Mass spectrometry-based proteomics approaches to interrogate skeletal muscle adaptations to exercise.

Acute exercise and chronic exercise training elicit beneficial whole-body changes in physiology that ultimately depend on profound alterations to the dynamics of tissue-specific proteins. Since the work accomplished during exercise owes predominantly to skeletal muscle, it has received the majority of interest from exercise scientists that attempt to unravel adaptive mechanisms accounting for salutary metabolic effects and performance improvements that arise from training. Contemporary scientists are also beginning to use mass spectrometry-based proteomics, which is emerging as a powerful approach to interrogate the muscle protein signature in a more comprehensive manner. Collectively, these technologies facilitate the analysis of skeletal muscle protein dynamics from several viewpoints, including changes to intracellular proteins (expression proteomics), secreted proteins (secretomics), post-translational modifications as well as fiber-, cell-, and organelle-specific changes. This review aims to highlight recent literature that has leveraged new workflows and advances in mass spectrometry-based proteomics to further our understanding of training-related changes in skeletal muscle. We call attention to untapped areas in skeletal muscle proteomics research relating to exercise training and metabolism, as well as basic points of contention when applying mass spectrometry-based analyses, particularly in the study of human biology. We further encourage researchers to couple the hypothesis-generating and descriptive nature of omics data with functional analyses that propel our understanding of the complex adaptive responses in skeletal muscle that occur with acute and chronic exercise.

Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M.

The discovery of exercise-regulated circulatory factors has fueled interest in organ crosstalk, especially between skeletal muscle and adipose tissue, and the role in mediating beneficial effects of exercise. We studied the adipose tissue transcriptome in men and women with normal glucose tolerance or type 2 diabetes following an acute exercise bout, revealing substantial exercise- and time-dependent changes, with sustained increase in inflammatory genes in type 2 diabetes. We identify oncostatin-M as one of the most upregulated adipose-tissue-secreted factors post-exercise. In cultured human adipocytes, oncostatin-M enhances MAPK signaling and regulates lipolysis. Oncostatin-M expression arises predominantly from adipose tissue immune cell fractions, while the corresponding receptors are expressed in adipocytes. Oncostatin-M expression increases in cultured human Thp1 macrophages following exercise-like stimuli. Our results suggest that immune cells, via secreted factors such as oncostatin-M, mediate a crosstalk between skeletal muscle and adipose tissue during exercise to regulate adipocyte metabolism and adaptation.

The effects of diet and chronic exercise on skeletal muscle ghrelin response.

Background: Recent findings indicate that ghrelin, particularly the unacylated form (UnAG), acutely stimulates skeletal muscle fatty acid oxidation (FAO) and can preserve insulin signaling and insulin-stimulated glucose uptake in the presence of high concentrations of saturated fatty acids. However, we recently reported that the stimulatory effect of ghrelin on FAO and subsequent ability to protect insulin stimulated glucose uptake was lost following 6-weeks (6w) of chronic high fat feeding. In the current study we examined the effects of both short-term 5 day (5d) and chronic 6w high-fat diet (HFD) on muscle ghrelin response, and whether exercise training could prevent the development of muscle ghrelin resistance with 6w of HFD. Methods and Results: Soleus muscle strips were isolated from male rats to determine the direct effects of acylated (AG) and UnAG isoforms on FAO and glucose uptake. A 5d HFD did not alter the response of soleus muscle to AG or UnAG. Conversely, 6w of HFD was associated with a loss of ghrelin's ability to stimulate FAO and protect insulin stimulated glucose uptake. Muscle response to UnAG remained intact following the 6w HFD with chronic exercise training. Unexpectedly, muscle response to both AG and UnAG was also lost after 6w of low-fat diet (LFD) consumption. Protein content of the classic ghrelin receptor, GHS-R1a, was not affected by diet or training. Corticotropin-releasing hormone receptor-2 (CRF-2R) content, a putative receptor for ghrelin in muscle, was significantly decreased in soleus from 6w HFD-fed animals and increased following exercise training. This may explain the protection of UnAG response with training in HFD-fed rats but does not explain why ghrelin response was also lost in LFD-fed animals. Conclusions: UnAG protects muscle glucose uptake during acute lipid oversupply, likely due to its ability to stimulate FAO. This effect is lost in 6w HFD-fed animals but protected with exercise training. Unexpectedly, ghrelin response was lost in 6w LFD-fed animals. The loss of ghrelin response in muscle with a LFD cannot be explained by a change in putative ghrelin receptor content. We believe that the sedentary nature of the animals is a major factor in the development of muscle ghrelin resistance and warrants further research.

