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ABSTRACT Adipocyte mitochondrial respiration may influence metabolic fuel partitioning into oxidation versus storage, with implications for whole-body energy expenditure. Although insulin

has been shown to influence mitochondrial respiration, the effects of dietary macronutrient composition have not been well characterized. The aim of this exploratory study was to test the

hypothesis that a high-carbohydrate diet lowers the oxygen flux of adipocyte mitochondria ex vivo. Among participants in a randomized-controlled weight-loss maintenance feeding trial, those

consuming a high-carbohydrate diet (60% carbohydrate as a proportion of total energy, _n_ = 10) had lower rates of maximal adipose tissue mitochondrial respiration than those consuming a

moderate-carbohydrate diet (40%, _n_ = 8, _p_ = 0.039) or a low-carbohydrate diet (20%, _n_ = 9, _p_ = 0.005) after 10 to 15 weeks. This preliminary finding may provide a mechanism for

postulated calorie-independent effects of dietary composition on energy expenditure and fat deposition, potentially through the actions of insulin on fuel partitioning. SIMILAR CONTENT BEING

VIEWED BY OTHERS DIETARY PROTEIN RESTRICTION ELEVATES FGF21 LEVELS AND ENERGY REQUIREMENTS TO MAINTAIN BODY WEIGHT IN LEAN MEN Article Open access 06 March 2025 A CRITICAL ASSESSMENT OF THE

ROLE OF CREATINE IN BROWN ADIPOSE TISSUE THERMOGENESIS Article 09 January 2023 TUNING FATTY ACID OXIDATION IN SKELETAL MUSCLE WITH DIETARY FAT AND EXERCISE Article 22 September 2020 The

hormone insulin plays a fundamental role in cellular nutrient signaling, including mitochondrial function. Joslin and Benedict observed in 1912 [1] that the metabolic rate in

insulin-deficient states (i.e., type 1 diabetes) is significantly higher than expected—a phenomenon subsequently shown to be correctable with administration of insulin [2]. A component of

this change in energy expenditure may involve insulin-induced changes in adipocyte mitochondrial bioenergetics. Dallon et al. [3] found that insulin produces a more tightly coupled state

between electron transport and oxidative phosphorylation in rodent brown adipose mitochondria, lowering mitochondrial respiration rate and energy expenditure. Moreover, evidence from rodent

models suggests that insulin has a suppressive effect on adipose tissue mitochondrial respiration, thereby lowering adipocyte energy expenditure [4]. While little is known about hormonal and

nutrient effects on mitochondrial respiration in human adipose tissue, a recent meta-analysis found that higher- versus lower-carbohydrate diets (which increase insulin secretion) reduce

total energy expenditure in humans after 2.5 weeks, with control for energy intake or body weight [5]. The aim of this exploratory study is to test the hypothesis that a high-carbohydrate

diet would lower measures of mitochondrial respiration in adipose tissue, consistent with the carbohydrate-insulin model of obesity [6]. MATERIALS AND METHODS STUDY DESIGN The analyses

presented here comprise an ancillary study of the Framingham State Food Study, in which the primary outcome was total energy expenditure [7]. Briefly, adults with BMI ≥ 25 were provided

prepared diets at 60% of estimated energy requirements to produce a 10–14% weight loss during the run-in phase. Individuals achieving the target weight loss were weight-stabilized by

adjustment of dietary energy and randomly assigned to low- (20%), moderate- (40%), or high- (60%) carbohydrate diets for a 20-week test diet phase. All diets had 20% protein, derived from

similar sources across diets, with the remainder of energy derived from fat. Energy provision during the weight-loss maintenance test diet phase was adjusted periodically, targeting ±2 kg of

the pre-randomization (post-weight loss) level. Additional detail on diets and experimental design were previously described [7]. A subset of participants in the parent trial was recruited

into this ancillary study prior to randomization, protecting against selection bias. Adipose biopsies were obtained following weight stabilization and again after 10–15 weeks on test diets,

in conjunction with another ancillary study focused on metabolic fuels [8]. PARTICIPANTS Participants in the first two of three cohorts in the parent trial were invited to opt-in, by

completing a telephone interview (_n_ = 51) followed by an in-person informational session (_n_ = 43). After considering additional exclusion criteria (history of allergy or prior reaction

to lidocaine, conditions or medications associated with increased risk for bleeding, infection or skin reaction following adipose tissue biopsy, inadequate weight loss in the parent study,

and scheduling conflicts with the ancillary study visits), 30 participants opted-in, were eligible for the ancillary study, and were among those randomized to the test diets in the parent

trial (see Table 1 for baseline characteristics and weight data). One participant in the low-carbohydrate group did not complete the post-intervention biopsy. Data from two participants were

