Disruption of dopamine receptor 1 localization to primary cilia impairs signaling in striatal neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G protein-coupled receptors (GPCRs) and their downstream effectors, suggesting they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT:Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G protein-coupled receptors (GPCRs), suggesting they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either abnormal accumulation of D1 in cilia or loss of D1 ciliary localization become obese. In both cases the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

Strain-specific differences in muscle Ca2+ transport and mitochondrial electron transport chain proteins between FVB/N and C57BL/6J mice.

Genetically engineered mouse models have been used to determine the role of sarcolipin (SLN) in muscle. However, a few studies had difficulty in detecting SLN in FBV/N mice and questioned its relevance to muscle metabolism. It is known that genetic alteration of proteins in different inbred mice strains produces dissimilar functional outcomes. Therefore, here we compared the expression of SLN and key proteins involved in Ca2+ handling and mitochondrial metabolism between FVB/N and C57BL/6J mouse strains. Data suggest that SLN expression is less abundant in the skeletal muscles of FVB/N mice than in the C57BL/6J strain. The expression of Ca2+ transporters in the mitochondrial membranes was also lower in FVB/N than in C57BL/6J mice. Similarly, electron transport chain proteins in the mitochondria were less abundant in FVB/N mice, which may contribute to differences in energy metabolism. Future studies using different mouse strains should take these differences into account when interpreting their data.

Loss of mitochondrial energetics is associated with poor recovery of muscle function but not mass following disuse atrophy.

Skeletal muscle atrophy is a clinically important outcome of disuse because of injury, immobilization, or bed rest. Disuse atrophy is accompanied by mitochondrial dysfunction, which likely contributes to activation of the muscle atrophy program. However, the linkage of muscle mass and mitochondrial energetics during disuse atrophy and its recovery is incompletely understood. Transcriptomic analysis of muscle biopsies from healthy older adults subject to complete bed rest revealed marked inhibition of mitochondrial energy metabolic pathways. To determine the temporal sequence of muscle atrophy and changes in intramyocellular lipid and mitochondrial energetics, we conducted a time course of hind limb unloading-induced atrophy in adult mice. Mitochondrial respiration and calcium retention capacity were diminished, whereas H2O2 emission was increased within 3 days of unloading before significant muscle atrophy. These changes were associated with a decrease in total cardiolipin and profound changes in remodeled cardiolipin species. Hind limb unloading performed in muscle-specific peroxisome proliferator-activated receptor-γ coactivator-1α/ß knockout mice, a model of mitochondrial dysfunction, did not affect muscle atrophy but impacted muscle function. These data suggest early mitochondrial remodeling affects muscle function but not mass during disuse atrophy. Early alterations in mitochondrial energetics and lipid remodeling may represent novel targets to prevent muscle functional impairment caused by disuse and to enhance recovery from periods of muscle atrophy.

Both brown adipose tissue and skeletal muscle thermogenesis processes are activated during mild to severe cold adaptation in mice.

Thermogenesis is an important homeostatic mechanism essential for survival and normal physiological functions in mammals. Both brown adipose tissue (BAT) (i.e. uncoupling protein 1 (UCP1)-based) and skeletal muscle (i.e. sarcolipin (SLN)-based) thermogenesis processes play important roles in temperature homeostasis, but their relative contributions differ from small to large mammals. In this study, we investigated the functional interplay between skeletal muscle- and BAT-based thermogenesis under mild versus severe cold adaptation by employing UCP1-/- and SLN-/- mice. Interestingly, adaptation of SLN-/- mice to mild cold conditions (16 °C) significantly increased UCP1 expression, suggesting increased reliance on BAT-based thermogenesis. This was also evident from structural alterations in BAT morphology, including mitochondrial architecture, increased expression of electron transport chain proteins, and depletion of fat droplets. Similarly, UCP1-/- mice adapted to mild cold up-regulated muscle-based thermogenesis, indicated by increases in muscle succinate dehydrogenase activity, SLN expression, mitochondrial content, and neovascularization, compared with WT mice. These results further confirm that SLN-based thermogenesis is a key player in muscle non-shivering thermogenesis (NST) and can compensate for loss of BAT activity. We also present evidence that the increased reliance on BAT-based NST depends on increased autonomic input, as indicated by abundant levels of tyrosine hydroxylase and neuropeptide Y. Our findings demonstrate that both BAT and muscle-based NST are equally recruited during mild and severe cold adaptation and that loss of heat production from one thermogenic pathway leads to increased recruitment of the other, indicating a functional interplay between these two thermogenic processes.

