A reduction of skeletal muscle DHA content does not result in impaired whole body glucose tolerance or skeletal muscle basal insulin signaling in otherwise healthy mice.

Delta-6 desaturase (D6D), encoded by the Fads2 gene, catalyzes the first step in the conversion of α-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ablation of D6D in whole body Fads2-/- knockout (KO) mice results in an inability to endogenously produce EPA and DHA. Evidence supports a beneficial role for EPA and DHA on insulin-stimulated glucose disposal in skeletal muscle in the context of a metabolic challenge; however, it is unknown how low EPA and DHA levels impact skeletal muscle fatty acid composition and insulin signaling in a healthy context. The objective of this study was to examine the impact of ablating the endogenous production of EPA and DHA on skeletal muscle fatty acid composition, whole body glucose and insulin tolerance, and a key marker of skeletal muscle insulin signaling (pAkt). Male C57BL/6J wild-type (WT), Fads2+/- heterozygous, and Fads2-/- KO mice were fed a low-fat diet (16% kcal from fat) modified to contain either 7% w/w lard or 7% w/w flaxseed for 21 wk. No differences in total phospholipid (PL), triacylglycerol, or reactive lipid content were observed between genotypes. As expected, KO mice on both diets had significantly less DHA content in skeletal muscle PL. Despite this, KO mice did not have significantly different glucose or insulin tolerance compared with WT mice on either diet. Basal pAktSer473 was not significantly different between the genotypes within each diet. Ultimately, this study shows for the first time, to our knowledge, that the reduction of DHA in skeletal muscle is not necessarily detrimental to glucose homeostasis in otherwise healthy animals.NEW & NOTEWORTHY Skeletal muscle is the primary location of insulin-stimulated glucose uptake. EPA and DHA supplementation has been observed to improve skeletal muscle insulin-stimulated glucose uptake in models of metabolic dysfunction. Fads2-/- knockout mice cannot endogenously produce long-chain n-3 polyunsaturated fatty acids. Our results show that the absence of DHA in skeletal muscle is not detrimental to whole body glucose homeostasis in healthy mice.

Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats.

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

Glucose-dependent insulinotropic polypeptide directly induces glucose transport in rat skeletal muscle.

Several gastrointestinal proteins have been identified to have insulinotropic effects, including glucose-dependent insulinotropic polypeptide (GIP); however, the direct effects of incretins on skeletal muscle glucose transport remain largely unknown. Therefore, the purpose of the current study was to examine the role of GIP on skeletal muscle glucose transport and insulin signaling in rats. Relative to a glucose challenge, a mixed glucose+lipid oral challenge increased circulating GIP concentrations, skeletal muscle Akt phosphorylation, and improved glucose clearance by ∼35% (P < 0.05). These responses occurred without alterations in serum insulin concentrations. In an incubated soleus muscle preparation, GIP directly stimulated glucose transport and increased GLUT4 accumulation on the plasma membrane in the absence of insulin. Moreover, the ability of GIP to stimulate glucose transport was mitigated by the addition of the PI 3-kinase (PI3K) inhibitor wortmannin, suggesting that signaling through PI3K is required for these responses. We also provide evidence that the combined stimulatory effects of GIP and insulin on soleus muscle glucose transport are additive. However, the specific GIP receptor antagonist (Pro(3))GIP did not attenuate GIP-stimulated glucose transport, suggesting that GIP is not signaling through its classical receptor. Together, the current data provide evidence that GIP regulates skeletal muscle glucose transport; however, the exact signaling mechanism(s) remain unknown.

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.

Adiponectin is sufficient, but not required, for exercise-induced increases in the expression of skeletal muscle mitochondrial enzymes.

