Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning.

OBJECTIVE: Norepinephrine stimulates the adipose tissue thermogenic program through a ß-adrenergic receptor (ßAR)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the ßAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood. METHODS: We used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the ßAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue. RESULTS: SIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after ßAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue. CONCLUSIONS: Taken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for ß-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.

Acetylation and phosphorylation changes to cardiac proteins in experimental HFpEF due to metabolic risk reveal targets for treatment.

AIMS: Despite the high prevalence of heart failure with preserved ejection fraction (HFpEF), the pathomechanisms remain elusive and specific therapy is lacking. Disease-causing factors include metabolic risk, notably obesity. However, proteomic changes in HFpEF are poorly understood, hampering therapeutic strategies. We sought to elucidate how metabolic syndrome affects cardiac protein expression, phosphorylation and acetylation in the Zucker diabetic fatty/Spontaneously hypertensive heart failure F1 (ZSF1) rat HFpEF model, and to evaluate changes regarding their potential for treatment. MAIN METHODS: ZSF1 obese and lean rats were fed a Purina diet up to the onset of HFpEF in the obese animals. We quantified the proteome, phosphoproteome and acetylome of ZSF1 obese versus lean heart tissues by mass spectrometry and singled out targets for site-specific evaluation. KEY FINDINGS: The acetylome of ZSF1 obese versus lean hearts was more severely altered (21 % of proteins changed) than the phosphoproteome (9 %) or proteome (3 %). Proteomic alterations, confirmed by immunoblotting, indicated low-grade systemic inflammation and endothelial remodeling in obese hearts, but low nitric oxide-dependent oxidative/nitrosative stress. Altered acetylation in ZSF1 obese hearts mainly affected pathways important for metabolism, energy production and mechanical function, including hypo-acetylation of mechanical proteins but hyper-acetylation of proteins regulating fatty acid metabolism. Hypo-acetylation and hypo-phosphorylation of elastic titin in ZSF1 obese hearts could explain myocardial stiffening. SIGNIFICANCE: Cardiometabolic syndrome alters posttranslational modifications, notably acetylation, in experimental HFpEF. Pathway changes implicate a HFpEF signature of low-grade inflammation, endothelial dysfunction, metabolic and mechanical impairment, and suggest titin stiffness and mitochondrial metabolism as promising therapeutic targets.

Raptor is critical for increasing the mitochondrial proteome and skeletal muscle force during hypertrophy.

Loss of skeletal muscle mass and force is of critical importance in numerous pathologies, like age-related sarcopenia or cancer. It has been shown that the Akt-mTORC1 pathway is critical for stimulating adult muscle mass and function, however, it is unknown if mTORC1 is the only mediator downstream of Akt and which intracellular processes are required for functional muscle growth. Here, we show that loss of Raptor reduces muscle hypertrophy after Akt activation and completely prevents increases in muscle force. Interestingly, the residual hypertrophy after Raptor deletion can be completely prevented by administration of the mTORC1 inhibitor rapamycin. Using a quantitative proteomics approach we find that loss of Raptor affects the increases in mitochondrial proteins, while rapamycin mainly affects ribosomal proteins. Taken together, these results suggest that mTORC1 is the key mediator of Akt-dependent muscle growth and its regulation of the mitochondrial proteome is critical for increasing muscle force.

Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones.

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B.

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.

An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.

Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.

Nicotinamide for the treatment of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.

Phosphoproteomics of the developing heart identifies PERM1 - An outer mitochondrial membrane protein.

Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1-/- mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

Exercise-dependent increases in protein synthesis are accompanied by chromatin modifications and increased MRTF-SRF signalling.

AIM: Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well-defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise. METHODS: We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans. RESULTS: We identified a new phosphorylation site on Myocardin-Related Transcription Factor B (MRTF-B), a co-activator of serum response factor (SRF). Phosphorylation of MRTF-B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high-intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF-target genes, and prevent increases in protein synthesis after exercise. CONCLUSION: Our results identify a new exercise signalling fingerprint in vivo, instrumental for exercise-induced protein synthesis and potentially muscle growth.

High-throughput proteomics fiber typing (ProFiT) for comprehensive characterization of single skeletal muscle fibers.

BACKGROUND: Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. METHODS: Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. RESULTS: Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes. CONCLUSION: Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups.

Skeletal muscle mTORC1 regulates neuromuscular junction stability.