Increased Reliance on Muscle-based Thermogenesis upon Acute Minimization of Brown Adipose Tissue Function.

Skeletal muscle has been suggested as a site of nonshivering thermogenesis (NST) besides brown adipose tissue (BAT). Studies in birds, which do not contain BAT, have demonstrated the importance of skeletal muscle-based NST. However, muscle-based NST in mammals remains poorly characterized. We recently reported that sarco/endoplasmic reticulum Ca(2+) cycling and that its regulation by SLN can be the basis for muscle NST. Because of the dominant role of BAT-mediated thermogenesis in rodents, the role of muscle-based NST is less obvious. In this study, we investigated whether muscle will become an important site of NST when BAT function is conditionally minimized in mice. We surgically removed interscapular BAT (iBAT, which constitutes ∼70% of total BAT) and exposed the mice to prolonged cold (4 °C) for 9 days. The iBAT-ablated mice were able to maintain optimal body temperature (∼35-37 °C) during the entire period of cold exposure. After 4 days in the cold, both sham controls and iBAT-ablated mice stopped shivering and resumed routine physical activity, indicating that they are cold-adapted. The iBAT-ablated mice showed higher oxygen consumption and decreased body weight and fat mass, suggesting an increased energy cost of cold adaptation. The skeletal muscles in these mice underwent extensive remodeling of both the sarcoplasmic reticulum and mitochondria, including alteration in the expression of key components of Ca(2+) handling and mitochondrial metabolism. These changes, along with increased sarcolipin expression, provide evidence for the recruitment of NST in skeletal muscle. These studies collectively suggest that skeletal muscle becomes the major site of NST when BAT activity is minimized.

Phospholamban and sarcolipin: Are they functionally redundant or distinct regulators of the Sarco(Endo)Plasmic Reticulum Calcium ATPase?

In muscle, the Sarco(Endo)plasmic Reticulum Calcium ATPase (SERCA) activity is regulated by two distinct proteins, PLB and SLN, which are highly conserved throughout vertebrate evolution. PLB is predominantly expressed in the cardiac muscle, while SLN is abundant in skeletal muscle. SLN is also found in the cardiac atria and to a lesser extent in the ventricle. PLB regulation of SERCA is central to cardiac function, both at rest and during extreme physiological demand. Compared to PLB, the physiological relevance of SLN remained a mystery until recently and some even thought it was redundant in function. Studies on SLN suggest that it is an uncoupler of the SERCA pump activity and can increase ATP hydrolysis resulting in heat production. Using genetically engineered mouse models for SLN and PLB, we showed that SLN, not PLB, is required for muscle-based thermogenesis. However, the mechanism of how SLN binding to SERCA results in uncoupling SERCA Ca(2+) transport from its ATPase activity remains unclear. In this review, we discuss recent advances in understanding how PLB and SLN differ in their interaction with SERCA. We will also explore whether structural differences in the cytosolic domain of PLB and SLN are the basis for their unique function and physiological roles in cardiac and skeletal muscle.

Uncoupling Protein 1 and Sarcolipin Are Required to Maintain Optimal Thermogenesis, and Loss of Both Systems Compromises Survival of Mice under Cold Stress.

The importance of brown adipose tissue as a site of nonshivering thermogenesis has been well documented. Emerging studies suggest that skeletal muscle is also an important site of thermogenesis especially when brown adipose tissue function is lacking. We recently showed that sarcolipin (SLN), an uncoupler of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) pump, could contribute to heat production in skeletal muscle. In this study, we sought to understand how loss of UCP1 or SLN is compensated during cold exposure and whether they are both necessary for thermogenesis. Toward this goal, we generated a UCP1;SLN double knock-out (DKO) mouse model and challenged the single and DKO mice to acute and long-term cold exposures. Results from this study show that there is up-regulation of SLN expression in UCP1-KO mice, and loss of SLN is compensated by increased expression of UCP1 and browning of white adipose tissue. We found that the DKO mice were viable when reared at thermoneutrality. When challenged to acute cold, the DKO were extremely cold-sensitive and became hypothermic. Paradoxically, the DKO mice were able to survive gradual cold challenge, but these mice lost significant weight and depleted their fat stores, despite having higher caloric intake. These studies suggest that UCP1 and SLN are required to maintain optimal thermogenesis and that loss of both systems compromises survival of mice under cold stress.

Sarcolipin Is a Key Determinant of the Basal Metabolic Rate, and Its Overexpression Enhances Energy Expenditure and Resistance against Diet-induced Obesity.

Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) in muscle. SLN binding to SERCA uncouples Ca(2+) transport from ATP hydrolysis. By this mechanism, SLN promotes the futile cycling of SERCA, contributing to muscle heat production. We recently showed that SLN plays an important role in cold- and diet-induced thermogenesis. However, the detailed mechanism of how SLN regulates muscle metabolism remains unclear. In this study, we used both SLN knockout (Sln(-/-)) and skeletal muscle-specific SLN overexpression (Sln(OE)) mice to explore energy metabolism by pair feeding (fixed calories) and high-fat diet feeding (ad libitum). Our results show that, upon pair feeding, Sln(OE) mice lost weight compared with the WT, but Sln(-/-) mice gained weight. Interestingly, when fed with a high-fat diet, Sln(OE) mice consumed more calories but gained less weight and maintained a normal metabolic profile in comparison with WT and Sln(-/-) mice. We found that oxygen consumption and fatty acid oxidation were increased markedly in Sln(OE) mice. There was also an increase in both mitochondrial number and size in Sln(OE) muscle, together with increased expression of peroxisome proliferator-activated receptor δ (PPARδ) and PPAR γ coactivator 1 α (PGC1α), key transcriptional activators of mitochondrial biogenesis and enzymes involved in oxidative metabolism. These results, taken together, establish an important role for SLN in muscle metabolism and energy expenditure. On the basis of these data we propose that SLN is a novel target for enhancing whole-body energy expenditure.

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport.

The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca(2+) transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues (2)ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.

Sarcoplasmic reticulum Ca2+ ATPase pump is a major regulator of glucose transport in the healthy and diabetic heart.

Despite intensive research, the pathways that mediate calcium (Ca(2+))-stimulated glucose transport in striated muscle remain elusive. Since the sarcoplasmic reticulum calcium ATPase (SERCA) pump tightly regulates cytosolic [Ca(2+)], we investigated whether the SERCA pump is a major regulator of cardiac glucose transport. We used healthy and insulin-deficient diabetic transgenic (TG) mice expressing SERCA1a in the heart. Active cell surface glucose transporter (GLUT)-4 was measured by a biotinylated photolabeled assay in the intact perfused myocardium and isolated myocytes. In healthy TG mice, cardiac-specific SERCA1a expression increased active cell-surface GLUT4 and glucose uptake in the myocardium, as well as whole body glucose tolerance. Diabetes reduced active cell-surface GLUT4 content and glucose uptake in the heart of wild type mice, all of which were preserved in diabetic TG mice. Decreased basal AS160 and increased proportion of calmodulin-bound AS160 paralleled the increase in cell surface GLUT4 content in the heart of TG mice, suggesting that AS160 regulates GLUT trafficking by a Ca(2+)/calmodulin dependent pathway. In addition, cardiac-specific SERCA1a expression partially rescues hyperglycemia during diabetes. Collectively, these data suggested that the SERCA pump is a major regulator of cardiac glucose transport by an AS160 dependent mechanism during healthy and insulin-deficient state. Our data further indicated that cardiac-specific SERCA overexpression rescues diabetes induced-alterations in cardiac glucose transport and improves whole body glucose homeostasis. Therefore, findings from this study provide novel mechanistic insights linking upregulation of the SERCA pump in the heart as a potential therapeutic target to improve glucose metabolism during diabetes.

Muscle damage, metabolism, and oxidative stress in mdx mice: Impact of aerobic running.

INTRODUCTION: We tested how a treadmill exercise program influences oxygen consumption, oxidative stress, and exercise capacity in the mdx mouse, a model of Duchenne muscular dystrophy. METHODS: At age 4 weeks mdx mice were subjected to 4 weeks of twice-weekly treadmill exercise. Sedentary mdx and wild-type mice served as controls. Oxygen consumption, time to exhaustion, oxidative stress, and myofiber damage were assessed. RESULTS: At age 4 weeks, there was a significant difference in exercise capacity between mdx and wild-type mice. After exercise, mdx mice had lower basal oxygen consumption and exercise capacity, but similar maximal oxygen consumption. Skeletal muscle from these mice displayed increased oxidative stress. Collagen deposition was higher in exercised versus sedentary mice. CONCLUSIONS: Exercised mdx mice exhibit increased oxidative stress, as well as deficits in exercise capacity, baseline oxygen consumption, and increased myofiber fibrosis. Muscle Nerve 54: 110-117, 2016.

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1.

AIMS: Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. METHODS AND RESULTS: In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex. CONCLUSIONS: Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients.

Obligatory role of neuronal nitric oxide synthase in the heart's antioxidant adaptation with exercise.