Insulin and 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Differentially Regulate the Skeletal Muscle Cell Secretome.

Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR-an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.

Regulation of adipose tissue lipolysis by ghrelin is impaired with high-fat diet feeding and is not restored with exercise.

Ghrelin is released from the stomach as an anticipatory signal prior to a meal and decreases immediately after. Previous research has shown that both acylated (AG) and unacylated (UnAG) ghrelin blunt adrenoreceptor-stimulated lipolysis in rat white adipose tissue (WAT) ex vivo. We investigated whether acute or chronic consumption of a high fat diet (HFD) impaired the ability of ghrelin to regulate adipose tissue lipolysis, and if this impairment could be restored with exercise. After 5 days (5d) of a HFD, or 6 weeks (6 w) of a HFD (60% kcal from fat) with or without exercise training, inguinal and retroperitoneal WAT was collected from anesthetized rats for adipose tissue organ culture. Samples were treated with 1 µM CL 316,243 (CL; lipolytic control), 1 µM CL+150 ng/ml AG or 1 µM CL+150 ng/ml UnAG. Incubation media and tissue were collected after 2 hours. Colorometric assays were used to determine glycerol and free fatty acid (FFA) concentrations in media. Western blots were used to quantify the protein content of lipolytic enzymes and ghrelin receptors in both depots. CL stimulated lipolysis was evidenced by increases in glycerol (p < 0.0001) and FFA (p < 0.0001) concentrations in media compared to control. AG decreased CL-stimulated glycerol release in inguinal WAT from 5d LFD rats (p = 0.0097). Neither AG nor UnAG blunted lipolysis in adipose tissue from 5d or 6 w HFD-fed rats, and exercise did not restore ghrelin's anti-lipolytic ability in 6 w HFD-fed rats. Overall, this study demonstrates that HFD consumption impairs ghrelin's ability to regulate adipose tissue lipolysis.

The torque-frequency relationship is impaired similarly following two bouts of eccentric exercise: No evidence of a protective repeated bout effect.

High-intensity eccentric exercise can lead to muscle damage and weakness. The 'repeated bout effect' (RBE) can attenuate these impairments when performing a subsequent bout. The influence of eccentric exercise-induced muscle damage on low-frequency force production is well-characterized; however, it is unclear how eccentric exercise and the RBE affect torque production across a range of stimulation frequencies (i.e., the torque-frequency relationship). We investigated the influence of an initial (Bout 1) and repeated bout (Bout 2) of eccentric exercise on the elbow flexor torque-frequency relationship. Eleven males completed two bouts of high-intensity eccentric elbow flexions, 4 weeks apart. Torque-frequency relationships were constructed at baseline and 0.5, 24, 48, 72, 96, and 168 h following both bouts via percutaneous stimulation at 1, 6, 10, 20, 30, 40, 50, and 100 Hz. Serum creatine kinase activity, self-reported muscle soreness, and isometric maximum voluntary contraction torque indirectly inferred the presence of muscle damage following Bout 1, and attenuation of muscle damage following Bout 2. Torque amplitude at all stimulation frequencies was impaired 30 min following eccentric exercise, however, torque at lower (1-10 Hz) and higher frequencies (40-100 Hz) recovered within 24 h while torque across the middle frequency range (20-30 Hz) recovered by 48 h. No between-bout differences were detected in absolute or normalized torque at any stimulation frequency, indicating no protective RBE on the elbow flexor torque-frequency relationship.

Power loss is attenuated following a second bout of high-intensity eccentric contractions due to the repeated bout effect's protection of rate of torque and velocity development.

