Vegans have a lower incidence of insulin resistance (IR)-associated diseases and a higher insulin sensitivity (IS) compared with omnivores. The aim of this study was to examine whether the higher IS in vegans relates to markers of mitochondrial biogenesis and to intramyocellular lipid (IMCL) content.
Eleven vegans and 10 matched (race, age, sex, body mass index, physical activity and energy intake) omnivorous controls were enrolled in a case–control study. Anthropometry, bioimpedance (BIA), ultrasound measurement of visceral and subcutaneous fat layer, parameters of glucose and lipid homeostasis, hyperinsulinemic euglycemic clamp and muscle biopsies were performed. Citrate synthase (CS) activity, mitochondrial DNA (mtDNA) and IMCL content were assessed in skeletal muscle samples.
Both groups were comparable in anthropometric and BIA parameters, physical activity and protein–energy intake. Vegans had significantly higher glucose disposal (M-value, vegans 8.11±1.51 vs controls 6.31±1.57 mg/kg/min, 95% confidence interval: 0.402 to 3.212, P=0.014), slightly lower IMCL content (vegans 13.91 (7.8 to 44.0) vs controls 17.36 (12.4 to 78.5) mg/g of muscle, 95% confidence interval: −7.594 to 24.550, P=0.193) and slightly higher relative muscle mtDNA amount (vegans 1.36±0.31 vs controls 1.13±0.36, 95% confidence interval:−0.078 to 0.537, P=0.135). No significant differences were found in CS activity (vegans 18.43±5.05 vs controls 18.16±5.41 μmol/g/min, 95% confidence interval: −4.503 to 5.050, P=0.906).
Vegans have a higher IS, but comparable mitochondrial density and IMCL content with omnivores. This suggests that a decrease in whole-body glucose disposal may precede muscle lipid accumulation and mitochondrial dysfunction in IR development.
Insulin resistance (IR)-associated diseases such as obesity and type 2 diabetes (T2DM) rank among the major risk factors of atherosclerosis, and its complications contribute greatly to cardiovascular mortality in developed countries. Composition of diet is considered to be one of the causes of IR. People consuming a strict plant-based diet (vegans) have more favorable parameters of glucose tolerance, lipid profile and lower IR compared with their counterparts without food restriction.1, 2, 3 The prevalence of T2DM is lower in vegan populations as well.4, 5 In line with these findings, the use of vegetarian diets in T2DM patients has been shown to improve diabetes compensation, lipid profile and to lower IR.6, 7
Development of IR is closely related to skeletal muscle metabolic function, as the skeletal muscle is normally responsible for up to 85% of insulin-stimulated glucose uptake.8 Insulin sensitivity (IS) of the muscle is also influenced by circulating the plasma free fatty acids (FFAs) and the related accumulation of intramyocellular lipids (IMCLs), and by mitochondrial function. IMCL likely interferes with intracellular insulin signaling, as the IMCL content has been shown to be positively correlated with IR.9, 10 However, higher IMCL content has also been found among endurance athletes11 who are generally insulin sensitive, which further complicates the causal model.
Previous studies have shown that IRs and IMCLs are correlated with reduced amounts of mitochondria and a related loss of oxidative capacity. Indeed, obese and T2DM patients have lower mitochondrial content12, 13 and lower skeletal muscle oxidative capacity; in addition, their mitochondrial cross-sectional area is reduced ∼30% (Kelly et al.14) and the electron transport chain activity is reduced as well.13 Nevertheless, the relationship between IR, IMCL and mitochondrial dysfunction is complex, causality is unclear and results from available studies are often controversial.15, 16
It has already been shown that vegans have higher IS and lower IMCL content compared with omnivores.2 There are currently no published data addressing mitochondrial function in vegans and a possible relationship between reported higher IS, lower IMCL and muscle mitochondrial function. The aim of this study was to assess whether higher IS and lower IMCL in vegans were associated with altered muscle mitochondrial density.
Subjects and methods
A total of 11 Caucasian vegan subjects (6 male and 5 female) comprised the first study group. These subjects had followed a strict vegan diet (no meat, no dairy products and no eggs) for more than 3 years before enrolling to the study (mean time on vegan diet was 8.05 years). The control group contained 10 subjects (6 male and 4 female) with no food restrictions, consuming meat and other animal products on a daily basis. The control group was selected to match the vegan group in race, gender, age, body mass index, anthropometry, bioimpedance (BIA) analysis, physical activity and energy intake.
