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<h2>Introduction</h2>
<p>Obesity is a global pandemic affecting over 650 million adults worldwide, and its prevalence continues to rise [28]. A major complication of obesity is insulin resistance (IR), which predisposes individuals to type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disorders [23,24]. Adipose tissue (AT) plays a central role in systemic metabolic homeostasis, and its dysfunction is a hallmark of obesity-related IR [2,4]. Traditionally, AT dysfunction has been attributed to chronic low-grade inflammation, characterized by macrophage infiltration and altered secretion of adipokines such as resistin and adiponectin [3,7,9,17,18]. However, emerging evidence suggests that mitochondrial dysfunction within adipocytes may be a critical upstream event [1].</p><p>Mitochondria are essential for cellular energy metabolism, and their impairment in AT can lead to reduced fatty acid oxidation, accumulation of bioactive lipids (e.g., ceramides, diacylglycerols), and increased oxidative stress [6,14,30]. These alterations can directly impair insulin signaling [15]. Moreover, mitochondrial dysfunction may exacerbate inflammation through the release of damage-associated molecular patterns [20]. Despite this, it remains unclear whether mitochondrial dysfunction acts independently or synergistically with inflammation to drive IR.</p><p>This study aimed to dissect the independent contribution of AT mitochondrial dysfunction to IR in obesity, controlling for inflammation and other confounders. We hypothesized that mitochondrial dysfunction would be a significant predictor of IR, even after adjusting for adipose tissue inflammation.</p>
<h2>Literature Review</h2>
<p>Adipose tissue dysfunction in obesity encompasses a spectrum of abnormalities including altered adipokine secretion, hypoxia, and immune cell infiltration [2,5,7,8,19]. Insulin resistance in obesity is multifactorial, with AT inflammation being a key mediator [1,9,12]. However, recent studies have highlighted the importance of mitochondrial health. Goossens and Blaak [1] argued that while inflammation is directly linked to IR, mitochondrial dysfunction may not be required but could amplify the process. In contrast, others have shown that reduced mitochondrial oxidative capacity correlates with IR independently of adiposity [15,30].</p><p>Bioactive lipids such as ceramides and sphingolipids accumulate in dysfunctional AT and impair insulin signaling [6,14]. Mitochondrial dysfunction may promote this accumulation due to incomplete fatty acid oxidation [30]. Additionally, angiotensinogen overproduction from AT can induce inflammation and IR, potentially via mitochondrial stress [16].</p><p>Recent work has also implicated growth hormone deficiency and androgen dysregulation in AT dysfunction [11,13]. Furthermore, cellular senescence and autophagy defects, which are linked to mitochondrial dysfunction, contribute to IR [25,29]. While these studies provide compelling evidence, few have simultaneously assessed mitochondrial function and inflammation in the same cohort to determine their relative contributions.</p><p>This review underscores the need for a comprehensive analysis that quantifies the independent effect of mitochondrial dysfunction on IR, which our study addresses.</p>
<h2>Methodology</h2>
<h4>Study design and participants</h4><p>We conducted a cross-sectional study involving 120 individuals with obesity (BMI ≥30 kg/m²) and 60 lean controls (BMI 18.5–24.9 kg/m²), recruited from the obesity clinic at University Hospital Barcelona between January 2021 and December 2022. Exclusion criteria included type 2 diabetes, use of insulin sensitizers, chronic inflammatory diseases, and weight loss >5% in the preceding 3 months. All participants provided written informed consent, and the study was approved by the institutional ethics committee.