Adiponectin (Ad) has been proposed to be a regulator of mitochondrial biogenesis in skeletal muscle, and necessary for exercise-induced increases in mitochondrial content. We first confirmed that Ad could acutely increase the expression of mitochondrial proteins during a 10 h incubation in isolated soleus and extensor digitorum longus (EDL) muscles. Next, we further examined the role of Ad as a regulator of mitochondrial content using Ad knockout (AdKO) mice. The AdKO animals showed no differences in resting VO2, respiratory exchange ratio, or in time to exhaustion during exercise when compared to wild-type (WT) mice. There was a reduction in resting palmitate oxidation in isolated soleus from AdKO animals (-23%, P < 0.05) but not EDL, and 5-aminoimidazole-4-carboxamide (AICAR)-stimulated palmitate oxidation was similar in both genotypes regardless of muscle. There were no differences in protein markers of mitochondrial content (COX4, CORE1, CS, PDHE1α) in red and white gastrocnemius between WT and AdKO animals. A single bout of treadmill running increased the phosphorylation of AMP-activated protein kinase (AMPK) and the mRNA expression of mitochondrial proteins in red and white gastrocnemius in both WT and AdKO animals, with no differences between genotypes. Finally, 8 weeks of chronic exercise training increased the protein content of mitochondrial markers similarly (∼25-35%) in red gastrocnemius from both WT and AdKO mice. Collectively, our results demonstrate that the absence of Ad is not accompanied by reductions in mitochondrial protein content, or a reduction in aerobic exercise capacity. We conclude that Ad is not required for the maintenance of mitochondrial content, or for exercise-induced increases in skeletal muscle mitochondrial proteins.

Feeding butter with elevated content of trans-10, cis-12 conjugated linoleic acid to lean rats does not impair glucose tolerance or muscle insulin response.

BACKGROUND: Numerous studies have investigated the effects of isolated CLA supplementation on glucose homeostasis in humans and rodents. However, both the amount and relative abundance of CLA isomers in supplemental form are not representative of what is consumed from natural sources. No study to date has examined the effects of altered CLA isomer content within a natural food source. Our goal was to increase the content of the insulin desensitizing CLAt10,c12 isomer relative to the CLAc9,t11 isomer in cow's milk by inducing subacute rumenal acidosis (SARA), and subsequently investigate the effects of this milk fat on parameters related to glucose and insulin tolerance in rats. METHODS: We fed female rats (~2.5 to 3 months of age) CLA t10,c12 -enriched (SARA) butter or non-SARA butter based diets for 4 weeks in either low (10% of kcal from fat; 0.18% total CLA by weight) or high (60% of kcal from fat; 0.55% total CLA by weight) amounts. In an effort to extend these findings, we then fed rats high (60% kcal) amounts of SARA or non-SARA butter for a longer duration (8 weeks) and assessed changes in whole body glucose, insulin and pyruvate tolerance in comparison to low fat and 60% lard conditions. RESULTS: There was a main effect for increased fasting blood glucose and insulin in SARA vs. non-SARA butter groups after 4 weeks of feeding (p < 0.05). However, blood glucose and insulin concentration, and maximal insulin-stimulated glucose uptake in skeletal muscle were similar in all groups. Following 8 weeks of feeding, insulin tolerance was impaired by the SARA butter, but not glucose or pyruvate tolerance. The non-SARA butter did not impair tolerance to glucose, insulin or pyruvate. CONCLUSIONS: This study suggests that increasing the consumption of a naturally enriched CLAt10,c12 source, at least in rats, has minimal impact on whole body glucose tolerance or muscle specific insulin response.

Regulation of peripheral tissue substrate metabolism by the gut-derived hormone ghrelin.

Ghrelin increases in the circulation prior to entrained mealtimes, with the acylated (AG) form functioning to stimulate food intake and growth hormone release. Acutely, AG induces whole-body insulin resistance, potentially to maintain glycemia between meals. Alternatively, chronic administration of both AG and the unacylated isoform of ghrelin (unAG) is associated with improved skeletal muscle insulin sensitivity as well as reduced intramuscular lipids and inflammation. This may be due to effects on lipid metabolism, with ghrelin promoting storage of fat in adipose and liver while stimulating oxidation in skeletal muscle, preventing ectopic lipid accumulation. This is of specific relevance in the handling of meal-derived lipids, as ghrelin rises preprandially with effects persisting for 2-3 h following exposure in skeletal muscle, coinciding with elevated plasma FFAs. We hypothesize that ghrelin acts as a preparatory signal for incoming lipids, as well as a regulatory hormone for their use and storage. The effects of ghrelin on skeletal muscle are lost with high fat diet feeding and physical inactivity, potentially being implicated in the pathogenesis of metabolic disease. This review summarizes the metabolic effects of both ghrelin isoforms on peripheral tissues including the pancreas, adipose, liver, and skeletal muscle. Additionally, we speculate on the physiological relevance of these effects in vivo and suggest that ghrelin may be a key regulatory hormone for nutrient handling in the postprandial state.