excluded due to loss of pre-intervention biopsy sample (_n_ = 1, high-carbohydrate) and development of hypothyroidism (_n_ = 1, low-carbohydrate). This report is based on the remaining 27

participants. The study was approved by the Institutional Review Boards at Boston Medical Center and Boston Children’s Hospital, as registered at www.ClinicalTrials.gov (NCT02235038). We

obtained written informed consent from participants. ADIPOSE BIOPSY Adipose tissue biopsies were performed at Boston Medical Center. Subcutaneous abdominal fat was sampled by needle biopsy

before and after the dietary intervention and following an overnight fast. Briefly, a sterile field was prepared, and an area 3 to 5 cm lateral to the umbilicus was isolated, cleaned, and

anesthetized with 1% lidocaine. A liposuction cannula (Unitech 1 or 3-hole cannula, 3 mm × 12 cm) was connected to a 60-mL syringe via a 24-inch segment of standard intravenous tubing primed

with a small volume of sterile 0.9% normal saline. The cannula was passed through a small cutaneous incision (<0.5 cm) and ~1–3 g of adipose tissue was aspirated under negative vacuum

pressure in the 60-mL syringe. Following biopsy, the tissue was frozen in liquid nitrogen and stored at −80 °C. To prepare for mitochondrial analysis, samples were thawed in ice-cold buffer

(60 mM K-MES, 35 mM KCl, 7.23 mM K2EGTA, 2.77 mM CaK2EGTA, 20 mM imidazole, 20 mM tuarine, 5.7 mM ATP, 15 mM PCr, 6.56 mM MgCl2−6H2O, pH 7.1) and trimmed of connective tissue. Samples were

transferred to a tube with chilled buffer and 50 μg/ml saponin, rocked at 4 °C for 30 min, and then washed (105 mM K-MES, 30 mM KCl, 10 mM KH2PO4, 5 mM MgCl2−6H2O, 0.5 mg/ml BSA, pH 7.1) at

4 °C for at least 15 min. Samples were stored at −70 °C until assay. MITOCHONDRIAL RESPIRATION ASSAY Before addition of sample into respiration chambers (Oroboros O2K, Innsbruck, Austria), a

baseline respiration rate was determined. After addition of sample, the chambers were hyperoxygenated to ~250 nmol/ml, as previously validated for quantification of mitochondrial

respiration rates in human adipose [9]. Respiration was determined by a substrate-uncoupler-inhibitor-titration protocol. Electron flow through complex I was supported by glutamate + malate

(10 and 2 mM, respectively) to determine leak oxygen consumption (GM). Following stabilization, ADP (2.5 mM) was added to determine oxidative phosphorylation capacity (GMD). Succinate (2.5 

mM) was added to determine complex I + II electron flow into the Q-junction (GMDS). Finally, the chemical uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (0.05 μM) was added

to determine full electron transport system capacity in cells over oxidative phosphorylation (FCCP). Mitochondrial membrane integrity was confirmed in all experiments by observing the effect

of adding cytochrome _c_ (not shown; 10 μM). Following the protocol, samples were lysed for protein quantification (BCA protein assay; Pierce). Respiration rates are shown relative to total

sample protein. Assays were batched by diet group assignments (designated by numerical codes), with laboratory staff masked to codes. STATISTICS Participant characteristics were summarized

with descriptive statistics (frequency and percentage for categorical variables, mean, and standard deviation for continuous variables with normal distribution, median and interquartile

range for continuous variables with skewed distribution). Mitochondrial respiration outcomes (pmol O2/sec/ug protein) were calculated as change from first biopsy pre-randomization

(post-weight loss) to second biopsy (after 10–15 weeks on weight-loss maintenance test diets). Data were analyzed using general linear models (GLM), with adjustment for sex, ethnicity

(Hispanic vs. non-Hispanic) and BMI pre-weight loss. Model assumptions and potential outliers were examined using residual QQ plots and studentized residuals. Statistical analyses were

performed using SAS 9.4 and statistical significance was defined as _P_ < 0.05 in a two-sided test. RESULTS AND DISCUSSION In the initial condition (GM), an assessment of mitochondrial

respiration in a basal state of complex I activation (via glutamate+malate), no differences in mitochondrial respiration were found by diet group. With addition of ADP (GMD) to engage

oxidative phosphorylation (via complex V), the high-carbohydrate group had lower respiration compared with the moderate-carbohydrate (_p_ = 0.021) but not with the low-carbohydrate group.

