Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Phospholamban deficiency does not alter skeletal muscle SERCA pumping efficiency or predispose mice to diet-induced obesity.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump is a major contributor to skeletal muscle Ca2+ homeostasis and metabolic rate. SERCA activity can become adaptively uncoupled by its regulator sarcolipin (SLN) to increase the energy demand of Ca2+ pumping, preventing excessive obesity and glucose intolerance in mice. Several other SERCA regulators bear structural and functional resemblance to SLN, including phospholamban (PLN). Here, we sought to examine whether endogenous levels of skeletal muscle PLN control SERCA Ca2+ pumping efficiency and whole body metabolism. Using PLN-null mice ( Pln-/-), we found that soleus (SOL) muscle's SERCA pumping efficiency (measured as an apparent coupling ratio: Ca2+ uptake/ATP hydrolysis) was unaffected by PLN. Expression of Ca2+-handling proteins within the SOL, including SLN, were comparable between Pln-/- and wild-type (WT) littermates, as were fiber-type characteristics. Not surprisingly then, Pln-/- mice developed a similar degree of diet-induced obesity and glucose intolerance as WT controls when given a "Western" high-fat diet. Lack of an excessively obesogenic phenotype of Pln-/- could not be explained by compensation from skeletal muscle SLN or brown adipose tissue uncoupling protein-1 content. In agreement with several other reports, our study lends support to the notion that PLN serves a functionally distinct role from that of SLN in skeletal muscle physiology.

Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle.

Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I + II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ-/- soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatß and Lpaatε in Lpaatδ-/- mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL.

Effects of sarcolipin deletion on skeletal muscle adaptive responses to functional overload and unload.

Overexpression of sarcolipin (SLN), a regulator of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), stimulates calcineurin signaling to enhance skeletal muscle oxidative capacity. Some studies have shown that calcineurin may also control skeletal muscle mass and remodeling in response to functional overload and unload stimuli by increasing myofiber size and the proportion of slow fibers. To examine whether SLN might mediate these adaptive responses, we performed soleus and gastrocnemius tenotomy in wild-type (WT) and Sln-null (Sln-/-) mice and examined the overloaded plantaris and unloaded/tenotomized soleus muscles. In the WT overloaded plantaris, we observed ectopic expression of SLN, myofiber hypertrophy, increased fiber number, and a fast-to-slow fiber type shift, which were associated with increased calcineurin signaling (NFAT dephosphorylation and increased stabilin-2 protein content) and reduced SERCA activity. In the WT tenotomized soleus, we observed a 14-fold increase in SLN protein, myofiber atrophy, decreased fiber number, and a slow-to-fast fiber type shift, which were also associated with increased calcineurin signaling and reduced SERCA activity. Genetic deletion of Sln altered these physiological outcomes, with the overloaded plantaris myofibers failing to grow in size and number, and transition towards the slow fiber type, while the unloaded soleus muscles exhibited greater reductions in fiber size and number, and an accelerated slow-to-fast fiber type shift. In both the Sln-/- overloaded and unloaded muscles, these findings were associated with elevated SERCA activity and blunted calcineurin signaling. Thus, SLN plays an important role in adaptive muscle remodeling potentially through calcineurin stimulation, which could have important implications for other muscle diseases and conditions.

Agpat4/Lpaatδ deficiency highlights the molecular heterogeneity of epididymal and perirenal white adipose depots.

Acylglycerophosphate acyltransferase 4 (AGPAT4)/lysophosphatidic acid acyltransferase delta catalyzes the formation of phosphatidic acid (PA), a precursor of triacyl-glycerol (TAG). We investigated the effect of Agpat4 gene ablation on white adipose tissue (WAT) after finding consistent expression across depots. Epididymal WAT mass was 40% larger in male Agpat4-/- mice than wild-type littermates, but unchanged in perirenal, retroperitoneal, and inguinal WAT and subscapular brown adipose tissue. Metabolic changes were identified in epididymal WAT that were not evident in perirenal WAT, which was analyzed for comparison. The total epididymal TAG content doubled, increasing adipocyte cell size without changing markers of differentiation. Enzymes involved in de novo lipogenesis and complex lipid synthesis downstream of phosphatidic acid production were also unchanged. However, total epididymal TAG hydrolase activity was reduced, and there were significant decreases in total ATGL and reduced phosphorylation of hormone-sensitive lipase at the S563 and S660 PKA-activation sites. Analysis of Agpats 1, 2, 3, and 5, as well as Gpats 1, 2, 3, and 4, demonstrated compensatory upregulation in perirenal WAT that did not occur in epididymal WAT. Our findings therefore indicate depot-specific differences in the redundancy of Agpat4 and highlight the molecular and metabolic heterogeneity of individual visceral depots.