Exclusion criteria for recruitment included the following: age under 18 years, obesity, any chronic disease related to energy metabolism (particularly diabetes, thyropathy, hypertension, dyslipidemia, atherosclerosis and so on), any chronic medication (including hormonal contraception), smoking (even in past medical history) and regular alcohol consumption. Subjects with close relatives suffering from T2DM and those contraindicated for muscle biopsy were also excluded.
The research protocol was approved by the Ethics Committee of the Third Faculty of Medicine of the Charles University, Prague, and the Ethics Committee of Faculty Hospital Kralovske Vinohrady in accordance with the Declaration of Helsinki. Each participant gave an informed consent before being enrolled in study.
Anthropometry and clinical examination
Each subject underwent a basic anthropometric examination (height (m), weight (kg), body mass index (kg/m2), waist circumference (cm) and waist–hip ratio). The arm, thigh and calf circumference and skinfold thickness were measured to calculate total muscle mass.17 Body composition was measured using BIA analysis (Nutriguard-M, Data Input GmbH, Frankfurt, Germany). The amount of visceral fat was assessed using ultrasonography (Philips iU22, Best, Netherlands) as the omental fat layer thickness (distance between the abdominal fascia and aorta at the umbilical level). Each of these measurements was performed three times and the mean value was recorded.
Each participant filled in a prospective questionnaire with dietary data from 3 days (2 working days and 1 weekend day). The NutriDan program was used for dietary intake calculations. As nutritional data for certain vegan products were not available in the database, vegans were asked to collect packages of these products and the manufacturer’s nutritional values were used for calculations. Carbohydrate, lipid and protein intakes were calculated separately.
Physical activity assessment
Physical activity was assessed using the Baecke questionnaire for habitual physical activity,18 the scores from which correlate well with maximum oxygen consumption (VO2max).19 Physical activity at work, leisure time and sport activities were assessed separately.
Peripheral venous blood sample was drawn from each subject after 12 h of fasting. Parameters of glucose homeostasis were assessed as follows: plasma glucose using the hexokinase reaction kit (KONELAB, Dreieich, Germany); C-peptide using solid-phase competitive chemiluminescent enzyme immunoassay (Immulite 2000, Los Angeles, CA, USA); HbA1c using high-pressure liquid boronate affinity chromatography (Primus Corporation, Kansas city, MO, USA); and insulin using solid-phase competitive chemiluminescent enzyme immunoassay (Immulite 2000). For the lipid profile, we measured total cholesterol and triglycerides using an enzymatic method kit (KONELAB); high-density lipoprotein–cholesterol measured using a polyethylene glycol-modified enzymatic assay kit (ROCHE, Basel, Switzerland); and low-density lipoprotein–cholesterol calculated using the standard Friedewald equation. Plasma levels of FFA were measured using the method described by Husek et al.20 FFAs were extracted together with neutral lipids into isooctane and cleaned using a reverse-extraction process. FFAs obtained in this way were derivatized to methyl esters and subsequently analyzed by gas chromatography.
IS was assessed in a 2-h hyperinsulinemic euglycemic clamp as described by DeFronzo et al.21 After 12 h of fasting, basal biochemical tests were performed in a blood sample (plasma glucose, insulin and C-peptide) and infusions of insulin (Humulin R, Eli Lilly, Prague, Czech Republic) in a standard dose of 1 mIU/kg/min and of 15% glucose solution were started. Blood glucose was measured every 5 min using a Precision PCX glucometer (Abbott Laboratories, Wiesbaden, Germany). Three consecutive blood tests were performed for plasma insulin during the last 30 min of the clamp protocol after a steady state had been reached. C-peptide was measured at the 120th minute as a confirmation of a sufficient suppression of endogenous insulin secretion. The mean steady-state infusion rate during the clamp (six consecutive measurements) was used for calculations. IS was then expressed as the glucose disposal rate (M-value, mg/kg/min) after a correction for changes in the glucose pool in the extracellular fluid (space correction).
