of the carbonyl group of the ester group in triglycerides and other esterified fatty acids, although it is not associated with obesity. Conclusions: Our data suggest that the mtDNA4977 deletion in visceral adipose tissues of obese individuals do not have a significant impact on the bioenergetic status. However, the increased accumulation of deletion may be associated with a specific change in the ester bond, indicating structural differences in the lipids. These findings shed light on our understanding of the tissue-specific distribution of mtDNA deletions and obesity-related adipose tissue pathogeneses.

Highlights of the Study

  • The adipose tissue of obese nondiabetic subjects is not prone to accumulation of mitochondrial DNA common deletion and change of the bioenergetic status compared to control subjects.

  • Fourier transform infrared spectroscopy revealed a connection between increased accumulation of deletion and changes in lipid composition.

  • Increased accumulation of mitochondrial DNA deletion might be associated with a specific change in the ester bond of triglycerides in visceral fat tissues.

It has been documented that adipose tissues overloaded with lipids can secrete a number of molecules that create a pro-inflammatory environment, which may disrupt metabolism and affect many organs due to the production of reactive oxygen species (ROS) and create oxidative stress in the body, particularly in (fat cells) adipocytes [1]. Mitochondria play major roles in energy metabolism. Oxidative stress caused by ROS may cause a loss of function by initiating several changes in the mitochondria [2]. Obese people have a higher fat content, and their body mass index (BMI) is in the normal range. Excessive nutrients may disrupt the mitochondrial respiratory chain and the Krebs cycle, which trigger even more ROS damage. The human body has two types of adipose tissues, white and brown adipose tissue (WAT and BAT, respectively). Although the number of mitochondria in WAT is far fewer than those in BAT, they are extremely crucial in maintaining the adipocyte functions such as maturation and differentiation [3]. While the preadipocytes undergo the differentiation process to form the mature adipocytes, the mitochondria must both carry out the routine functions of the cell and sustain ATP production to fulfill the energy needs of lipogenesis [2, 3]. In addition, mitochondria in adipose tissues have adipocyte-specific functions and are intimately integrated with critical adipocyte biology, including adipogenesis, lipid metabolism, and thermogenesis. Recent studies suggest that they may play substantial roles in regulating whole-body energy, insulin sensitivity, and glucose metabolism [4]. Therefore, any disruption in the mitochondrial function can spoil the function and structure of fat tissues. Indeed, it has been shown that the loss of function of the mitochondria leads to defective oxidation of fatty acids, uncontrolled secretion of adipokines, and broken glucose balance. In addition, oxidative phosphorylation and oxidation in WAT can be affected by changes in the number, mass, and activity of the mitochondria, as well as alterations in the copy numbers of mitochondrial DNA (mtDNA) [3, 5]. Therefore, mitochondrial dysfunction has been suggested to play a role in the development of obesity and comorbidities such as type 2 diabetes [5]. Whether altered mitochondrial function plays a causative or adaptive role in obesity, as well as various metabolic disorders, and the important factors contributing to mitochondrial dysfunction in adipose tissue remain unclear.

As the mitochondrion has a limited ability to repair its DNA, oxidative stress can inflict considerable damage to mtDNA, and the newly formed deletions and mutations can accumulate and cause diseases originating in the mitochondria [6]. Of these, a large deletion between ND5 and ATPse8 genes that spans almost a 5 kb region is among the most investigated deletions (mtDNA4977). This large deletion eliminates five tRNA genes; four complex I subunit genes (ND3, ND4, ND4L, and some part of ND5); one complex IV subunit (COXIII); and two complex V subunits (ATPases 6 and 8) [6, 7]. These large deletions may break the respiratory chain that keeps a delicate balance. Large and point mutations may result in the development of metabolic and neurodegenerative diseases and even cancer [8].

