Despite being recognized as a key element in the etiology of type 2 diabetes, skeletal muscle insulin resistance lacks a unifying mechanism that can be consistently translated to human disease or pathobiology. Moreover, whether improvements in insulin sensitivity with weight loss and exercise share common underpinnings is unclear. In this context, bariatric surgery–induced weight loss, with or without concomitant exercise training, provides a conceptual and practical framework to examine important questions about treatment for insulin resistance. We recently reported that insulin resistance is not generally normalized in the weeks to months after RYGB surgery and that regular exercise can further improve peripheral insulin sensitivity. In the present study, we examined whether alterations in mitochondrial energetics or intramyocellular lipids correspond with improved insulin sensitivity following RYGB surgery with or without regular exercise.
A key finding was that surgery-induced weight loss, particularly with adjunct exercise, imparted distinct effects on mitochondrial energetics in the absence of any notable alterations in mitochondrial content. Much controversy exists regarding the role of mitochondria in muscle insulin resistance and how weight loss affects this relationship. Calorie restriction–induced weight loss in humans, an intervention that improves peripheral insulin resistance, has been reported to elicit muscle mitochondrial biogenesis, reduce mitochondrial respiration, or have no effect on mitochondrial content and ETC activity. In the context of bariatric surgery–induced weight loss, only a handful of studies have examined skeletal muscle. These studies show changes in gene expression related to glucose metabolism and mitochondrial function in muscle biopsy samples and myotubes derived from biopsy specimens, changes that may be mediated by an altered epigenome. Others have shown that the expression of genes associated with mitochondrial biogenesis in skeletal muscle are enhanced and related to improved insulin sensitivity after bariatric surgery. The few studies that examined mitochondrial respiratory performance after bariatric surgery produced equivocal results.[19,20]
We used a number of analytical approaches to assess mitochondrial function more comprehensively. Our observation that surgery-induced weight loss did not alter markers of mitochondrial content is in line with a growing consensus that weight loss alone does not induce mitochondrial biogenesis.[15,18] Surgery-induced weight loss, however, did induce a robust increase in CI P /CI&II P control ratios, indicating increased capacity for OXPHOS without increased ETS capacity. Weight loss in the CON group also increased the CI P /CI&II P control ratio, indicating a relative increase in the contribution of electron flow from CI to maximal OXPHOS respiration. Taken together, these data suggest a unique remodeling of mitochondrial respiratory parameters with surgery-induced weight loss that occur along with improved insulin sensitivity.
The exercise program further improved SI compared with surgery-induced weight loss alone, concomitant with distinct improvements in mitochondrial respiration. Abundant evidence indicates that aerobic exercise induces muscle mitochondrial biogenesis and improves oxidative capacity,[23,29] but few exercise studies with a focus on mitochondria have been conducted in obese patients,[29,30] and none have been conducted in patients after bariatric surgery. We found no increase in OXPHOS protein or total CL content after exercise (a surprising observation that may be due to massive weight loss modulating the effect of exercise on mitochondria), whereas mitochondrial enzymes, including citrate synthase and NADH oxidase activities, increased robustly.
Exercise induced a remodeling of CL species, a phospholipid that is primarily localized to the inner mitochondrial membrane and plays a key role in maintaining OXPHOS complex integrity and function. We observed a distinct pattern of remodeling, with species containing the polyunsaturated linoleoyl-CoA [CL-(C18:2)4] becoming more abundant and CL-(C18:2)3(C18:1)1, CL-(C18:2)2(C18:1)2, and CL-(C18:2)3(C18:0)1 decreasing with exercise. The increase in tetralinoleic CL may reflect lower exposure of CL to oxidative stress because polyunsaturated acyl chains in CL are particularly vulnerable to oxidative modification by reactive oxygen species. This would be in line with the increase in mitochondrial antioxidant capacity that has been reported with regular exercise. The increase in tetralinoleic CL also reflects a more mature CL pool, meaning that the immature form of CL is characterized by a random assortment of attached acyl chains that are saturated and variable in length. A mature CL pool has structural uniformity and contains predominantly tetralinoleoyl acyl chain features that are essential to its function to maintain the integrity of the OXPHOS capacity. The importance of CL to mitochondrial function is highlighted by studies of patients with mutations in the tafazzin gene (Barth syndrome) and studies in rodents where relatively small changes in CL profile reduce mitochondrial respiratory capacity in cardiomyocytes. Further investigations are needed to interrogate the role of CL in improved insulin resistance.
The differential response of various indices of mitochondrial content and CL remodeling to exercise may relate to the fact that obese individuals may not adapt to contractile activity in a manner consistent with the plasticity of mitochondria.[36,37] The impact of varying intensity and volume of exercise on mitochondrial remodeling, particularly in obese individuals, may also play a role. Overall, these data suggest that exercise-induced remodeling of the various components of the mitochondria may occur to different degrees in obese individuals. Congruent with a more mature CL pool, we observed an increase CI P , CI&II P , and CI&II and FAO P respiration after the exercise program. Although these effects have been previously reported for exercise,[29,33] the present report is the first in obese patients following bariatric surgery. For the first time to our knowledge, the data link exercise-induced CL remodeling with improved mitochondrial OXPHOS and insulin sensitivity during RYGB surgery–induced weight loss.