Muscle biopsy, IMCLs and mitochondrial density in skeletal muscle
A biopsy of the vastus lateralis muscle was performed using the Bergström technique22 under basal fasting conditions and ∼200 mg of muscle was obtained. The sample was immediately microdissected under a binocular microscope, weighed, divided for respective analyses and frozen in liquid nitrogen.
Mitochondrial density was assessed using citrate synthase (CS) activity and the relative amount of mitochondrial DNA (mtDNA). CS activity was determined in muscle homogenates using the CS assay kit (Sigma, St Louis, MO, USA). Briefly, ∼20 mg of muscle was homogenized in 400 μl of CelLytic MT lysis buffer (Sigma) using 2 ml Dounce homogenizer. Homogenates were centrifuged at 14 000 g (10 min, 4 °C) and the enzyme activity in the supernatant was assessed. CS activity was measured spectrophotometrically at 25 °C and 412 nm, and was expressed in μmol/min/g of tissue.
Relative amounts of mtDNA to nuclear DNA were determined using semiquantitative PCR to assess the amount of mtDNA per cell. Genomic and mtDNA were isolated from ∼20 mg of muscle sample using the DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The tissue protocol was used, as the protocol for DNA extraction of cultured cells involves an isolation of nuclei and hence the loss of mtDNA. A 141-bp fragment of nuclear DNA and a 221-bp fragment of mtDNA were amplified from 15 ng of total DNA per tube. The nuclear DNA target primer sequences were as follows: forward primer 5′-IndexTermCGAGTAAGAGACCATTGTGGCAG-3′, reverse primer 5′-IndexTermGGGGCTTGTAGGCATTTGCT-3′; and for mtDNA target: forward primer 5′-IndexTermTTTCATCATGCGGAGATGTTGGATGG-3′, reverse primer 5′-IndexTermCCCCACAAACCCCATTACTAAACCCA-3′. The number of cycles for both fragments was determined using a cycle test to keep the amplification rate within the exponential range of the PCR. The total amount of isolated DNA and PCR products were quantified fluorometrically using a Qubit dsDNA HS Assay Kit and a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA).
IMCL content was assessed using gas chromatography to measure fatty acid content in muscle fibers. The method described by Lepage and Roy23 with modifications by Rodrigues-Palmero et al.24 was used for profiling and measuring the amount of FFA in samples. In brief, the method involves a chloroform/methanol extraction of freeze-dried muscle to isolate lipids and subsequent transesterification or esterification of FFA bound to lipids to form methyl esters, which were then analyzed using gas chromatography.
Data were tested for normality using the Shapiro–Wilk test. For normally distributed data, samples were compared using the Student’s t-test to test the statistical significance of differences between independent groups. The Pearson’s correlation coefficient R was calculated to express relationships between variables. The Mann–Whitney test was used for not normally distributed data. Data are presented in text and tables as means±s.d. with a 95% confidence interval for the difference; not normally distributed data are presented as medians and ranges. The differences at P<0.05 were considered statistically significant. Statistica 9.0 (StatSoft, Inc., Tulsa, OK, USA) was used to perform all statistical procedures.
Table 1 summarizes characteristics of both groups. Both groups were statistically comparable in terms of race, gender, anthropometry (body mass index and the waist–hip ratio) and BIA characteristics, that is, fat mass and fat-free mass. There was an apparent trend toward higher age in the vegan group (difference of means, 2.51 years, P=0.084). In addition, vegans had a trend toward a higher visceral (1.15 cm, P=0.181) and lower subcutaneous fat amount (−0.64 cm, P=0.178), and higher physical activity (1.01 points, P=0.09). Groups did not differ significantly in overall energy intake (carbohydrates, lipids and proteins).
Plasma lipid profile
Fasting plasma lipid profiles are presented in Table 2. Vegans had lower plasma level of total cholesterol (−0.72 mmol/l, P=0.038). Other parameters (plasma levels of triacylglycerols, low-density lipoprotein– and high-density lipoprotein–cholesterol) were not significantly different. Vegans had higher levels of total plasma polyunsaturated fatty acid (25.09 μg/ml, P=0.001) and a clear trend toward higher total plasma FFA (53.05 μg/ml, P=0.089). The results showed higher levels of omega-3 α-linolenic fatty acid (ALA; 1.20 μg/ml, P=0.009) and omega-6 linoleic acid (23.81 μg/ml, P=0.001), eicosadienoic acid (0.15 μg/ml, P=0.009) and dihomo-γ-linolenic (0.15 μg/ml, P=0.038) in vegans.