Adipose tissue has its own microenvironment, and metabolic events such as oxidative stress and inflammation are more frequent in obese people, which may trigger mitochondrial dysfunction. In light of the given information, one may expect that mtDNA deletions over tolerable limits may bring cellular homeostasis to a halt, and this disrupted homeostasis can cause the accumulation of mtDNA deletions, eventually creating a vicious circle. Considering that mtDNA4977 deletion may cause some changes in cell metabolism such as energy production and lipid synthesis, and because a high-fat diet may disturb the mitochondrial redox balance [9], one can suggest that these changes may disrupt the biomolecular structure and composition of adipose tissues. However, this suggestion has not been confirmed. In addition, it is uncertain whether obesity definitely causes mtDNA4977 deletion in adipocytes. Therefore, we aimed to determine the common mitochondrial deletion in obese visceral adipose tissues in terms of mitochondrial function and tissue-specific composition and structure of macromolecules by using Fourier transform infrared (FTIR) spectroscopy.

Case Selection

This study enrolled thirty morbidly obese and twelve healthy subjects who attended the General Surgery Department in the Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa. Abdominal visceral adipose tissue was obtained from subjects who were undergoing laparoscopic gastric bypass for the treatment of obesity (BMI >35 kg/m2). In addition, visceral adipose tissue samples were taken from normal-weight individuals (BMI <25 kg/m2) who had undergone laparoscopic cholecystectomy (n = 5) and hiatal hernia repair (n = 7) (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000535443). None of the participants had cardiovascular disease or any active inflammatory disease; they had not taken any therapy that might affect lipid metabolism and had not participated in any weight loss program within the past 6 months. The Local Ethics Committee gave its approval for this study, which was carried out in conformity with the Helsinki Declaration. All individuals taking part in the study gave their informed consent. Every technique was used in compliance with the rules and regulations that were applicable.

Extraction of DNA from Adipose Tissues

DNA from adipose tissues was extracted using a DNA isolation kit (Roche, High Pure PCR Template Preparation Kit, Mannheim, Germany). The samples were kept at −80°C until further testing and DNA concentration was measured using a NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Analysis of the Extent of the mtDNA4977 bp Deletion by Polymerase Chain Reaction

Quantitative polymerase chain reaction (qPCR) was used to evaluate the amount of mitochondrial reference fragment and mtDNA4977 deletion in tissue sample-derived mtDNA, with a few changes from the methods previously described [10, 11]. In amplifying DNA fragments by PCR, the primers representing deletion were chosen as shown in online supplementary Figure 1, specifically targeting the edge of the deletion. These primers only bind to mtDNA that has undergone the deletion, whereas the primers representing the normal mtDNA were selected in the mutation-free D-loop region. We employed the following primer sets: the forward and reverse bases for normal mtDNA (internal control, mtDNAn) are 5′-AAC​ATA​CCC​ATG​GCC​AAC-3′ and 5′-TCA​GCG​AAG​GGT​TGT​AGT​AGC-3′, respectively. The forward and reverse bases for mtDNA4977 deletion are 5′-TAT​GGC​CCA​CCA​TAA​TTA​CCC-3′ and 5′-AAG​CGA​GGT​TGA​CCT​GTT​AGG 3′. Following the manufacturer’s instructions, purified PCR products were cloned into the pGEM-T Easy vector (Promega Corporation, Madison, WI, USA). For qPCR optimization and sensitivity analysis, dose-dependent plasmid-constructed (mtDNAn and mtDNA4977) standards were employed in PCR experiments. The ABI Prism 3130XL Genetic Analyzer and the BigDye Terminator v3.1 Ready Reaction Kit (Applied Biosystems; Thermo Fisher Scientific, MA, USA) were used to confirm the plasmids using the Sanger sequencing method. Using the EvaGreen 2X qPCR MasterMix-ROX from Abm (Canada), DNA samples were run in qPCR using the CFX ConnectTM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) at 94°C for 15 s and 60°C for 1 min, for a total of 40 cycles. The PCR was performed using a 20 ng DNA template and a 10 nmol/L concentration of each primer, in a total volume of 20 µL.

The mtDNA content and deletion load in samples were determined using the average quantitation cycle values. The relevant plasmid clones were serially diluted at a known concentration and used for standardization of each measurement, which was carried out two or three times. The amount of each target gene in the samples was then determined using the associated standard curve. The mtDNA and nuclear DNA copy numbers were determined using the formula provided by the Genomics and Sequencing Center of the University of Rhode Island (Kingston, RI, USA; cels.uri.edu/gsc/cndna.html). Number of copies = (amount of DNA × 6.022 × 1023)/(length of amplicon × 1 × 109 × 650) was the formula employed. The percentage ratio of the deleted mtDNA copy number to the total mtDNA copy number was used to indicate the amount of mtDNA4977 deletion.