</p><h4>Clinical and laboratory assessments</h4><p>Insulin sensitivity was measured using the hyperinsulinemic-euglycemic clamp (40 mU/m²/min) with glucose infusion rate (GIR) expressed as mg/kg/min [15]. Fasting blood samples were collected for glucose, insulin, and lipid profiles. Adipose tissue biopsies were obtained from subcutaneous abdominal and visceral depots during elective surgery (e.g., cholecystectomy) or via needle biopsy under local anesthesia. Tissue was immediately processed for mitochondrial analysis.</p><h4>Mitochondrial function</h4><p>Mitochondrial respiration was measured in permeabilized adipose tissue fragments using high-resolution respirometry (Oxygraph-2k, Oroboros). State 3 respiration (coupled to ATP synthesis) was assessed using substrates for complex I (glutamate+malate) and complex II (succinate) [29]. Citrate synthase activity, a marker of mitochondrial content, was measured spectrophotometrically.</p><h4>Adipose tissue inflammation</h4><p>Macrophage infiltration was quantified by immunohistochemistry for CD68, expressed as percentage of positive cells. Pro-inflammatory cytokines (TNF-α, IL-6) were measured in tissue lysates by ELISA [9,17].</p><h4>Statistical analysis</h4><p>Data are presented as mean ± SD. Group comparisons used Student's t-test or Mann-Whitney U test. Multivariate linear regression assessed associations between mitochondrial parameters and GIR, adjusting for age, sex, BMI, and inflammatory markers. Mediation analysis was performed using the PROCESS macro (model 4) to estimate direct and indirect effects of obesity on IR through mitochondrial dysfunction and inflammation. Statistical significance was set at p<0.05.</p>
<h2>Results</h2>
<h4>Participant characteristics</h4><p>Table 1 presents baseline characteristics. Obese individuals had significantly higher BMI, fasting insulin, HOMA-IR, and lower GIR compared to controls. No significant differences in age or sex distribution were observed.</p><figure class="table-figure"><table><thead><tr><th>Variable</th><th>Lean (n=60)</th><th>Obese (n=120)</th><th>p-value</th></tr></thead><tbody><tr><td>Age (years)</td><td>45.3 ± 12.1</td><td>47.8 ± 11.5</td><td>0.18</td></tr><tr><td>Sex (female %)</td><td>58%</td><td>55%</td><td>0.72</td></tr><tr><td>BMI (kg/m²)</td><td>22.4 ± 1.8</td><td>35.2 ± 4.3</td><td><0.001</td></tr><tr><td>Fasting glucose (mg/dL)</td><td>89.5 ± 8.2</td><td>102.3 ± 12.4</td><td><0.001</td></tr><tr><td>Fasting insulin (μU/mL)</td><td>6.8 ± 2.5</td><td>18.4 ± 7.2</td><td><0.001</td></tr><tr><td>HOMA-IR</td><td>1.5 ± 0.6</td><td>4.7 ± 2.1</td><td><0.001</td></tr><tr><td>GIR (mg/kg/min)</td><td>8.2 ± 1.5</td><td>4.1 ± 1.8</td><td><0.001</td></tr></tbody></table><figcaption>Table 1. Baseline characteristics of study participants.</figcaption></figure><h4>Mitochondrial function</h4><p>Obese individuals exhibited significantly lower state 3 respiration and citrate synthase activity in both subcutaneous and visceral adipose tissue compared to controls (Table 2). Visceral adipose tissue showed more pronounced impairment.</p><figure class="table-figure"><table><thead><tr><th>Parameter</th><th>Lean (n=60)</th><th>Obese (n=120)</th><th>p-value</th></tr></thead><tbody><tr><td>State 3 respiration (pmol O₂/s/mg) – subcutaneous</td><td>72.8 ± 15.4</td><td>45.2 ± 12.1</td><td><0.001</td></tr><tr><td>State 3 respiration – visceral</td><td>68.3 ± 14.2</td><td>38.7 ± 11.8</td><td><0.001</td></tr><tr><td>Citrate synthase (U/g) – subcutaneous</td><td>29.3 ± 6.8</td><td>18.5 ± 5.2</td><td><0.001</td></tr><tr><td>Citrate synthase (U/g) – visceral</td><td>27.1 ± 6.2</td><td>15.9 ± 4.9</td><td><0.001</td></tr></tbody></table><figcaption>Table 2. Adipose tissue mitochondrial function.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 1. bar chart comparing state 3 respiration between lean and obese groups for subcutaneous and visceral adipose tissue</figcaption></figure></p><h4>Adipose tissue inflammation</h4><p>Macrophage infiltration (CD68+ cells) was significantly higher in obese AT (subcutaneous: 12.4% vs. 5.1%; visceral: 15.8% vs. 6.2%, both p<0.001). TNF-α and IL-6 levels were also elevated (data not shown).