High-intensity unaccustomed eccentric contractions result in weakness and power loss because of fatigue and muscle damage. Through the repeated bout effect (RBE), adaptations occur, then damage and weakness are attenuated following a subsequent bout. However, it is unclear whether the RBE protects peak power output. We investigated the influence of the RBE on power production and estimated fatigue- and damage-induced neuromuscular impairments following repeated high-intensity eccentric contractions. Twelve healthy adult males performed 5 sets of 30 maximal eccentric elbow flexions and repeated an identical bout 4 weeks later. Recovery was tracked over 7 days following both bouts. Reduced maximum voluntary isometric contraction torque, and increased serum creatine kinase and self-reported soreness indirectly inferred muscle damage. Peak isotonic power, time-dependent measures - rate of velocity development (RVD) and rate of torque development (RTD) - and several electrophysiological indices of neuromuscular function were assessed. The RBE protected peak power, with a protective index of 66% 24 h after the second eccentric exercise bout. The protection of power also related to preserved RVD (R2 = 0.61, P < 0.01) and RTD (R2 = 0.39, P < 0.01). Furthermore, the RBE's protection against muscle damage permitted the estimation of fatigue-associated neuromuscular performance decrements following eccentric exercise. Novelty: The repeated bout effect protects peak isotonic power. Protection of peak power relates to preserved rates of torque and velocity development, but more so rate of velocity development. The repeated bout effect has little influence on indices of neuromuscular fatigue.

Unacylated ghrelin stimulates fatty acid oxidation to protect skeletal muscle against palmitate-induced impairment of insulin action in lean but not high-fat fed rats.

BACKGROUND: Ghrelin is a gut hormone that spikes in circulation before mealtime. Recent findings suggest that both ghrelin isoforms stimulate skeletal muscle fatty acid oxidation, lending to the possibility that it may regulate skeletal muscle's handling of meal-derived substrates. It was hypothesized in the current study that ghrelin may preserve muscle insulin response during conditions of elevated saturated fatty acid (palmitate) availability by promoting its oxidation. METHODS AND RESULTS: Soleus muscle strips were isolated from male rats to determine the direct effects of ghrelin isoforms on fatty acid oxidation, glucose uptake and insulin signaling. We demonstrate that unacylated ghrelin (UnAG) is the more potent stimulator of skeletal muscle fatty acid oxidation. Both isoforms of ghrelin generally protected muscle from impaired insulin-mediated phosphorylation of AKT Ser473 and Thr308, as well as downstream phosphorylation of AS160 Ser588 during high palmitate exposure. However, only UnAG was able to preserve insulin-stimulated glucose uptake during exposure to high palmitate concentrations. The use of etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase (CPT-1) abolished this protection, strongly suggesting that UnAG's stimulation of fatty acid oxidation may be essential to this protection. To our knowledge, we are also the first to investigate the impact of a chronic high-fat diet on ghrelin's actions in muscle. Following 6 wks of a high-fat diet, UnAG was unable to preserve insulin-stimulated signaling or glucose transport during an acute high palmitate exposure. UnAG was also unable to further stimulate 5' AMP-activated protein kinase (AMPK) or fatty acid oxidation during high palmitate exposure. Corticotropin-releasing hormone receptor-2 (CRF-2R) content was significantly decreased in muscle from high-fat fed animals, which may partially account for the loss of UnAG's effects. CONCLUSIONS: UnAG is able to protect muscle from acute lipid exposure, likely due to its ability to stimulation fatty acid oxidation. This effect is lost in high-fat fed animals, implying a resistance to ghrelin at the level of the muscle. The underlying mechanisms accounting for ghrelin resistance in high fat-fed animals remain to be discovered.

Regulation of adipose tissue and skeletal muscle substrate metabolism by the stomach-derived hormone, ghrelin.