Measurement of ATP and ATP Production Potential

To determine the level of ATP activity in the tissue samples, we used the ATP Bioluminescence Assay Kit CLS II (Roche). The manufacturer’s recommendations were followed. The modified Lowry method [12] was employed to determine the protein concentration in the samples. The inclusion of 2.5 mM ADP to the ATP-measuring procedure allowed for the determination of ATP production potential.

Measurement of Lactate

Using the L-Lactate Colorimetric Assay Kit and adhering to the manufacturer’s instructions, measurements of adipose tissue lactate were carried out (AAT Bioquest, Sunnyvale, CA, USA). The modified Lowry method [12] was used to calculate the protein concentrations in the samples, and the lactate content was represented as nmol/mg protein.

ATR-FTIR Spectroscopy

Infrared spectra of adipose tissues were obtained using the ZnSe ATR crystal (Perkin Elmer Inc., Norwalk, CT, USA). Samples were dried onto the crystal with a gentle stream of dry nitrogen gas for 5 min. Air was taken as a reference for the background spectrum before each sample. Background and sample measurements were taken at room temperature (21–23°C) with a spectral resolution of 4 cm−1 in the range of 4,000 and 650 cm−1. Each sample was scanned thrice and the average values were recorded. The data obtained were analyzed using the OPUS program. The second derivative process was applied to the mean band data of the tissue samples to minimize baseline variability and background effects in the spectra. Apart from the second derivative process, vector normalization was used to eliminate the differences that may arise from sample thickness variations, which ensured that the variability in absorbance values was almost the same, and to minimize experimental errors that can be affected by the variation in sample and test conditions. Band intensities were measured in the regions of lipid and protein bands, within the spectral range of 4,000–650 cm−1, and band positions and shifts were compared. Lipid/protein ratio, lipid peroxidation, and saturated-unsaturated lipid ratio were evaluated except for band intensities in FTIR spectroscopy. Changes in lipid and protein metabolism due to the changing macromolecular structure were determined using these parameters.

Statistical Analysis

The results are expressed as mean ± standard deviation. The differences were analyzed statistically using Student t, Mann-Whitney U, χ2, and multivariate analysis of variance (MANOVA) tests. The relationship between the measurement values was determined by Spearman correlation analysis. GraphPad Prism 5.0 (GraphPad, San Diego, CA) and Statistical Package for SocialSciences version 21 (IBM SPSS, Armonk, NY, USA) were used for calculations. A post-hoc power analysis (G*Power software, package version 3.1.4) was also performed to determine the power of the study on deletion load data in the adipose tissue as no prior studies exist to inform a pre-hoc power analysis. A p value less than or equal to 0.05 was considered statistically significant.

Demographic Data

Age, sex, and BMI values were used for clinical parameters in patient and healthy subject groups. The mean age and BMI values of the obese and control groups were 53.08 ± 8.18 and 51.14 ± 7.74 years and 39.76 ± 3.73 and 21.47 ± 2.68 kg/m2, respectively. Male/female distribution was 56.66/43.34% and 41.66/58.34% for obese and control groups, respectively. There were no significant differences in terms of age and gender between the patient and control groups (p > 0.05) (online suppl. Table 1).

MtDNA4977 Deletion Load and Bioenergetic Status of the Adipose Tissue

Adipose tissue mtDNA function parameter results are presented in Table 1. There were no significant changes in mtDNA4977 deletion (%), ATP, ATP production potential, and lactate values between obese and control groups.

Table 1.

mtDNA function parameter results of the obese and control groups

ParametersObese group (N = 30)Control group (N = 12)p value
mtDNA 4,977 deletion, % 53.02±25.76 60.68±20.26 0.496 
ATP, pmol/g protein 22.23±37.34 8.75±3.15 0.992 
ATP production, nmol/g protein 40.71±43.89 20.66±16.56 0.248 
Lactate, nmol/mg protein 86.37±46.75 285.00±237.60 0.063 
ParametersObese group (N = 30)Control group (N = 12)p value
mtDNA 4,977 deletion, % 53.02±25.76 60.68±20.26 0.496 
ATP, pmol/g protein 22.23±37.34 8.75±3.15 0.992 
ATP production, nmol/g protein 40.71±43.89 20.66±16.56 0.248 
Lactate, nmol/mg protein 86.37±46.75 285.00±237.60 0.063 

The results are expressed as mean ± standard deviation.