Glucose homeostasis parameters are summarized in Table 3. Fasting plasma glucose was lower in vegans (−0.64 mmol/l, P=0.04), whereas insulin and glycosylated hemoglobin showed no significant differences. Glucose consumption during the steady-state hyperinsulinemic clamp is shown in Figure 1. Vegans had a higher M-value compared with controls (1.82 mg/kg/min, P=0.023), as well as M-values adjusted for total skeletal muscle mass (calculated from the arm, thigh and calf circumferences (1.80 mg/kg/min, P=0.014)). Both groups reached comparable steady-state insulinemia, which was sufficient to suppress endogenous insulin production (data not shown).
Mitochondrial density and IMCL content
Results are summarized in Table 4. Markers of mitochondrial density (CS activity and relative amount of mtDNA to nuclear DNA) did not show significant differences, although there was a trend toward a higher relative mtDNA content in vegans (0.23, P=0.135). There was also an apparent trend toward a lower IMCL content in vegans (−3.45 mg/g, P=0.193). Vegans had lower amounts of pentadecanoic (−0.04 mg/g, P=0.004) and docosahexaenoic (DHA; −0.08 mg/g, P=0.003) in muscle.
We analyzed correlations of data pooled from all subjects and found that plasma insulin levels were negatively correlated with mtDNA amount (R=0.55, P=0.01) and there were trends toward a negative correlation between glycemia and mtDNA (R=0.30, P=0.180), a positive correlation between M-values and mtDNA (R=0.30, P=0.191), a positive correlation between the Baecke score and mtDNA (R=0.34, P=0.07) and a positive correlation between CS and mtDNA (R=0.33, P=0.15). Surprisingly, in vegan sample the M-value was highly positively correlated with IMCL content (R=0.85, P<0.001).
In this study, we investigated the influence of a vegan diet on IS and phenomena associated with IR in skeletal muscle, particularly mitochondrial density. It has been previously established that vegans are more insulin sensitive than omnivores and have a lower IMCL content.1, 2, 3 We hypothesized that the reported higher IS and lower IMCL content in vegans are associated with a higher mitochondrial density. A major finding of the current study is that vegans are more insulin sensitive, but we found only statistically insignificant differences in IMCL content or mitochondrial density compared with omnivores.
We found that vegans have lower fasting plasma glucose and insulin levels, and higher insulin-stimulated glucose disposal. This is a plausible explanation for the lower incidence of IR-related diseases in vegans and similar data have been previously published.1, 2 However, to our knowledge the current study was the first to use hyperinsulinemic euglycemic clamp for the assessment of IS.
Plasma lipid and FFA profile
A survey of the literature shows that vegans have more favorable lipid profiles, have lower levels of total and LDL plasma cholesterol and lower triglycerides.3, 25 Our results showed significantly lower total cholesterol levels in vegans and we found that vegans have higher plasma levels of total polyunsaturated fatty acids and higher amounts of omega-6 fatty acids linoleic acid, eicosadienoic acid and dihomo-γ-linolenic in their FFA profiles. We also found higher plasma levels of omega-3 ALA in vegans, which has not been previously reported. Published studies suggest that vegans have lower circulating EPA and DHA levels.26 Indeed, vegan’s intake of EPA and DHA is very low and, therefore, these FA are mainly synthesized endogenously from ALA. Unfortunately, we cannot comment on EPA and DHA levels in circulation because the detection limit of the method used in this study for EPA and DHA was 1.0 and 2.2 μg/ml, respectively, and none of the subjects in our study exceeded these limits.