Ghrelin is a stomach-derived hormone and a potent appetite stimulant. Ghrelin has recently harbored interest as a potential regulator of carbohydrate and lipid metabolism in skeletal muscle and adipose tissue; however, in vivo ghrelin administration is confounded by secondary effects. The assessment of the direct metabolic effects of acylated (AG) and unacylated (UnAG) ghrelin is a relatively new area of research. In isolated adipocytes and muscle, ghrelin has demonstrated antilipolytic effects. In muscle, ghrelin has been shown to acutely stimulate fat oxidation, which may protect the muscle from the insulin-desensitizing effects of high fatty acid concentrations. The effects of ghrelin directly on muscle glucose uptake are controversial. Whether ghrelin can be utilized therapeutically for conditions such as type 2 diabetes will depend on our better understanding of ghrelin's independent effects on muscle and adipose tissue metabolism, and whether this can predict ghrelin's effects when administered in vivo.

Resting metabolic rate and skeletal muscle SERCA and Na+ /K+ ATPase activities are not affected by fish oil supplementation in healthy older adults.

Omega-3 polyunsaturated fatty acids (PUFAs) have unique properties purported to influence several aspects of metabolism, including energy expenditure and protein function. Supplementing with n-3 PUFAs may increase whole-body resting metabolic rate (RMR), by enhancing Na+ /K+ ATPase (NKA) activity and reducing the efficiency of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) activity by inducing a Ca2+ leak-pump cycle. The purpose of this study was to examine the effects of fish oil (FO) on RMR, substrate oxidation, and skeletal muscle SERCA and NKA pump function in healthy older individuals. Subjects (n = 16 females; n = 8 males; 65 ± 1 years) were randomly assigned into groups supplemented with either olive oil (OO) (5 g/day) or FO (5 g/day) containing 2 g/day eicosapentaenoic acid and 1 g/day docosahexaenoic acid for 12 weeks. Participants visited the laboratory for RMR and substrate oxidation measurements after an overnight fast at weeks 0 and 12. Skeletal muscle biopsies were taken during weeks 0 and 12 for analysis of NKA and SERCA function and protein content. There was a main effect of time with decrease in RMR (5%) and fat oxidation (18%) in both the supplementation groups. The kinetic parameters of SERCA and NKA maximal activity, as well as the expression of SR and NKA proteins, were not affected after OO and FO supplementation. In conclusion, these results suggest that FO supplementation is not effective in altering RMR, substrate oxidation, and skeletal muscle SERCA and NKA protein levels and activities, in healthy older men and women.

Ghrelin stimulates fatty acid oxidation and inhibits lipolysis in isolated muscle from male rats.

Ghrelin is classically known as a central appetite-stimulating hormone but has recently been recognized to have a significant role in peripheral tissue energy metabolism. However, the direct effects of ghrelin on skeletal muscle, a major site for glucose and lipid disposal, remain understudied. We found that the two major ghrelin isoforms, acylated and unacylated ghrelin, were able to significantly increase skeletal muscle fatty acid oxidation (~20%) while incorporation of fatty acids into major lipid pools remained unchanged. The increase in fatty acid oxidation was accompanied by increases in acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK. Ghrelin isoforms had no independent effect on lipolysis under unstimulated conditions, but nearly completely abolished epinephrine-stimulated lipolysis. This effect was generally, but not consistently related to a blunting in the phosphorylation of HSL activation sites, Ser660 and 563. Taken together, these findings suggest that ghrelin isoforms have a direct, acute effect on fatty acid oxidation and lipolysis.

No effect of beetroot juice supplementation on exercise economy and performance in recreationally active females despite increased torque production.