Moreover, the effect of age, gender, and BMI on mtDNA deletion load was evaluated using the MANOVA test, which did not reveal any significant effect. When post-hoc power analysis was performed based on the deletion load data in the adipose tissue for all obese and control subjects, the obese subjects with high deletion and all control subjects, and the obese subjects with high and low deletion, statistical powers (1-β) were obtained 24, 83, and 99%, respectively.

FTIR Results of Adipose Tissues

In our study, the FTIR spectra, which aimed to determine obesity-related alterations and the effects of mtDNA mutation load on the concentration and composition of biomolecules, in the adipose tissues were evaluated in the 4,000–650 cm−1 region. In order to obtain more homogeneous data in FTIR results, taking into account the 50% cut-off value, two subgroups were created as high and low mtDNA deletion load of obese patients, and they were compared with the control group. The wavenumber and detailed spectral band assignments of adipose tissues are given in online supplementary Table 2. Additionally, FTIR band area values of ten spectral bands were evaluated in the analysis: unsaturated lipid (3,017–3,000 cm−1), lipid CH3 antisymmetric (2,975–2,952 cm−1), lipid CH2 antisymmetric (2,943–2,910 cm−1), lipid CH3 symmetric (2,877–2,868 cm−1), lipid CH2 symmetric (2,865–2,841 cm−1), ester (1,765–1,731 cm−1), amid-I (1,700–1,592 cm−1), amid-II (1,577–1,498 cm−1), lipid-I (1,478–1,435 cm−1), and lipid-II (1,181–1,131 cm−1), and are shown in Table 2.

Table 2.

General band areas of the FTIR spectrum in adipose tissues in the obese and control groups

ParametersObese group (N = 12)Control group (N = 12)p value
Unsaturated lipid A3017-3000 0.121±0.077 0.210±0.150 0.213 
Lipid CH3 antisymmetric A2975-2952 0.326±0.097 0.358±0.163 0.173 
Lipid CH2 antisymmetric A2943-2910 4.771±1.856 5.905±3.427 0.678 
Lipid CH3 symmetric A2877-2868 0.017±0.008 0.012±0.008 0.279 
Lipid CH2 symmetric A2865-2841 2.767±1.232 3.452±2.009 0.815 
Ester A1765-1731 4.829±2.671 6.636±3.250 0.249 
Amid-1 A1700-1592 78.146±2.464 75.207±3.693 0.032 
Amid-2 A1577-1498 24.687±4.224 22.437±5.033 0.365 
Lipid-1 A1478-1435 6.719±1.106 6.899±1.824 0.849 
Lipid-2 A1181-1131 3.608±1.886 4.966±2.169 0.179 
ParametersObese group (N = 12)Control group (N = 12)p value
Unsaturated lipid A3017-3000 0.121±0.077 0.210±0.150 0.213 
Lipid CH3 antisymmetric A2975-2952 0.326±0.097 0.358±0.163 0.173 
Lipid CH2 antisymmetric A2943-2910 4.771±1.856 5.905±3.427 0.678 
Lipid CH3 symmetric A2877-2868 0.017±0.008 0.012±0.008 0.279 
Lipid CH2 symmetric A2865-2841 2.767±1.232 3.452±2.009 0.815 
Ester A1765-1731 4.829±2.671 6.636±3.250 0.249 
Amid-1 A1700-1592 78.146±2.464 75.207±3.693 0.032 
Amid-2 A1577-1498 24.687±4.224 22.437±5.033 0.365 
Lipid-1 A1478-1435 6.719±1.106 6.899±1.824 0.849 
Lipid-2 A1181-1131 3.608±1.886 4.966±2.169 0.179 

The results are expressed as mean ± standard deviation. A: band area, “A” is the area obtained by “method B” integration bands in OPUS. The min-max normalized data were used for area calculation.