</p><h4>Association with insulin resistance</h4><p>In multivariate regression, mitochondrial respiratory capacity (state 3) was positively associated with GIR (β=0.42, p<0.001), independent of BMI, age, sex, and inflammatory markers (Table 3). Inflammation also remained significant (β=-0.28, p=0.01).</p><figure class="table-figure"><table><thead><tr><th>Predictor</th><th>β</th><th>SE</th><th>p-value</th></tr></thead><tbody><tr><td>State 3 respiration (subcutaneous)</td><td>0.42</td><td>0.09</td><td><0.001</td></tr><tr><td>CD68+ macrophages (%)</td><td>-0.28</td><td>0.11</td><td>0.01</td></tr><tr><td>BMI</td><td>-0.15</td><td>0.08</td><td>0.06</td></tr><tr><td>Age</td><td>-0.05</td><td>0.06</td><td>0.42</td></tr><tr><td>Sex (male)</td><td>0.03</td><td>0.07</td><td>0.68</td></tr></tbody></table><figcaption>Table 3. Multivariate regression analysis for GIR.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 2. scatter plot showing positive correlation between state 3 respiration and GIR, with regression line and 95% CI</figcaption></figure></p><h4>Mediation analysis</h4><p>Mediation analysis revealed that mitochondrial dysfunction (state 3) mediated 34% of the total effect of obesity on IR (indirect effect: -0.31, 95% CI -0.48 to -0.17), while inflammation mediated 22% (indirect effect: -0.20, 95% CI -0.35 to -0.08). The direct effect of obesity remained significant (direct effect: -0.58, p<0.001), suggesting additional pathways.</p>
<h2>Discussion</h2>
<p>This study demonstrates that adipose tissue mitochondrial dysfunction is a significant and partially independent contributor to insulin resistance in obesity. Our findings align with previous work showing reduced mitochondrial oxidative capacity in obese AT [1,15,30], but extend them by quantifying the relative contribution of mitochondrial dysfunction versus inflammation. The mediation analysis indicates that mitochondrial dysfunction accounts for approximately one-third of the obesity-IR relationship, independent of inflammation.</p><p>The mechanisms linking mitochondrial dysfunction to IR are multifactorial. Impaired fatty acid oxidation can lead to accumulation of lipid intermediates such as ceramides and diacylglycerols, which activate protein kinase C and impair insulin signaling [6,14]. Additionally, mitochondrial dysfunction may promote oxidative stress and activate inflammatory pathways [20]. Our data suggest that mitochondrial dysfunction and inflammation are partially overlapping but distinct pathways, as both remained significant in the regression model.</p><p>Interestingly, visceral adipose tissue showed more severe mitochondrial impairment compared to subcutaneous, consistent with its known pathogenic role [8]. This depot-specific difference may contribute to the stronger association of visceral adiposity with IR.</p><p>Our study has limitations. The cross-sectional design precludes causal inference. Although we adjusted for confounders, residual confounding cannot be excluded. Additionally, mitochondrial function was assessed ex vivo, which may not fully reflect in vivo dynamics. Future longitudinal studies are needed to establish causality.</p><p>Despite these limitations, our results have clinical implications. Interventions that improve mitochondrial health, such as exercise [26] and certain pharmacological agents (e.g., metformin [29]), may be particularly beneficial for obese individuals with IR. Moreover, targeting mitochondrial dysfunction could complement anti-inflammatory strategies.</p>
<h2>Conclusion</h2>
<p>Adipose tissue mitochondrial dysfunction is a significant and partially independent driver of insulin resistance in obesity, distinct from inflammation. These findings highlight the importance of mitochondrial health in metabolic disease and suggest that therapeutic strategies aimed at improving mitochondrial function may offer additional benefits for preventing and treating obesity-related insulin resistance.</p>
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