Deletion of ARNT/HIF1ß in pancreatic beta cells does not impair glucose homeostasis in mice, but is associated with defective glucose sensing ex vivo.

AIMS/HYPOTHESIS: It has been suggested that the transcription factor ARNT/HIF1ß is critical for maintaining in vivo glucose homeostasis and pancreatic beta cell glucose-stimulated insulin secretion (GSIS). Our goal was to gain more insights into the metabolic defects seen after the loss of ARNT/HIF1ß in beta cells. METHODS: The in vivo and in vitro consequences of the loss of ARNT/HIF1ß were investigated in beta cell specific Arnt/Hif1ß knockout mice (ß-Arnt (fl/fl/Cre) mice). RESULTS: The only in vivo defects found in ß-Arnt (fl/fl/Cre) mice were significant increases in the respiratory exchange ratio and in vivo carbohydrate oxidation, and a decrease in lipid oxidation. The mitochondrial oxygen consumption rate was unaltered in mouse ß-Arnt (fl/fl/Cre) islets upon glucose stimulation. ß-Arnt (fl/fl/Cre) islets had an impairment in the glucose-stimulated increase in Ca(2+) signalling and a reduced insulin secretory response to glucose in the presence of KCl and diazoxide. The glucose-stimulated increase in the NADPH/NADP(+) ratio was reduced in ß-Arnt (fl/fl/Cre) islets. The reduced GSIS and NADPH/NADP(+) levels in ß-Arnt (fl/fl/Cre) islets could be rescued by treatment with membrane-permeable tricarboxylic acid intermediates. Small interfering (si)RNA mediated knockdown of ARNT/HIF1ß in human islets also inhibited GSIS. These results suggest that the regulation of GSIS by the KATP channel-dependent and -independent pathways is affected by the loss of ARNT/HIF1ß in islets. CONCLUSIONS/INTERPRETATION: This study provides three new insights into the role of ARNT/HIF1ß in beta cells: (1) ARNT/HIF1ß deletion in mice impairs GSIS ex vivo; (2) ß-Arnt (fl/fl/Cre) mice have an increased respiratory exchange ratio; and (3) ARNT/HIF1ß is required for GSIS in human islets.

Sarcoplasmic Reticulum Phospholipid Fatty Acid Composition and Sarcolipin Content in Rat Skeletal Muscle.

In a previous study, we reported lower sarcoplasmic reticulum (SR) Ca(2+) pump ionophore ratios in rat soleus compared to red and white gastrocnemius (RG, WG) muscles which may be indicative of greater SR Ca(2+) permeability in soleus. Here we assessed the lipid composition of the SR membranes obtained from these muscles to determine if SR docosahexaenoic acid (DHA) content and fatty acid unsaturation could help to explain the previously observed differences in SR Ca(2+) permeability. Since we have shown previously that sarcolipin may also influence SR Ca(2+) permeability, we also examined the levels of sarcolipin in rat muscle. We found that SR membrane DHA content was significantly higher in soleus (5.3 ± 0.2 %) compared to RG (4.2 ± 0.2 %) and WG (3.3 ± 0.2 %). Likewise, total SR membrane unsaturation and unsaturation index (UI) were significantly higher in soleus (% unsaturation: 59.1 ± 2.4; UI: 362.9 ± 0.8) compared to RG (% unsaturation: 55.3 ± 1.0; UI: 320.9 ± 2.5) and WG (% unsaturation: 52.6 ± 1.1; UI: 310. ± 2.2). Sarcolipin protein was 17-fold more abundant in rat soleus compared to RG and was not detected in WG; however, comparisons between soleus, RG, and WG in sarcolipin-null mice revealed that, in the absence of sarcolipin, ionophore ratios are still lowest in soleus and highest in WG. Overall, our results suggest that SR membrane DHA content and unsaturation, and, in part, sarcolipin expression may contribute to SR Ca(2+) permeability and, in turn, may have implications in muscle-based metabolism and diet-induced obesity.