The nine spectra of the obese and control groups were quite similar. However, the area of the amide-I band, which gives information about the total protein concentration and conformation, showed a significant increase in the obese group compared to the control group (p = 0.032) (Table 2). The area of comparison of the FTIR spectrum according to the mtDNA4977 deletion load did not reveal any significant differences between the patients with high and low mtDNA deletion levels, as well as those with high deletion mtDNA levels and control subjects (online suppl. Tables 3, 4).

In order to obtain information about the structure of adipose tissue, the total fat and protein contents were also examined by analyzing the alterations in various ratio values presented in online supplementary Table 5. As shown in online supplementary Figure 2, 3, there was no significant difference between the evaluated compositional spectral parameters, such as the total lipid and protein contents, lipid/protein ratio, aliphatic chain length, triglyceride-cholesterol ester amount, protein structural and conformational changes, and protein concentration values in the obese and control groups. In addition, the evaluation of these spectral parameters according to the mtDNA4977 deletion percentage did not show a significant difference between the high deletion and low deletion obese groups, as well as the high deletion and control groups.

The examination of the band shifts of the FTIR spectrum showed that there was a significant shift in lipid CH2 antisymmetric and lipid bands in the obese group compared to the control group (p = 0.037 and p = 0.020, respectively). The shifts in other bands were not significant (Table 3). The comparison of band shifts according to the mtDNA4977 deletion load showed statistically nonsignificant alterations between the patients with high and low levels of mtDNA4977 deletion, and the patients with high mtDNA4977 deletion levels and control subjects (online suppl. Tables 6, 7). Also, MANOVA test was performed to analyze the data for verification of the influence of the set of variables in relation to the outcome. There was no significant difference (p = 0.156) for Wilks’ Lambda of MANOVA among all molecular subtypes and controls in all studied FTIR spectrum data.

Table 3.

Shifting of the FTIR band in obese and control groups

ParametersObese group (N = 12)Control group (N = 12)p value
Unsaturated lipid band 3,011.500±3.177 3,008.667±0.516 0.057 
Lipid CH3 antisymmetric band 2,927.083±6.230 2,922.833±0.408 0.253 
Lipid CH2 antisymmetric band 2,962.250±4.288 2,958.000±0.000 0.037 
Lipid CH3 symmetric band 2,876.333±2.462 2,874.000±0.000 0.085 
Lipid CH2 symmetric band 2,856.500±4.079 2,853.000±0.000 0.126 
Ester band 1,744.083±0.900 1,744.333±0.516 0.591 
Amid-1 band 1,635.167±3.689 1,636.333±3.669 0.409 
Amid-2 band 1,551.917±2.314 1,551.167±3.125 0.722 
Lipid band 1,163.250±4.048 1,161.000±1.095 0.020 
ParametersObese group (N = 12)Control group (N = 12)p value
Unsaturated lipid band 3,011.500±3.177 3,008.667±0.516 0.057 
Lipid CH3 antisymmetric band 2,927.083±6.230 2,922.833±0.408 0.253 
Lipid CH2 antisymmetric band 2,962.250±4.288 2,958.000±0.000 0.037 
Lipid CH3 symmetric band 2,876.333±2.462 2,874.000±0.000 0.085 
Lipid CH2 symmetric band 2,856.500±4.079 2,853.000±0.000 0.126 
Ester band 1,744.083±0.900 1,744.333±0.516 0.591 
Amid-1 band 1,635.167±3.689 1,636.333±3.669 0.409 
Amid-2 band 1,551.917±2.314 1,551.167±3.125 0.722 
Lipid band 1,163.250±4.048 1,161.000±1.095 0.020 

The results were expressed as mean ± standard deviation.