Stimulation of fat oxidation in rat muscle by unacylated ghrelin persists for 2-3 hours, but is independent of fatty acid transporter translocation.

While a definitive mechanism-of-action remains to be identified, recent findings indicate that ghrelin, particularly the unacylated form (UnAG), stimulates skeletal muscle fatty acid oxidation. The biological importance of UnAG-mediated increases in fat oxidation remains unclear, as UnAG peaks in the circulation before mealtimes, and decreases rapidly during the postprandial situation before increases in postabsorptive circulating lipids. Therefore, we aimed to determine if the UnAG-mediated stimulation of fat oxidation would persist long enough to affect the oxidation of meal-derived fatty acids, and if UnAG stimulated the translocation of fatty acid transporters to the sarcolemma as a mechanism-of-action. In isolated soleus muscle strips from male rats, short-term pre-treatment with UnAG elicited a persisting stimulus on fatty acid oxidation 2 h after the removal of UnAG. UnAG also caused an immediate phosphorylation of AMPK, but not an increase in plasma membrane FAT/CD36 or FABPpm. There was also no increase in AMPK signaling or increased FAT/CD36 or FABPpm content at the plasma membrane at 2 h which might explain the sustained increase in fatty acid oxidation. These findings confirm UnAG as a stimulator of fatty acid oxidation and provide evidence that UnAG may influence the handling of postprandial lipids. The underlying mechanisms are not known.

Fish oil prevents high-saturated fat diet-induced impairments in adiponectin and insulin response in rodent soleus muscle.

High saturated fatty acid (SFA) diets contribute to the development of insulin resistance, whereas fish oil-derived n-3 polyunsaturated fatty acids (PUFA) increase the secretion of adiponectin (Ad), an adipocyte-derived protein that stimulates fatty acid oxidation (FAO) and improves skeletal muscle insulin response. We sought to determine whether fish oil could prevent and/or restore high SFA diet-induced impairments in Ad and insulin response in soleus muscle. Sprague-Dawley rats were fed 1) a low-fat control diet (CON group), 2) high-SFA diet (SFA group), or 3) high SFA with n-3 PUFA diet (SFA/n-3 PUFA group). At 4 wk, CON and SFA/n-3 PUFA animals were terminated, and SFA animals were either terminated or fed SFA or SFA/n-3 PUFA for an additional 2 or 4 wk. The effect of diet on Ad-stimulated FAO, insulin-stimulated glucose transport, and expression of Ad, insulin and inflammatory signaling proteins was determined in the soleus muscle. Ad stimulated FAO in CON and 4 wk SFA/n-3 PUFA (+36%, +39%, respectively P ≤ 0.05) only. Insulin increased glucose transport in CON, 4 wk SFA/n-3 PUFA, and 4 wk SFA + 4 wk SFA/n-3 PUFA (+82%, +33%, +25%, respectively P ≤ 0.05); this effect was lost in all other groups. TLR4 expression was increased with 4 wk of SFA feeding (+24%, P ≤ 0.05), and this was prevented in 4 wk SFA/n-3 PUFA. Suppressor of cytokine signaling-3 expression was increased in SFA and SFA/n-3 PUFA (+33 and +18%, respectively, P ≤ 0.05). Our results demonstrate that fish oil can prevent high SFA diet-induced impairments in both Ad and insulin response in soleus muscle.

Exercise restores insulin, but not adiponectin, response in skeletal muscle of high-fat fed rodents.