Delay in post-ovariectomy estrogen replacement negates estrogen-induced augmentation of post-exercise muscle satellite cell proliferation.

This study examined the effects of a delay in post-ovariectomy replacement of 17ß-estradiol (estrogen) on the post-exercise proliferation of muscle satellite cells. Nine-week-old, ovariectomized, female Sprague-Dawley rats (n = 64) were distributed among 8 groups based on estrogen status (0.25 mg estrogen pellet or sham), exercise status (90 min run at 17 m·min(-1) and a grade of -13.5° or unexercised), and estrogen replacement ("proximal", estrogen replacement within 2 weeks; or "delayed", estrogen replacement at 11 weeks following ovariectomy). Significant increases in satellite cells were found in the soleus and white gastrocnemius muscle (immunofluorescent colocalization of nuclei with Pax7) 72 h following eccentric exercise (p < 0.05) in all exercised groups. Proximal E2 replacement resulted in a further augmentation of muscle satellite cells in exercised rats (p < 0.05) relative to the delayed estrogen replacement group. Expression of PI3K was unaltered and phosphorylation of Akt relative to total Akt increased following estrogen supplementation and exercise. Exercise alone did not alter the expression levels of Akt. An 11 week delay in post-ovariectomy estrogen replacement negated the augmenting influence seen with proximal (2 week delay) post-ovariectomy estrogen replacement on post-exercise muscle satellite cell proliferation. This effect appears to be independent of the PI3K-Akt signaling pathway.

Sarcolipin trumps ß-adrenergic receptor signaling as the favored mechanism for muscle-based diet-induced thermogenesis.

Sarcolipin (SLN) regulates muscle-based nonshivering thermogenesis and is up-regulated with high-fat feeding (HFF). To investigate whether other muscle-based thermogenic systems compensate for a lack of Sln and to firmly establish SLN as a mediator of diet-induced thermogenesis (DIT), we measured muscle and whole-body energy expenditure in chow- and high-fat-fed Sln(-/-) and wild-type (WT) mice. Following HFF, resting muscle metabolic rate (VO2, µl/g/s) was increased similarly in WT (0.28±0.02 vs. 0.31±0.03) and Sln(-/-) (0.23±0.03 vs. 0.35±0.02) mice due to increased sympathetic nervous system activation in Sln(-/-) mice; however, whole-body metabolic rate (VO2, ml/kg/h) was lower in Sln(-/-) compared with WT mice following HFF but only during periods when the mice were active in their cages (WT, 2894±87 vs. Sln(-/-), 2708±61). Treatment with the ß-adrenergic receptor (ß-AR) antagonist propranolol during HFF completely prevented muscle-based DIT in Sln(-/-) mice; however, it had no effect in WT mice, resulting in greater differences in whole-body metabolic rate and diet-induced weight gain. Our results suggest that ß-AR signaling partially compensates for a lack of SLN to activate muscle-based DIT, but SLN is the primary and more effective mediator.

Sarcolipin provides a novel muscle-based mechanism for adaptive thermogenesis.

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) transports Ca into the sarcoplasmic reticulum lumen and contributes significantly to skeletal muscle metabolic rate. Sarcolipin (SLN) has been shown recently to uncouple Ca transport from adenosine triphosphate hydrolysis by SERCA. We have hypothesized that SLN provides a novel mechanism of adaptive thermogenesis within skeletal muscle and protects against diet-induced obesity.

The role of estrogen receptor-α in estrogen-mediated regulation of basal and exercise-induced Hsp70 and Hsp27 expression in rat soleus.

We examined the influence of estrogen receptor-alpha (ERα) activation on estrogen-mediated regulation of heat shock proteins 70 (Hsp70) and 27 (Hsp27) in soleus. Ovariectomized rats received estrogen (EST), an ERα agonist (propyl pyrazole triol, PPT), both (EST+PPT), or a sham, and they served as either unexercised controls or were subjected to exercise by having to run downhill (17 m/min, -13.5° grade) for 90 min. At 72 h postexercise, soleus muscles were removed and either immunohistochemically stained for Hsp70 and myosin heavy chain or homogenized for Western blotting for Hsp70 and Hsp27. Elevated (p < 0.05) basal Hsp70 in both type I and II fibres in the unexercised EST, PPT, and EST+PPT groups relative to unexercised sham animals was noted. Compared with Hsp70 levels in the unexercised animals, that in exercised animals was elevated (p < 0.05) in both sham and PPT groups but not in EST and EST+PPT groups. Western blot determined that Hsp27 levels were not significantly different between groups. Hence, the ability of estrogen to augment resting type I and type II muscle fibre Hsp70 content is primarily mediated via muscle ERα. However, the blunted Hsp70 response following damaging exercise in estrogen-supplemented animals does not appear to be fully accounted for by ERα-mediated effects.