This study investigated the effects of acute and chronic beetroot juice (BRJ) supplementation on submaximal exercise oxygen uptake (VO2 ), time trial (TT) performance, and contractile properties of the plantar flexors in females. Study 1: Using a double blind, randomized, crossover design, 12 recreationally active females using hormonal contraceptives supplemented acutely (2.5 h) and chronically (8 days) with 280 mL BRJ/d (~26 mmoles nitrate [ NO3- ]) or a NO3- -free placebo (PLA). On days 1 and 8, participants cycled for 10 min at 50% and 70% VO2peak and completed a 4 kJ/kg body mass TT. Plasma [ NO3- ] and nitrite ([NO2- ]) increased significantly following BRJ supplementation versus PLA. There was no effect of BRJ supplementation on VO2 at 50% or 70% VO2peak , or TT performance. Study 2: 12 recreationally active females (n = 7 from Study 1) using hormonal contraceptives participated in a baseline visit and were supplemented acutely (2.5 h) and chronically (8 days) with 280 mL BRJ/d. Maximum voluntary strength (MVC) of the plantar flexors was assessed and a torque-frequency curve performed. BRJ had no effect on MVC, voluntary activation, peak twitch torque, time to peak torque, or half relaxation time. Following both acute (46.6 ± 4.9% of 100 Hz torque) and chronic (47.2 ± 4.4%) supplementation, 10 Hz torque was significantly greater compared to baseline (32.9 ± 2.6%). In summary, BRJ may not be an effective ergogenic aid in recreationally active females as it did not reduce submaximal exercise VO2 or improve aerobic TT performance despite increasing low frequency torque production.

Acylated and unacylated ghrelin directly regulate ß-3 stimulated lipid turnover in rodent subcutaneous and visceral adipose tissue ex vivo but not in vivo.

Ghrelin has garnered interest as a gut-derived regulator of lipid metabolism, beyond its classical roles in driving appetite and growth hormone release. Ghrelin's circulating concentrations follow an ultradian rhythm, peak immediately before a meal and point towards a potential metabolic role in reducing the mobilization of fatty acid stores in preparation for the storage of ingested food. Here, we demonstrate that both acylated and unacylated ghrelin have physiological roles in attenuating lipolysis in mature subcutaneous and visceral adipose tissue depots of rats. Ghrelin blunted the ß3-induction (CL 316, 243) of glycerol release (index of lipolysis) which coincided with a reduced activation of the key lipid hydrolase HSL at two of its serine residues (Ser563/660). Furthermore, ghrelin appeared to inhibit fatty acid reesterification in the presence of CL such that fatty acid concentrations in the surrounding media were maintained in spite of a reduction in lipolysis. Importantly, these aforementioned effects were not observed following ghrelin injection in vivo, as there was no attenuation of CL-induced glycerol release. This highlights the importance of exercising caution when interpreting the effects of administering ghrelin in vivo, and the necessity for uncovering the elusive mechanisms by which ghrelin regulates lipolysis and fatty acid reesterification. We conclude that both acylated and unacylated ghrelin can exert direct inhibitory effects on lipolysis and fatty acid reesterification in adipose tissue from rats. However, these effects are not observed in vivo and outline the complexity of studying ghrelin's effects on fatty acid metabolism in the living animal.

Acylated and unacylated ghrelin do not directly stimulate glucose transport in isolated rodent skeletal muscle.

Emerging evidence implicates ghrelin, a gut-derived, orexigenic hormone, as a potential mediator of insulin-responsive peripheral tissue metabolism. However, in vitro and in vivo studies assessing ghrelin's direct influence on metabolism have been controversial, particularly due to confounding factors such as the secondary rise in growth hormone (GH) after ghrelin injection. Skeletal muscle is important in the insulin-stimulated clearance of glucose, and ghrelin's exponential rise prior to a meal could potentially facilitate this. This study was aimed at elucidating any direct stimulatory action that ghrelin may have on glucose transport and insulin signaling in isolated rat skeletal muscle, in the absence of confounding secondary factors. Oxidative soleus and glycolytic extensor digitorum longus skeletal muscles were isolated from male Sprague Dawley rats in the fed state and incubated with various concentrations of acylated and unacylated ghrelin in the presence or absence of insulin. Ghrelin did not stimulate glucose transport in either muscle type, with or without insulin. Moreover, GH had no acute, direct stimulatory effect on either basal or insulin-stimulated muscle glucose transport. In agreement with the lack of observed effect on glucose transport, ghrelin and GH also had no stimulatory effect on Ser473 AKT or Thr172 AMPK phosphorylation, two key signaling proteins involved in glucose transport. Furthermore, to our knowledge, we are among the first to show that ghrelin can act independent of its receptor and cause an increase in calmodulin-dependent protein kinase 2 (CaMKII) phosphorylation in glycolytic muscle, although this was not associated with an increase in glucose transport. We conclude that both acylated and unacylated ghrelin have no direct, acute influence on skeletal muscle glucose transport. Furthermore, the immediate rise in GH in response to ghrelin also does not appear to directly stimulate glucose transport in muscle.