High-fat (HF) diets impair skeletal muscle response to the insulin-sensitizing adipokine adiponectin (Ad) in rodents, preceding the development of insulin resistance. Skeletal muscle insulin response in HF-fed rats can be restored with chronic exercise; whether recovery of skeletal muscle Ad response is necessary for the exercise-induced recovery of insulin-stimulated glucose transport is not known. In the current study, insulin and Ad resistance were induced in rodents with 4 wk of HF feeding (HF(4); low-fat fed animals used as control). Rats were then treadmill-exercised (HF(5)EX(1), HF(6)EX(2)) or supplemented orally with the pharmacological agent ß-guadinoproprionic acid (GPA; HF(5)GPA(1), HF(6)GPA(2)) for 1 or 2 wk with continued HF feeding. Insulin and Ad responses (glucose transport and palmitate oxidation, respectively) were assessed 48 h after the last exercise bout ex vivo in isolated solei. Insulin response was impaired following 4 wk of HF feeding and improved with 1 and 2 wk of exercise and ß-GPA supplementation (HF(5)EX(1), HF(6)EX(2), HF(5)GPA(1), and HF(6)GPA(2)). The recovery of insulin response generally coincided with improved Akt Thr(308) phosphorylation in HF(5)GPA(1), HF(6)EX(2), and HF(6)GPA(2), although not in HF(5)EX(1). Ad-stimulated palmitate oxidation was not restored with either treatment. Total protein contents of AdipoR1, AdipoR2, APPL1, and APPL2, as well as total and phosphorylated AMPK and ACC were unaltered by diet, exercise, and ß-GPA at the assessed time points. We conclude that the exercise and pharmacologically (ß-GPA)-induced recovery of skeletal muscle insulin response after HF feeding is not dependent on the restoration of Ad response, as assessed ex vivo.

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.

A single prior bout of exercise protects against palmitate-induced insulin resistance despite an increase in total ceramide content.

Ceramide accumulation has been implicated in the impairment of insulin-stimulated glucose transport in skeletal muscle following saturated fatty acid (FA) exposure. Importantly, a single bout of exercise can protect against acute lipid-induced insulin resistance. The mechanism by which exercise protects against lipid-induced insulin resistance is not completely known but may occur through a redirection of FA toward triacylglycerol (TAG) and away from ceramide and diacylglycerol (DAG). Therefore, in the current study, an in vitro preparation was used to examine whether a prior bout of exercise could confer protection against palmitate-induced insulin resistance and whether the pharmacological [50 µM fumonisin B(1) (FB1)] inhibition of ceramide synthesis in the presence of palmitate could mimic the protective effect of exercise. Soleus muscle of sedentary (SED), exercised (EX), and SED in the presence of FB1 (SED+FB1) were incubated with or without 2 mM palmitate for 4 h. This 2-mM palmitate exposure impaired insulin-stimulated glucose transport (-28%, P < 0.01) and significantly increased ceramide, DAG, and TAG accumulation in the SED group (P < 0.05). A single prior bout of exercise prevented the detrimental effects of palmitate on insulin signaling and caused a partial redistribution of FA toward TAG (P < 0.05). However, the net increase in ceramide content in response to palmitate exposure in the EX group was not different compared with SED, despite the maintenance of insulin sensitivity. The incubation of soleus from SED rats with FB1 (SED+FB1) prevented the detrimental effects of palmitate and caused a redirection of FA toward TAG accumulation (P < 0.05). Therefore, this research suggests that although inhibiting ceramide accumulation can prevent the detrimental effects of palmitate, a single prior bout of exercise appears to protect against palmitate-induced insulin resistance, which may be independent of changes in ceramide content.

Recovered insulin response by 2 weeks of leptin administration in high-fat fed rats is associated with restored AS160 activation and decreased reactive lipid accumulation.