Ca2+ dysregulation in Ryr1(I4895T/wt) mice causes congenital myopathy with progressive formation of minicores, cores, and nemaline rods.

Ryr1(I4895T/wt) (IT/+) mice express a knockin mutation corresponding to the human I4898T EC-uncoupling mutation in the type 1 ryanodine receptor/Ca(2+) release channel (RyR1), which causes a severe form of central core disease (CCD). IT/+ mice exhibit a slowly progressive congenital myopathy, with neonatal respiratory stress, skeletal muscle weakness, impaired mobility, dorsal kyphosis, and hind limb paralysis. Lesions observed in myofibers from diseased mice undergo age-dependent transformation from minicores to cores and nemaline rods. Early ultrastructural abnormalities include sarcomeric misalignment, Z-line streaming, focal loss of cross-striations, and myofibrillar splitting and intermingling that may arise from defective myofibrillogenesis. However, manifestation of the disease phenotype is highly variable on a Sv129 genomic background. Quantitative RT-PCR shows an equimolar ratio of WT and mutant Ryr1 transcripts within IT/+ myofibers and total RyR1 protein expression levels are normal. We propose a unifying theory in which the cause of core formation lies in functional heterogeneity among RyR1 tetramers. Random combinations of normal and either leaky or EC-uncoupled RyR subunits would lead to spatial differences in Ca(2+) transients; the resulting heterogeneity of contraction among myofibrils would lead to focal, irreversible tearing and shearing, which would, over time, enlarge to form minicores, cores, and nemaline rods. The IT/+ mouse line is proposed to be a valid model of RyR1-related congenital myopathy, offering high potential for elucidation of the pathogenesis of skeletal muscle disorders arising from impaired EC coupling.

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice.

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.

ATP consumption by sarcoplasmic reticulum Ca2+ pumps accounts for 50% of resting metabolic rate in mouse fast and slow twitch skeletal muscle.

In this study, we aimed to directly quantify the relative contribution of Ca(2+) cycling to resting metabolic rate in mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) skeletal muscle. Resting oxygen consumption of isolated muscles (Vo(2), microl.g wet wt(-1).s(-1)) measured polarographically at 30 degrees C was approximately 25% higher in soleus (0.61 +/- .03) than in EDL (0.46 +/- .03). To quantify the specific contribution of Ca(2+) cycling to resting metabolic rate, cyclopiazonic acid (CPA), a highly specific inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPases (SERCAs), was added to the bath at different concentrations (1, 5, 10, and 15 microM). There was a concentration-dependent effect of CPA on Vo(2), with increasing CPA concentrations up to 10 microM resulting in progressively greater reductions in muscle Vo(2). There were no differences between 10 and 15 microM CPA, indicating that 10 microM CPA induces maximal inhibition of SERCAs in isolated muscle preparations. Relative reduction in muscle Vo(2) in response to CPA was nearly identical in EDL (1 microM, 10.6 +/- 3.0%; 5 microM, 33.2 +/- 3.4%; 10 microM, 49.2 +/- 2.9%; 15 microM, 50.9 +/- 2.1%) and soleus (1 microM, 11.2 +/- 1.5%; 5 microM, 37.7 +/- 2.4%; 10 microM, 50.0 +/- 1.3%; 15 microM, 49.9 +/- 1.6%). The results indicate that ATP consumption by SERCAs is responsible for approximately 50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30 degrees C. Thus SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.

Prolonged moderate-intensity aerobic exercise does not alter apoptotic signaling and DNA fragmentation in human skeletal muscle.