Associations between mtDNA4977 Deletion and FTIR Parameters

Subsequent correlation analyses of the mtDNA deletion load and FTIR values showed a negative correlation between mtDNA4977 deletion level percentage and shift of the ester band in both obese and control groups (p < 0.001, r = −0.486 and p < 0.0001 r = −0.828, respectively). Additionally, there were positive correlations between mtDNA4977 deletion level and shift of the amid-I and shift of the lipid CH2 antisymmetric band (p = 0.016, r = 0.943 and p < 0.0001, r = 0.926, respectively) in the obese group with high deletion levels, even though there was a negative correlation between mtDNA4977 deletion (%) and shift of the lipid band (p < 0.0001, r = −0.778) in this group. On the other hand, there were negative correlations between the deletion frequency and shift of the lipid CH3 antisymmetric band, shift of the lipid CH2 antisymmetric band, and shift of the lipid CH3 symmetric band (p < 0.0001, r = −0.845; p < 0.0001, r = −0.971; and p < 0.0001, r = −0.926, respectively) in obese group with low deletion levels. However, simple correlation analyses also demonstrated that there was no significant relationship between the accumulation of mtDNA4977 and other evaluated FTIR values (Table 4).

Table 4.

Significant correlation values in obese and control groups

ParametersCorrelations
Obese group (N = 12) 
 mtDNA 4,977 deletion (%) versus shift of the ester band r = −0.486; p = 0.0015 
Obese group with high deletion levels (N = 6) 
 Deletion (%) versus shift of the amid-I band r = 0.943; p = 0.0167 
 Deletion (%) versus shift of the lipid CH2 antisymmetric band r = 0.926; p < 0.0001 
 Deletion (%) versus shift of the lipid band r = −0.778; p < 0.0001 
Obese group with low deletion levels (N = 6) 
 Deletion (%) versus shift of the lipid CH3 antisymmetric band r = −0.845; p < 0.0001 
 Deletion (%) versus shift of the lipid CH2 antisymmetric band r = −0.971; p < 0.0001 
 Deletion (%) versus shift of the lipid CH3 symmetric band r = −0.926; p < 0.0001 
Control group (n = 12) 
 mtDNA 4,977 deletion (%) versus shift of the ester band r = −0.828; p < 0.0001 
ParametersCorrelations
Obese group (N = 12) 
 mtDNA 4,977 deletion (%) versus shift of the ester band r = −0.486; p = 0.0015 
Obese group with high deletion levels (N = 6) 
 Deletion (%) versus shift of the amid-I band r = 0.943; p = 0.0167 
 Deletion (%) versus shift of the lipid CH2 antisymmetric band r = 0.926; p < 0.0001 
 Deletion (%) versus shift of the lipid band r = −0.778; p < 0.0001 
Obese group with low deletion levels (N = 6) 
 Deletion (%) versus shift of the lipid CH3 antisymmetric band r = −0.845; p < 0.0001 
 Deletion (%) versus shift of the lipid CH2 antisymmetric band r = −0.971; p < 0.0001 
 Deletion (%) versus shift of the lipid CH3 symmetric band r = −0.926; p < 0.0001 
Control group (n = 12) 
 mtDNA 4,977 deletion (%) versus shift of the ester band r = −0.828; p < 0.0001 

There is much evidence that various genetic abnormalities cause metabolic disorders associated with abnormalities of lipid metabolism and storage and that some cases are associated with abnormal mtDNA and systemic mitochondrial dysfunction [13, 14]. However, whether mitochondrial dysfunction is a cause or consequence of metabolic disorders is the chicken-or-egg causality dilemma. The heteroplasmy is not sufficient to explain the numerous cases in which there is no correlation between the percentages of mutant mtDNA and phenotype. Nevertheless, according to some studies, the A to G transition at nucleotide 3,243 in the mitochondrial tRNA Leu-(UUR) gene results in intracellular triglyceride droplets and reduced beta-oxidation [15], which may increase the redistribution of body fat to lipomas [16].