Exercise training is an effective alternative to estrogen supplementation for improving glucose homeostasis in ovariectomized rats.

The irreversible loss of estrogen (specifically 17-ß-estradiol; E2) compromises whole-body glucose tolerance in women. Hormone replacement therapy (HRT) is frequently prescribed to treat estrogen deficiency, but has several deleterious side effects. Exercise has been proposed as an HRT substitute, however, their relative abilities to treat glucose intolerance are unknown. Thirty ovariectomized (OVX) and 20 SHAM (control) rats underwent glucose tolerance tests (GTT) 10 weeks post surgery. Area under the curve (AUC) for OVX rats was 60% greater than SHAM controls (P = 0.0005). Rats were then randomly assigned to the following treatment groups: SHAM sedentary (sed) or exercise (ex; 60 min, 5×/weeks), OVX sed, ex, or E2 (28 µg/kg bw/day) for 4 weeks. OVX ex rats experienced a ~45% improvement in AUC relative to OVX sed rats, whereas OVX E2 underwent a partial reduction (17%; P = 0.08). Maximal insulin-stimulated glucose uptake in soleus and EDL was not impaired in OVX rats, or augmented with exercise or E2. Akt phosphorylation did not differ in soleus, EDL, or liver of any group. However, OVX ex and OVX E2 experienced greater increases in p-Akt Ser473 in VAT and SQ tissues compared with SHAM and OVX sed groups. Mitochondrial markers CS, COXIV, and core1 were increased in soleus posttraining in OVX ex rats. The content of COXIV was reduced by 52% and 61% in SQ of OVX sed and E2 rats, compared to SHAM controls, but fully restored in OVX ex rats. In summary, exercise restores glucose tolerance in OVX rats more effectively than E2. This is not reflected by alterations in muscle maximal insulin response, but increased insulin signaling in adipose depots may underlie whole-body improvements.

Feeding butter with elevated content of trans-10, cis-12 conjugated linoleic acid to obese-prone rats impairs glucose and insulin tolerance.

BACKGROUND: We recently demonstrated that feeding a natural CLAt10,c12-enriched butter to lean female rats resulted in small, but significant increases in fasting glucose and insulin concentrations, and impaired insulin tolerance. Our goal was to extend these findings by utilizing the diabetes-prone female fatty Zucker rat. Rats were fed custom diets containing 45 % kcal of fat derived from control and CLAt10,c12-enriched butter for 8 weeks. METHODS: CLA t10,c12-enriched butter was prepared from milk collected from cows fed a high fermentable carbohydrate diet to create subacute rumen acidosis (SARA); control (non-SARA) butter was collected from cows fed a low grain diet. Female fatty Zucker rats (10 weeks old) were randomly assigned to one of four diet treatments: i) low fat (10 % kcal), ii) 45 % kcal lard, iii) 45 % kcal SARA butter, or iv) 45 % kcal non-SARA butter. A low fat fed lean Zucker group was used as a control group. After 8 weeks, i) glucose and insulin tolerance tests, ii) insulin signaling in muscle, adipose and liver, and iii) metabolic caging measurements were performed. RESULTS: Glucose and insulin tolerance were significantly impaired in all fatty Zucker groups, but to the greatest extent in the LARD and SARA conditions. Insulin signaling (AKT phosphorylation) was impaired in muscle, visceral (perigonadal) adipose tissue and liver in fatty Zucker rats, but was generally similar across dietary groups. Physical activity, oxygen consumption, food intake and weight gain were also similar amongst the various fatty Zucker groups. CONCLUSIONS: Increasing the consumption of a food naturally enriched with CLAt10,c12 significantly worsens glucose and insulin tolerance in a diabetes-prone rodent model. This outcome is not explained by changes in tissue insulin signaling, physical activity, energy expenditure, food intake or body mass.