Apoptosis in skeletal muscle plays an important role in age- and disease-related tissue dysfunction. Physical activity can influence apoptotic signaling; however, this process has not been well studied in human skeletal muscle. The purpose of this study was to perform a comprehensive analysis of apoptosis-related proteins/enzymes, DNA fragmentation, and oxidative stress in skeletal muscle of humans during an acute bout of prolonged moderate-intensity exercise. Eight healthy, recreationally active individuals (age 20.8 +/- 0.5 yr, Vo(2peak) 51.2 +/- 0.9 ml . kg(-1) . min(-1), BMI 21.5 +/- 0.8 kg/m(2)) exercised on a cycle ergometer at approximately 60% Vo(2peak) for 2 h. Muscle biopsies were obtained at rest as well as at 60 and 120 min of exercise. Although exercise was associated with a significant whole body and muscle metabolic response, there were no significant changes in the content of antiapoptotic (ARC, Bcl-2, Hsp70, XIAP) and proapoptotic (AIF, Bax, Smac) proteins, activity of proteolytic enzymes (caspase-3, caspase-8, caspase-9), DNA fragmentation, or TUNEL-positive nuclei in skeletal muscle. Furthermore, the protein levels of several antioxidant enzymes (catalase, CuZnSOD, MnSOD), concentrations of GSH and GSSG, and degree of ROS generation in skeletal muscle were not altered by exercise. Fiber type-specific analysis also revealed that ARC (P < 0.001) and Hsp70 (P < 0.05) protein were significantly higher in type I compared with type IIA and type IIAX/X fibers; however, protein levels were not affected by exercise. These findings suggest that a single bout of prolonged moderate-intensity aerobic exercise is not sufficient to alter apoptotic signaling in skeletal muscle of healthy humans.

The Loss of ARNT/HIF1ß in Male Pancreatic ß-Cells Is Protective Against High-Fat Diet-Induced Diabetes.

The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1ß (ARNT/HIF1ß) plays a key role in maintaining ß-cell function and has been shown to be one of the most downregulated transcription factors in islets from patients with type 2 diabetes. We have shown a role for ARNT/HIF1ß in glucose sensing and insulin secretion in vitro and no defects in in vivo glucose homeostasis. To gain a better understanding of the role of ARNT/HIF1ß in the development of diabetes, we placed control (+/+/Cre) and ß-cell-specific ARNT/HIF1ß knockout (fl/fl/Cre) mice on a high-fat diet (HFD). Unlike the control (+/+/Cre) mice, HFD-fed fl/fl/Cre mice had no impairment in in vivo glucose tolerance. The lack of impairment in HFD-fed fl/fl/Cre mice was partly due to an improved islet glucose-stimulated NADPH/NADP+ ratio and glucose-stimulated insulin secretion. The effects of the HFD-rescued insulin secretion in fl/fl/Cre islets could be reproduced by treating low-fat diet (LFD)-fed fl/fl/Cre islets with the lipid signaling molecule 1-monoacylglcyerol. This suggests that the defects seen in LFD-fed fl/fl/Cre islet insulin secretion involve lipid signaling molecules. Overall, mice lacking ARNT/HIF1ß in ß-cells have altered lipid signaling in vivo and are resistant to an HFD's ability to induce diabetes.

Sarcolipin expression is not required for the mitochondrial enzymatic response to physical activity or diet.

In mice, transgenic manipulation of Ca2+-handling proteins is sufficient to alter the metabolic phenotype of muscle. We have previously shown that ablation of sarcolipin (SLN), a regulatory protein and uncoupler of sarco(endo)plasmic reticulum Ca2+-ATPases, leads to excessive diet-induced obesity and glucose intolerance in mice. However, it is unclear how loss of SLN per se affects muscle oxidative capacity and the ability of mitochondria to adapt to physiological stimuli, such as exercise training or calorie overload. To address this question, Sln-/- and wild-type (WT) littermates were given access to voluntary running wheels or underwent a treadmill training protocol for 8 wk. Furthermore, a separate group of mice were given a high-fat diet (42% kcal from fat for 8 wk) to determine whether the excessively obese phenotype of Sln-/- mice is associated with altered oxidative capacity. While voluntary running was insufficient to elicit mitochondrial adaptations, treadmill-trained mice showed significant increases (P < 0.05) in the maximal activities of succinate dehydrogenase (+11%), citrate synthase (+12%), cytochrome oxidase (COX: +17%), along with increased protein expression of cytochrome c (+34%) and COX IV (+28%), which were irrespective of SLN expression. Lastly, no changes in the activities of mitochondrial marker enzymes existed with high-fat feeding, regardless of genotype. Together, these findings indicate that SLN is not required for the regulation of oxidative capacity in response to physiological stress, namely exercise or caloric surfeit.NEW & NOTEWORTHY Sarcolipin (SLN) has gained considerable attention for its uncoupling role of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Because of SLN's ability to alter both cellular energy use and cytosolic [Ca2+], the potential exists for a regulatory role of mitochondrial biogenesis. Herein, we show skeletal muscle oxidative capacity to be unaltered in mice lacking SLN following exercise training or high-fat feeding. Our results contrast with published studies of SLN-overexpressing mice, possibly owing to supraphysiological uncoupling of SERCA.