Surgery-induced weight loss reduced IMTG in type I and II fibers, a finding in line with that of Gray et al. and others who have shown that calorie restriction–induced weight loss also reduces IMTG.[18,40] The present study is the first to comprehensively compare skeletal muscle sphingolipid and DAG species after bariatric surgery. The sphingolipid ceramide has been touted as a key mediator of insulin resistance through inhibition of Akt/protein kinase B signaling and mediation of inflammation (tumor necrosis factor-α). Associations between muscle ceramide content and insulin resistance in obesity have been reported in some[9,10,13] but not all studies. A limitation in the current literature is that many previous studies only measure total ceramide or a small number of molecular species. However, the identity of sphingolipid species that is associated with improvements in insulin sensitivity with weight loss has not been adequately examined. In this study, we used state-of-the-art lipidomics to quantify individual molecular species of sphingolipid (17 total, including ceramide) in the muscle of RYGB surgery patients during weight loss and provide strong evidence that ceramide is reduced concomitant with improved insulin sensitivity. Of note, the reduction in ceramide may also be involved in the observed alterations in mitochondria respiration because ceramide can suppress the ETS at CI and CIII, leading to oxidative stress, and modulate mitochondrial permeability transition pore opening. However, in two previous studies, diet-induced calorie restriction and RYGB- and laproscopic gastric banding–induced weight loss (20%) did not alter intramyocellular ceramide. Although reconciling the present data to these findings is difficult, the data underscore the need for further studies to evaluate the importance of intramyocellular ceramide in improved insulin sensitivity with weight loss.
Exercise also induced distinct intramyocellular lipid partitioning and prevented the RYGB surgery–induced decrease in IMTG in type I myofibers, an observation that echoes the athlete's paradox and is consistent with observations made during calorie-induced weight loss with exercise. Exercise also resulted in greater selective reductions in a number of individual ceramide species with long acyl chains (C18, C18:1, C24:1, C22:1) and total sphingolipid and ceramide content. This is consistent with our previous reports of individual ceramide species being elevated in obesity and insulin resistance[13,14] and being reduced with exercise, further supporting a role for ceramide in human muscle insulin resistance.
A significant body of research in animal models supports a role for DAG as a mediator of muscle insulin resistance. However, evidence from studies of human muscle remain equivocal. Here, we did not observe changes in intramyocellular DAG during RYGB surgery–induced weight loss with or without exercise, indicating no relationship with improved insulin sensitivity. This result is in line with our previous reports that whole-muscle DAG does not play a role in insulin sensitivity in human muscle[10,13,14] and a report that intramyocellular DAG does not change after surgery-induced weight loss. Of note, Jocken et al. reported paradoxically lower levels of DAG in obese compared with lean subjects. Although the consensus in human studies thus far suggests that whole-muscle levels of DAG are not associated with insulin resistance, this does not preclude the possibility that organelle/lipid membrane–specific accumulation of DAG species plays an important role in mediating insulin resistance in human obesity.
Finally, the expression of GLUT proteins did not appear to explain the exercise-induced improvements in SI. GLUT4 is the key GLUT responsible for insulin- and contraction-stimulated glucose transport in skeletal muscle. Expression of GLUT4 is not different between healthy individuals with insulin resistance and those with type 2 diabetes, and caloric restriction has no effect on GLUT expression in muscle. However, many studies have demonstrated that increased GLUT4 expression is a key adaptation to long-term exercise[54,55] and that GLUT4 expression increases in muscle of obese patients with type 2 diabetes after exercise. Fewer studies have examined the effect of exercise on GLUT1 and 12 isoforms, which are expressed at lower levels than GLUT4. It is possible that similar to the lack of effect on mitochondria biogenesis or content in the present study, the dose of exercise was insufficient to elicit changes in GLUT proteins [exercise-induced expression of GLUT4 is regulated in parallel with mitochondrial biogenesis through the same signaling pathways]. It is also possible that these patients are resistant to these exercise improvements for reasons not explored in this study.
A distinguishing feature of the present study was that we used a randomized exercise trial to determine the additional effects of exercise postsurgery, so we did not capture changes that could have initially occurred after surgery. Presurgery data would have allowed us to examine potentially important changes in myocellular metabolism during the initial period after surgery. Another limitation was that nutritional intake was not controlled or monitored and may represent an important factor that affects outcome measures, including intramyocellular lipids, mitochondrial function, and SI. Moreover, future studies should examine other potential mediators of insulin resistance and their response to surgery-induced weight loss and exercise (e.g., acylcarntines, amino acids, gut microbiome).
In summary, this randomized exercise intervention study provides the first evidence in RYGB surgery patients to show distinct weight loss and exercise effects on mitochondrial respiration and CL remodeling independent of mitochondrial content and reductions in IMTG and intramyocellular ceramide but not DAG. RYGB surgery–induced weight loss enhanced specific aspects of mitochondrial OXPHOS, a novel observation that may prove to be a valuable target for therapy. Exercise further improved SI along with improved respiratory capacity, remodeling of the CL profile, and further reductions in specific ceramide species. Thus, several facets of myocellular energetics are not rectified with substantial weight loss from bariatric surgery alone and further support the implementation of an adjunct exercise program after bariatric surgery. These data provide valuable mechanistic insight into how both surgery-induced weight loss and exercise are complementary therapies to improve the metabolic profile in severe obesity.
Clinical trial reg. no. NCT00692367, clinicaltrials.gov.
The authors gratefully appreciate the contributions of the study participants and acknowledge the excellent technical assistance of Steven Anthony for administering the exercise and nutrition interventions at the University of Pittsburgh; Alex Despines, of the University of Pittsburgh, for histological analysis; and the staff at the Clinical Translational Research Center, University of Pittsburgh. They also appreciate the excellent technical assistance of the East Carolina Clinical team. The authors also recognize the laboratory of Darrell Neufer at East Carolina University for technical assistance with mitochondrial respirometry assays and Ashley Cowart and the expertise at the Medical University of South Carolina Lipidomics core.
Parts of this study were presented in abstract form at the Cell Symposia on Exercise Metabolism, Amsterdam, the Netherlands, 12–14 July 2015.
Diabetes. 2015;64(11):3737-3750. © 2015 American Diabetes Association, Inc.