When complex II-supported respiration was tested with the addition of succinate (GMDS), the high-carbohydrate group had lower respiration versus moderate- (_p_ = 0.035) or low- (_p_ = 0.038)

carbohydrate groups. Finally, the addition of FCCP, which elicits a maximal respiratory response (i.e., mitochondrial uncoupling), produced a robust difference between the high- versus

moderate- (_p_ = 0.039) or low- (_p_ = 0.005) carbohydrate groups (Fig. 1). Our study suggests that a high-carbohydrate diet, possibly through an increase in insulin secretion [8], lowers

mitochondrial respiratory function—a metabolic state that would favor deposition rather than oxidation of fat and predispose to weight gain. This finding is consistent with longer-term

feeding trials examining the effects of dietary carbohydrate, as a proportion of total energy intake, on total energy expenditure [5], and a recent study demonstrating adverse effects of

carbohydrate overfeeding on cellular redox states [10]. A mechanistic role for insulin secretion on weight gain is supported by a Mendelian randomization study, a two-cohort prospective

study, and several clinical trials showing effect modification of dietary carbohydrate by insulin secretion [7, 11,12,13,14]. Nevertheless, causal roles for insulin secretion and high

glycemic load diets in human obesity have not been established. As the specific mitochondrial assays were not pre-specified, and the methods have not been standardized among laboratories

(including sample storage stability), these findings should be considered preliminary. In addition, we cannot directly translate our findings to whole-body energetics. White adipose tissue

is less metabolically active than lean mass, and our sample may not reflect mitochondrial activity in all body fat depots. Nevertheless, even a small shift in substrate partitioning from

oxidation to storage, on the order of 1 g body fat per day, could have a major impact on obesity predisposition over the long term. Furthermore, we cannot definitively attribute the observed

effects to insulin reduction per se, as other aspects of diet (e.g., fatty acid profile) or indirect influences (e.g., the microbiome) may be mechanistically involved. Additional research

is needed to replicate these findings, conduct quantitative energetic studies (e.g., ATP generation), examine generalizability to other populations and experimental conditions, and explore

translation to the prevention and treatment of obesity. DATA AVAILABILITY The protocol and dataset are available at Open Science Framework (https://osf.io/rvbuy/). REFERENCES * Benedict FG,

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obese young adults: a randomized trial. JAMA. 2007;297:2092–102. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Kimberly Greco for reproducing the statistical

analyses. FUNDING The trial considered here was funded by Nutrition Science Initiative (made possible by gifts from Arnold Ventures and Robert Lloyd Corkin Charitable Foundation), New

Balance Foundation, Many Voices Foundation, and Blue Cross Blue Shield. This work was also supported by the Boston Nutrition and Obesity Research Center (P30DK046200). KJS was supported by a

Pediatric Endocrine Society fellowship. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Cell Biology and Physiology, Brigham Young University, Provo, UT, USA Benjamin T. Bikman,

 Erin R. Saito & Chase M. Walton * New Balance Foundation Obesity Prevention Center, Boston Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA, USA Kim

J. Shimy, S. Yu, Cara B. Ebbeling & David S. Ludwig * Division of Endocrinology, Children’s National Hospital, Washington, DC, USA Kim J. Shimy * Division of Endocrinology, Diabetes, and

Hypertension, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA, USA Caroline M. Apovian Authors * Benjamin T. Bikman View author publications You

can also search for this author inPubMed Google Scholar * Kim J. Shimy View author publications You can also search for this author inPubMed Google Scholar * Caroline M. Apovian View author

publications You can also search for this author inPubMed Google Scholar * S. Yu View author publications You can also search for this author inPubMed Google Scholar * Erin R. Saito View

author publications You can also search for this author inPubMed Google Scholar * Chase M. Walton View author publications You can also search for this author inPubMed Google Scholar * Cara

B. Ebbeling View author publications You can also search for this author inPubMed Google Scholar * David S. Ludwig View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS BTB supervised the mitochondrial studies and co-wrote the first draft of the manuscript. KJS helped design and conduct the trial and revised the manuscript. CMA

helped conduct the trial and revised the manuscript. SY conducted the statistical analysis and revised the manuscript. ERS and CMW helped conduct the mitochondrial assays. CBE helped design

the trial and revised the manuscript. DSL helped design the trial, co-wrote the first draft of the manuscript, and provided supervision. All authors approved the final manuscript.

CORRESPONDING AUTHOR Correspondence to Benjamin T. Bikman. ETHICS DECLARATIONS COMPETING INTERESTS BTB received royalties for a book about insulin resistance. CMA. received royalties for

books that recommend a low-carbohydrate diet. DSL received royalties for books that recommend a low-glycemic load diet; his spouse owns a nutrition education and consulting business. No

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ARTICLE Bikman, B.T., Shimy, K.J., Apovian, C.M. _et al._ A high-carbohydrate diet lowers the rate of adipose tissue mitochondrial respiration. _Eur J Clin Nutr_ 76, 1339–1342 (2022).

https://doi.org/10.1038/s41430-022-01097-3 Download citation * Received: 16 November 2021 * Revised: 01 February 2022 * Accepted: 04 February 2022 * Published: 17 February 2022 * Issue Date:

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