Leptin is an adipokine that increases fatty acid (FA) oxidation, decreases intramuscular lipid stores, and improves insulin response in skeletal muscle. In an attempt to elucidate the underlying mechanisms by which these metabolic changes occur, we administered leptin (Lep) or saline (Sal) by miniosmotic pumps to rats during the final 2 wk of a 6-wk low-fat (LF) or high-fat (HF) diet. Insulin-stimulated glucose transport was impaired by the HF diet (HF-Sal) but was restored with leptin administration (HF-Lep). This improvement was associated with restored phosphorylation of Akt and AS160 and decreased in reactive lipid species (ceramide, diacylglycerol), known inhibitors of the insulin-signaling cascade. Total muscle citrate synthase (CS) activity was increased by both leptin and HF diet, but was not additive. Leptin increased subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria CS activity. Total muscle, sarcolemmal, and mitochondrial (SS and IMF) FA transporter (FAT/CD36) protein content was significantly increased with the HF diet, but not altered by leptin. Therefore, the decrease in reactive lipid stores and subsequent improvement in insulin response, secondary to leptin administration in rats fed a HF diet was not due to a decrease in FA transport protein content or altered cellular distribution.

Restoration of skeletal muscle leptin response does not precede the exercise-induced recovery of insulin-stimulated glucose uptake in high-fat-fed rats.

Leptin administration increases fatty acid (FA) oxidation rates and decreases lipid storage in oxidative skeletal muscle, thereby improving insulin response. We have previously shown high-fat (HF) diets to rapidly induce skeletal muscle leptin resistance, prior to the disruption of normal muscle FA metabolism (increase in FA transport; accumulation of triacylglycerol, diacylglycerol, ceramide) that occurs in advance of impaired insulin signaling and glucose transport. All of this occurs within a 4-wk period. Conversely, exercise can rapidly improve insulin response, in as little as one exercise bout. Thus, if the early development of leptin resistance is a contributor to HF diet-induced insulin resistance (IR) in skeletal muscle, then it is logical to predict that the rapid restoration of insulin response by exercise training would be preceded by the recovery of leptin response. In the current study, we sought to determine 1) whether 1, 2, or 4 wk of exercise training was sufficient to restore leptin response in isolated soleus muscle of rats already consuming a HF diet (60% kcal), and 2) whether this preceded the training-induced corrections in FA metabolism and improved insulin-stimulated glucose transport. In the low-fat (LF)-fed control group, insulin increased glucose transport by 153% and leptin increased AMPK and ACC phosphorylation and the rate of palmitate oxidation (+73%). These responses to insulin and leptin were either severely blunted or absent following 4 wk of HF feeding. Exercise intervention decreased muscle ceramide content (-28%) and restored insulin-stimulated glucose transport to control levels within 1 wk; muscle leptin response (AMPK and ACC phosphorylation, FA oxidation) was also restored, but not until the 2-wk time point. In conclusion, endurance exercise training is able to restore leptin response, but this does not appear to be a necessary precursor for the restoration of insulin response.

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.

Skeletal muscle inflammation is not responsible for the rapid impairment in adiponectin response with high-fat feeding in rats.

Adiponectin (Ad) is an insulin-sensitizing adipokine known to stimulate fatty acid (FA) oxidation in skeletal muscle. Skeletal muscle can become resistant to Ad very rapidly, after only 3 days of high saturated fat feeding in rats. Whether the same occurs following a high polyunsaturated fat diet is unknown. Obesity, insulin resistance, and hyperlipidemia are recognized as low-grade inflammatory diseases; therefore, we hypothesized that high-fat feeding induces inflammation, which interferes with Ad action at skeletal muscle. To this end, rats were placed into one of three dietary groups, control (CON, 10% kcal from fat), high saturated (SAT), or high polyunsaturated (PUFA) fat (60% kcal from fat) for 3 days to determine whether Ad resistance develops. Half of the animals from each group were further supplemented with aspirin, a common anti-inflammatory drug. Ad stimulated FA metabolism, Ad signaling intermediates [AdipoR1, APPL1, LKB1, AMPK, and acetyl-CoA carboxylase (ACC)], and inflammatory proteins [Toll-like receptor (TLR4), IKK alpha/beta, IkappaB alpha, NF-kappaB, suppressor of cytokine signaling-3 (SOCS3), and JNK] were measured in soleus muscle. Three days of SAT feeding induced Ad resistance in soleus muscle, assessed as an inability of Ad to phosphorylate ACC and increase FA oxidation. In PUFA-fed animals, Ad-stimulated FA oxidation and ACC phosphorylation to the same degree as CON animals (FA oxidation: +35%, +41%; pACC +29%, +19%; CON, PUFA, P < 0.05). However, neither SAT nor PUFA feeding for 3 days induced skeletal muscle inflammation. Surprisingly, aspirin prevented Ad-stimulated increases in FA oxidation. In conclusion, FA type is critical in the development of Ad resistance, but this does not appear to be mediated by inflammation.