In addition, mtDNA4977 deletion was observed in 25% of muscle biopsy samples from a patient with multiple symmetric lipomatosis. The most frequent large loss in the mitochondrial genome is this one. In addition, recent research supports the idea that this deletion increases with age, specifically in post-mitotic tissues that are engaged in oxidative metabolism, such as the skeletal muscle, lung, colon, and brain [17‒19]. The ATP/ADP ratio of related cells is thought to decrease if the ratio of deletions to wild-type mtDNA for a cell or tissue surpasses a certain threshold. This is probably due to a decrease in mitochondrial ATP synthesis. A higher percentage of common mtDNA deletion is linked to issues with mitochondria, including lactic acidosis and a general decrease in energy supply [19]. Studies on obese patients have shown that adipose tissue is an active region where lactate production is increased [20, 21]. With regard to obesity-induced changes in the adipose tissue microenvironment, such as inflammation and oxidative stress, which can lead to mitochondrial dysfunction, the accumulation of mtDNA deletions in the tissue may disrupt metabolic homeostasis and vice versa. Therefore, our initial expectation was that a possible increase in mtDNA4977 levels in the adipose tissue might be associated with obesity. However, although we detected mtDNA4977 deletion in all examined adipose tissue samples from metabolically healthy obese subjects, there was no significant difference in terms of the accumulation rate of this mutation among the obese nondiabetic and control subjects. The current data, however, revealed that in obese people without diabetes, there was 50–75% interindividual heterogeneity in terms of mtDNA4977 deletion. Similarly, the findings with respect to blood as a fast-replicating tissue of Ahmadi et al. [22] also showed that the abundance of mtDNA4977 deletion has no significant relationship with BMI. It is known that age is a candidate causative factor for this interindividual variation. However, the lack of correlation with simple correlation analysis and observing interindividual variation in a narrow age group suggest that this variation in the present study is not related. Further research is required to elucidate the sources of interindividual variability observed in the mtDNA deletion rate of adipose tissues.

We also examined the bioenergetic effects of accumulating mtDNA4977 deletion levels in adipose tissues in the context of obesity by measuring the ATP and ADP contents. We did this as an increased frequency of mtDNA4977 deletion in cells raises the possibility of reduced ATP production in cells [23]. According to the findings of this study, the bioenergetic condition of the tissue does not appear to be negatively affected by the mutation load of the adipose tissue of obese individuals. In contrast, experimental data show that the glycolytic process is far more significant than the oxidative phosphorylation respiratory pathway in creating energy in a variety of tissues, including the soleus muscle, spleen, diaphragm, blood, and intestinal tract [24, 25]. However, this deletion can alter mitochondrial metabolism, and accumulation of the deleted mtDNA in any tissue may upset the metabolic balance by resulting in mitochondrial malfunction [19]. Monitoring lactate levels in cells or tissue can help detect imbalances in the energy metabolism between aerobic glycolysis and mitochondrial oxidation [26, 27]. Therefore, we also examined lactate levels in tissue homogenates. It has been demonstrated that subjects with mitochondrial disease have lactic acidosis in more than 50% of patients [19].

Our results showed that lactate levels in adipose tissues were not significantly higher in obese nondiabetic subjects compared with controls, and the abundance of mtDNA4977 deletion was not correlated with tissue lactate levels, suggesting that this deletion status in adipose tissues may not cause a shift toward glycolysis, which provides less efficient ATP for cellular processes than oxidative phosphorylation. Certainly, this finding will require further replication in larger cohorts.

Adipose tissue expansion and adipogenesis are not only dependent on single targets or biomolecules but rather involve sophisticated interactions between compounds and biomolecules (such as lipids, carbohydrates, proteins, and nucleic acids). The molecular structure and chemical make-up of biological samples, as well as the characterization of proteins, lipids, nucleic acids, and carbohydrates, can all be successfully revealed by FTIR spectroscopy. Additionally, FTIR spectroscopy has been used in obesity studies in the past to identify the structural and functional alterations in adipose tissues brought by obesity. As a result of disease-related global changes in the biochemical content of biological materials like cells, tissues, and even biofluids, FTIR is also capable of identifying changes in molecular compositions. Thus, it discriminates between diseases and healthy conditions.

Therefore, in the second part of the study, we used FTIR spectroscopy to characterize the chemical and structural changes in adipose tissues caused by obesity in the context of deletion load. Variability in metabolic effects has been shown in clinical and experimental studies of hereditary disorders linked to mitochondrial malfunction in humans. In general, phenotypic and biochemical mitochondrial dysfunction is only observed when a certain mutational threshold is crossed [28, 29]. Based on a 50% cut-off value, we also created a threshold, grouped the patients into low- and high-level mutation rate groups, identified the FTIR spectra in the grouped samples, and performed relevant analysis and statistics accordingly.