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.

The C-terminal calcium-sensitive disordered motifs regulate isoform-specific polymerization characteristics of calsequestrin.

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C-terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn-motif and DEXn-motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C-terminal motifs possess Ca(2+)-sensitivity and affect protein function. Sequence analysis shows that both the Dn- and DEXn-motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca(2+)-mediated folding suggesting that these disordered motifs are Ca(2+)-sensitivity. We generated chimeric proteins by swapping the C-terminal portions between CASQ1 and CASQ2. Our studies show that the C-terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C-terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca(2+)-sensitivity IDRs located at the back-to-back dimer interface influence isoform-specific Ca(2+)-dependent polymerization properties of CASQ.

Cold adaptation overrides developmental regulation of sarcolipin expression in mice skeletal muscle: SOS for muscle-based thermogenesis?

Neonatal mice have a greater thermogenic need than adult mice and may require additional means of heat production, other than the established mechanism of brown adipose tissue (BAT). We and others recently discovered a novel mediator of skeletal muscle-based thermogenesis called sarcolipin (SLN) that acts by uncoupling sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA). In addition, we have shown that SLN expression is downregulated during neonatal development in rats. In this study we probed two questions: (1) is SLN expression developmentally regulated in neonatal mice?; and (2) if so, will cold adaptation override this? Our data show that SLN expression is higher during early neonatal stages and is gradually downregulated in fast twitch skeletal muscles. Interestingly, we demonstrate that cold acclimation of neonatal mice can prevent downregulation of SLN expression. This observation suggests that SLN-mediated thermogenesis can be recruited to a greater extent during extreme physiological need, in addition to BAT.

Sarcolipin is a novel regulator of muscle metabolism and obesity.

Obesity is increasing at an alarming rate, both in adults and adolescents, across the globe due to increased consumption of caloric rich diet. Obesity and its associated complications appear to be major contributing factors not only to diabetes/heart disease but also to cancer, and neurological diseases causing a huge burden on the health care system. To date, there are no effective treatments to reduce weight gain, other than caloric restriction and exercise which are often difficult to enforce. There are very few drugs available for treating obesity and those that are available only reduce obesity by ∼ 10%. Identifying mechanisms to increase energy expenditure, on top of the increase elicited by exercise, would be more beneficial to control weight gain. The purpose of this review is to highlight the role of sarcolipin (SLN), a regulator of SERCA pump, in muscle thermogenesis and metabolism. We will further discuss if enhancing SLN activity could be an effective mechanism to increase energy expenditure and control weight gain. We will also discuss the merits of adaptive thermogenesis in muscle and brown fat as potential mechanisms to increase energy expenditure during caloric overload. That said, there is still a great need for further research into the mechanism of diet induced thermogenesis and its relevance to overall metabolism and obesity.

Sarcolipin protein interaction with sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is distinct from phospholamban protein, and only sarcolipin can promote uncoupling of the SERCA pump.

Sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547-554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575-1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca(2+) causes uncoupling of the SERCA pump and increased heat production.

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart.

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.

Effects of insulin resistance on skeletal muscle growth and exercise capacity in type 2 diabetic mouse models.

Type 2 diabetes mellitus is associated with an accelerated muscle loss during aging, decreased muscle function, and increased disability. To better understand the mechanisms causing this muscle deterioration in type 2 diabetes, we assessed muscle weight, exercise capacity, and biochemistry in db/db and TallyHo mice at prediabetic and overtly diabetic ages. Maximum running speeds and muscle weights were already reduced in prediabetic db/db mice when compared with lean controls and more severely reduced in the overtly diabetic db/db mice. In contrast to db/db mice, TallyHo muscle size dramatically increased and maximum running speed was maintained during the progression from prediabetes to overt diabetes. Analysis of mechanisms that may contribute to decreased muscle weight in db/db mice demonstrated that insulin-dependent phosphorylation of enzymes that promote protein synthesis was severely blunted in db/db muscle. In addition, prediabetic (6-wk-old) and diabetic (12-wk-old) db/db muscle exhibited an increase in a marker of proteasomal protein degradation, the level of polyubiquitinated proteins. Chronic treadmill training of db/db mice improved glucose tolerance and exercise capacity, reduced markers of protein degradation, but only mildly increased muscle weight. The differences in muscle phenotype between these models of type 2 diabetes suggest that insulin resistance and chronic hyperglycemia alone are insufficient to rapidly decrease muscle size and function and that the effects of diabetes on muscle growth and function are animal model-dependent.

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.