Oral administration of a PPAR-delta agonist to rodents worsens, not improves, maximal insulin-stimulated glucose transport in skeletal muscle of different fibers.

Agonists targeting the nuclear receptor peroxisome proliferator-activated receptors (PPAR)-delta may be potential therapeutic agents for insulin-resistant related conditions, as they may be able to stimulate fatty acid (FA) oxidation and attenuate the accumulation of harmful lipid species in skeletal muscle. Several reports have demonstrated that PPAR-delta agonists improve whole body insulin sensitivity. However, whether these agonists exert their direct effects on glucose and FA metabolism in skeletal muscle, and specifically with different fiber types, is unknown. This study was undertaken to determine the effects of oral treatment with the PPAR-delta agonist, GW 501516, in conjunction with the administration of a high-saturated-fat diet on insulin-stimulated glucose transport in isolated oxidative (soleus) and glycolytic (epitrochlearis) rodent skeletal muscle in vitro. High-fat feeding significantly decreased maximal insulin-stimulated glucose transport in soleus, but not epitrochlearis muscle, and was associated with increased skeletal muscle diacylglycerol and ceramide content. Unexpectedly, treatment with the PPAR-delta agonist significantly reduced insulin-stimulated glucose transport in both soleus and epitrochlearis muscles, regardless of dietary fat content. The reduction in insulin-stimulated glucose transport induced by the agonist was associated with large increases in total muscle fatty acid translocase (FAT)/CD36protein content, but not diacylglycerol or ceramide contents. Agonist treatment did not alter the protein content of PPAR-delta, GLUT4, or insulin-signaling proteins (IRS-1, p85 PI3-K, Akt). Agonist treatment led to a small, but significant increase, in the oxidative capacity of glycolytic but not oxidative muscle. We propose that chronic treatment with the PPAR-delta agonist GW 501516 may induce or worsen insulin resistance in rodent skeletal muscle by increasing the capacity for FA transport across the sarcolemma without a sufficient compensatory increase in FA oxidation. However, an accumulation of diacylglycerol and ceramide, while associated with diet-induced insulin resistance, does not appear to be responsible for the agonist-induced reduction in insulin-stimulated glucose transport.

The interaction between adipokines, diet and exercise on muscle insulin sensitivity.

PURPOSE OF REVIEW: High-fat diets lead to obesity and increase the risk of developing insulin resistance and type 2 diabetes. Adipose tissue and skeletal muscle act as endocrine organs, and produce various cytokines that can potentially alter peripheral insulin sensitivity. The purpose of the present review is to briefly summarize the effects of major cytokines (leptin, adiponectin, tumor necrosis factor-alpha, and interleukin-6) on muscle metabolism and insulin response, with a focus on the effects of diet and exercise. RECENT FINDINGS: Leptin and adiponectin improve insulin sensitivity. However, in obesity there is a diminished response to these adipokines. This resistance can be induced very rapidly and may lead to subsequent impairments in insulin response. Tumor necrosis factor-alpha is a proinflammatory cytokine that has been implicated as a mediator of insulin resistance, particularly in obesity. Interleukin-6 was the first identified myokine. There is evidence to implicate interleukin-6 both as a mediator of impaired insulin action in obesity, and also as a facilitator of increased fuel metabolism during exercise. The effect of each of these cytokines on muscle insulin sensitivity can be modulated by diet and exercise. SUMMARY: Much of the information summarized in the present review focuses on the effects of various cytokines in isolation, although in vivo there can be considerable interaction with each other. Future research should consider these potential interactions.

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.

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.