Previous studies reported that IMCL content correlates with IR in healthy non-diabetic subjects9 and that vegans have lower IMCL values compared with omnivores.2 However, a lower IMCL content in vegans was found only in the (oxidative) soleus muscle and not in the (glycolytic) tibialis anterior muscle. We obtained samples from the vastus lateralis, which is glycolytic, and we did not find any statistically significant differences among the groups even though there was a trend toward a lower IMCL content and a positive correlation between IMCL content and glucose disposal (M-value) in vegans. Biochemical methods of IMCL detection are burdened by potential contamination with subcutaneous and extramyocellular fat tissue, and therefore we performed a microdissection of the muscle samples immediately after biopsy, using a binocular microscope to minimize this potential confounder. The advantage of a biochemical assessment of IMCL is the possibility to measure individual FFA content in the lipid extract. We found that vegans had lower DHA in their muscles compared with omnivores, although the ALA and EPA content was similar (data not shown). As already mentioned, circulating EPA and DHA levels are lower in vegans due to their lower intake.26 To our knowledge, this study is the first to show a decreased DHA content in muscle tissue samples. The clinical impact of the previously reported lower circulating EPA and DHA levels and lower muscle DHA content in vegans deserves further investigation.
Mitochondrial aerobic capacity in skeletal muscle is potentially a major contributor to whole-body IS with skeletal muscles responsible for almost 85% of insulin-stimulated glucose uptake.8 It was previously reported that insulin-resistant subjects have a lower mitochondrial oxidative capacity13, 27 and lower mitochondrial content.12, 13 There are currently no published data addressing the effect of a vegan diet on mitochondrial density in skeletal muscle. We assessed the mitochondrial density by measuring CS activity (nuclear-encoded mitochondrial enzyme) and relative amounts of mtDNA in skeletal muscle. There was a trend toward a higher relative amount of mtDNA in vegans and a correlation between mtDNA and CS, which would be in line with literature,28 but these differences did not reach statistical significance. The relative amount of mtDNA was negatively correlated with plasma insulin levels and there was a trend toward a positive correlation between mtDNA and glucose disposal (M-value) across the whole sample. This suggests that a reduction in IS and the related elevation of plasma insulin levels may be linked with decreased amounts of mtDNA and a possible loss of mitochondrial oxidative capacity.
Limitations of this study
The main limitation of this study was its relatively small sample size. Because of strict inclusion criteria, we were not able to enroll more eligible subjects into the study. Groups were intentionally matched so that the maximum number of variables associated with IR could be controlled. In spite of the intention to match groups in baseline characteristics, there were small differences between groups, namely in age, visceral and subcutaneous fat and physical activity. We cannot exclude that these differences could have had some influence on results. Vegans were on average 2.51 years older than omnivores in our sample. However, we do not believe this age difference had a significant effect on the tested variables, as both groups were relatively young.
Physical activity and VO2max has an important role in the development of IR, as well as in muscle lipid accumulation and mitochondrial biogenesis. Physical activity represents a major potential confounder when it comes to the development of IR. Therefore, the aim of the initial matching of groups was to use the same level of physical activity between groups. In this study, VO2max was not assessed and the Baecke questionnaire of habitual physical activity was used instead. Although VO2max is a more accurate parameter for describing physiological changes induced by physical activity, it has been shown that Baecke questionnaire scores correlate well with VO2max.19
We found that vegans have more favorable glucose homeostasis and plasma lipid profile, which is in line with previous studies. We also found that vegans have higher plasma levels of polyunsaturated fatty acid, more precisely linoleic acid, ALA, eicosadienoic acid and dihomo-γ-linolenic, and lower skeletal muscle DHA content. We also demonstrated that vegans had a significantly higher insulin-stimulated glucose uptake; however, we found only small or no differences in IMCL, mtDNA and CS activity compared with their matched omnivorous counterparts. These findings suggest that a decrease in whole-body glucose disposal precedes muscle lipid accumulation and mitochondrial bioenergetic failure in the development of IR. Therefore, IMCL accumulation and mitochondrial dysfunction may be consequences or epiphenomena associated with IR, and may contribute to its progression rather than the initial trigger.
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This study was funded within the scientific framework of research programs of the Charles University in Prague, PRVOUK-P31 and UNCE 204015, and was supported by a Grant of the Ministry of Health of the Czech Republic, number NT/14416.
The authors declare no conflict of interest.
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Gojda, J., Patková, J., Jaček, M. et al. Higher insulin sensitivity in vegans is not associated with higher mitochondrial density. Eur J Clin Nutr 67, 1310–1315 (2013). https://doi.org/10.1038/ejcn.2013.202
- insulin resistance
- mitochondrial density
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