When we investigated obese and control adipose tissues, the changes in absorbance intensity and transfer of major absorption bands present in all of the measured ranges were typically similar (Table 2; online suppl. Tables 3, 4). However, as can be seen from Table 3; online supplementary Figure 2d, the frequency of the CH2 antisymmetric stretching band shifted significantly to higher values in the obese group, along with a decreased unsaturated/saturated ratio. The shifts in the frequencies of the CH2 stretching vibrations provide information about the changes in lipid order [30, 31]. This shifting indicates a decrease in the lipid order, which results in a less rigid membrane structure and potentially decreased thickness of the lipid bilayer [32]. This difference is not related to the mutation load. In addition, the comparison of the spectral profiles of controls with the obese group, including their classification according to the low and high mutation load, did not find any differences.

The simple correlation rate with the overall mutation rate and all the analyzed spectra for the obese and control groups showed only one correlation between a band around 1,744 cm−1 and mtDNA deletion load. The C = O stretching mode of the ester group in triglycerides and other esterified fatty acids is connected with the signal at 1,744 cm−1 [30]. The exact frequency of the ester carbonyl groups found in lipid molecules is greatly determined by the lipid acyl chain and headgroup packing, as well as the hydration state of the headgroups [33]. Frequency shifting is always an indicator of structural changes, and if there is a structural change, we can expect a change in function [34]. Our results revealed that the increase in the relative amount of studied mtDNA deletion in adipose tissues correlates with the shifting to a significantly higher wavenumber of the band at 1,744 cm−1. However, this shift is not associated with obesity. Although it has been demonstrated that the amount of unsaturated fat is correlated with the age-related increase in the relative amount of deleted mtDNA [35], this statement is far from explaining the shift to higher wavenumbers, which is an indicator of structural change. Therefore, the exact reason for this observed association with increased accumulation of adipose tissues is open to speculation.

Our study has some limitations. Although statistical power analysis revealed that the number of patients in our study group is adequate for showing statistical significance, a larger group would yield more robust results. Another limitation is that our study group included only patients with a BMI of 25–35 kg/m2. Nevertheless, we did not group the female participants according to their pre- and post-menopausal status. While some patients tended to develop diabetes, their number was small, so this group was not included in the statistics.

Our findings showed that the levels of mtDNA4977 deletion in visceral adipose tissues from obese subjects without diabetes, while similar to nonobese subjects, did not appear to have a significant impact on total energetic reserves, despite significant interindividual variability. Therefore, our results indicate that the levels of mtDNA4977 deletion in visceral adipose tissues from obese subjects without DM did not have a significant impact on the bioenergetic status. However, FTIR results suggest that increased deletion accumulation may be associated with a specific change in the ester bond of triglycerides, indicating structural differences of lipids in adipose tissues related to mitochondrial deletion. Even though our study group is small, and therefore statistical significance is far from desired, our results may be considered exploratory. Our initial findings may contribute to better understanding of the tissue-specific distribution of mtDNA deletions and obesity-related adipose tissue pathogenesis.

This study protocol was reviewed and approved by the Ethics Committee of the Cerrahpasa Medical Faculty of Istanbul University-Cerrahpasa, approval number 422872. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and/or with the World Medical Association Declaration of Helsinki. Informed consent or substitute for it was obtained from all participants for being included in the study.

All the authors declare that they have no competing interests.

The present study was supported by the Research Fund of the Istanbul University-Cerrahpasa (Grant No. 25381).

Ayda Yılmaz, Nurten Bahtiyar, Ayça Doğan Mollaoğlu, Onur Baykara, Turgut Ulutin, and Ilhan Onaran conceived and designed the experiments. Kagan Zengin and Halit Eren Taskin enrolled patients. Ayda Yılmaz, Nurten Bahtiyar, Ayça Doğan Mollaoğlu, Ayla Karimova, and Ilhan Onaran analyzed the data. Ilhan Onaran, Nurten Bahtiyar, and Onur Baykara wrote the first draft of the manuscript. All authors contributed to final version of the manuscript. All the authors have read and confirmed that they meet the ICMJE criteria for authorship.

Data are available on request from the corresponding author.

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