Prior Endurance Training Enhances Beta-Adrenergic Signaling in Epidydimal Adipose from Mice Fed a High-Fat Diet.

OBJECTIVE: Adipose tissue beta-adrenergic signaling is attenuated in obesity and insulin resistance. It has been previously demonstrated that prior exercise training protects against short-term, high-fat diet (HFD)-induced weight gain and glucose intolerance. This study aimed to determine whether prior exercise training results in altered beta-adrenergic and lipolytic signaling in adipose tissue when challenged with a HFD. METHODS: Male C57BL/6J mice underwent 4 weeks of treadmill training (1 h/d, 5 d/wk). Twenty-four hours after the final bout of exercise, mice were fed a HFD (60% kcal lard) for 4 days. RESULTS: Serum fatty acids, beta-adrenergic signaling (phosphorylated ERK, hormone-sensitive lipase, and p38), and perilipin 1 content were greater in epididymal white adipose tissue (eWAT) from previously trained mice. These changes were not evident in eWAT from trained mice prior to the HFD and were not secondary to alterations in insulin responsiveness or catecholamine concentrations. CL 316,243-mediated increases in hormone-sensitive lipase phosphorylation and fatty acid accumulation in the media were greater in adipose tissue explants from previously trained mice fed a HFD. CONCLUSIONS: These findings suggest that previous training increases adipose tissue beta-adrenergic responsiveness to a short-term HFD. This may help to explain the protective effect of prior exercise training against the deleterious effects of a HFD.

Sarcolipin deletion exacerbates soleus muscle atrophy and weakness in phospholamban overexpressing mice.

Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that regulate the sarco(endo)plasmic reticulum Ca2+-ATPase pumps. In a recent study, we discovered that Pln overexpression (PlnOE) in slow-twitch type I skeletal muscle fibers drastically impaired SERCA function and caused a centronuclear myopathy-like phenotype, severe muscle atrophy and weakness, and an 8 to 9-fold upregulation of SLN protein in the soleus muscles. Here, we sought to determine the physiological role of SLN upregulation, and based on its role as a SERCA inhibitor, we hypothesized that it would represent a maladaptive response that contributes to the SERCA dysfunction and the overall myopathy observed in the PlnOE mice. To this end, we crossed Sln-null (SlnKO) mice with PlnOE mice to generate a PlnOE/SlnKO mouse colony and assessed SERCA function, CNM pathology, in vitro contractility, muscle mass, calcineurin signaling, daily activity and food intake, and proteolytic enzyme activity. Our results indicate that genetic deletion of Sln did not improve SERCA function nor rescue the CNM phenotype, but did result in exacerbated muscle atrophy and weakness, due to a failure to induce type II fiber compensatory hypertrophy and a reduction in total myofiber count. Mechanistically, our findings suggest that impaired calcineurin activation and resultant decreased expression of stabilin-2, and/or impaired autophagic signaling could be involved. Future studies should examine these possibilities. In conclusion, our study demonstrates the importance of SLN upregulation in combating muscle myopathy in the PlnOE mice, and since SLN is upregulated across several myopathies, our findings may reveal SLN as a novel and universal therapeutic target.

Diaphragm assessment in mice overexpressing phospholamban in slow-twitch type I muscle fibers.

AIMS: Phospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (Pln (OE)), SERCA function was impaired, dynamin 2 (3-5 fold) and SLN (7-9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the Pln (OE) mouse would exhibit CNM pathology and muscle weakness. METHODS: Diaphragm muscles from Pln (OE) and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca(2+)-ATPase and Ca(2+) uptake assays, Western blotting, and in vitro electrical stimulation. RESULTS: Our results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca(2+) but did not reduce maximal SERCA activity or rates of Ca(2+) uptake. SLN was upregulated 2.5-fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of Pln (OE) diaphragm strips revealed no reductions in force-generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged. CONCLUSIONS: Therefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.