BACKGROUND: Skeletal muscle is a plastic tissue that can adapt to different stimuli. It is well established that Mammalian Target of Rapamycin Complex 1 (mTORC1) signalling is a key modulator in mediating increases in skeletal muscle mass and function. However, the role of mTORC1 signalling in adult skeletal muscle homeostasis is still not well defined. METHODS: Inducible, muscle-specific Raptor and mTOR k.o. mice were generated. Muscles at 1 and 7 months after deletion were analysed to assess muscle histology and muscle force. RESULTS: We found no change in muscle size or contractile properties 1 month after deletion. Prolonging deletion of Raptor to 7 months, however, leads to a very marked phenotype characterized by weakness, muscle regeneration, mitochondrial dysfunction, and autophagy impairment. Unexpectedly, reduced mTOR signalling in muscle fibres is accompanied by the appearance of markers of fibre denervation, like the increased expression of the neural cell adhesion molecule (NCAM). Both muscle-specific deletion of mTOR or Raptor, or the use of rapamycin, was sufficient to induce 3-8% of NCAM-positive fibres (P < 0.01), muscle fibrillation, and neuromuscular junction (NMJ) fragmentation in 24% of examined fibres (P < 0.001). Mechanistically, reactivation of autophagy with the small peptide Tat-beclin1 is sufficient to prevent mitochondrial dysfunction and the appearance of NCAM-positive fibres in Raptor k.o. muscles. CONCLUSIONS: Our study shows that mTOR signalling in skeletal muscle fibres is critical for maintaining proper fibre innervation, preserving the NMJ structure in both the muscle fibre and the motor neuron. In addition, considering the beneficial effects of exercise in most pathologies affecting the NMJ, our findings suggest that part of these beneficial effects of exercise are through the well-established activation of mTORC1 in skeletal muscle during and after exercise.

Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder, which causes dysfunction/loss of lower motor neurons and muscle weakness as well as atrophy. While SMA is primarily considered as a motor neuron disease, recent data suggests that survival motor neuron (SMN) deficiency in muscle causes intrinsic defects. We systematically profiled secreted proteins from control and SMN deficient muscle cells with two combined metabolic labeling methods and mass spectrometry. From the screening, we found lower levels of C1q/TNF-related protein 3 (CTRP3) in the SMA muscle secretome and confirmed that CTRP3 levels are indeed reduced in muscle tissues and serum of an SMA mouse model. We identified that CTRP3 regulates neuronal protein synthesis including SMN via mTOR pathway. Furthermore, CTRP3 enhances axonal outgrowth and protein synthesis rate, which are well-known impaired processes in SMA motor neurons. Our data revealed a new molecular mechanism by which muscles regulate the physiology of motor neurons via secreted molecules. Dysregulation of this mechanism contributes to the pathophysiology of SMA.

A kindlin-3-leupaxin-paxillin signaling pathway regulates podosome stability.

Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation, requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3-null cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation and downstream signaling.

Single Muscle Fiber Proteomics Reveals Distinct Protein Changes in Slow and Fast Fibers during Muscle Atrophy.

Skeletal muscles are composed of heterogeneous collections of fibers with different metabolic profiles. With varied neuronal innervation and fiber-type compositions, each muscle fulfils specific functions and responds differently to stimuli and perturbations. We assessed individual fibers by mass spectrometry to dissect protein changes after loss of neuronal innervation due to section of the sciatic nerve in mice. This proteomics approach enabled us to quantify ∼600 proteins per individual soleus and EDL (extensor digitorum longus) muscle fiber. Expression of myosin heavy chain (MyHC) in individual fibers enabled clustering of specific fiber types; comparison of fibers from control and denervated muscles with the same MyHC expression revealed restricted regulation of a total of 240 proteins in type-I, -IIa, or -IIb fibers 7 days after denervation. The levels of several mitochondrial and proteasomal proteins were significantly altered, indicating rapid adaption of metabolic processes after denervation. Furthermore, we observed fiber-type-specific regulation of proteins involved in calcium ion binding and transport, such as troponins, parvalbumin, and ATP2A2, indicating marked remodeling of muscle contractility after denervation. This study provides novel insight into how different muscle fiber types remodel their proteomes during muscular atrophy.

Dynamic changes in the mouse skeletal muscle proteome during denervation-induced atrophy.

Loss of neuronal stimulation enhances protein breakdown and reduces protein synthesis, causing rapid loss of muscle mass. To elucidate the pathophysiological adaptations that occur in atrophying muscles, we used stable isotope labelling and mass spectrometry to quantify protein expression changes accurately during denervation-induced atrophy after sciatic nerve section in the mouse gastrocnemius muscle. Additionally, mice were fed a stable isotope labelling of amino acids in cell culture (SILAC) diet containing 13C6-lysine for 4, 7 or 11 days to calculate relative levels of protein synthesis in denervated and control muscles. Ubiquitin remnant peptides (K-ε-GG) were profiled by immunoaffinity enrichment to identify potential substrates of the ubiquitin-proteasomal pathway. Of the 4279 skeletal muscle proteins quantified, 850 were differentially expressed significantly within 2 weeks after denervation compared with control muscles. Moreover, pulse labelling identified Lys6 incorporation in 4786 proteins, of which 43 had differential Lys6 incorporation between control and denervated muscle. Enrichment of diglycine remnants identified 2100 endogenous ubiquitination sites and revealed a metabolic and myofibrillar protein diglycine signature, including myosin heavy chains, myomesins and titin, during denervation. Comparative analysis of these proteomic data sets with known atrogenes using a random forest approach identified 92 proteins subject to atrogene-like regulation that have not previously been associated directly with denervation-induced atrophy. Comparison of protein synthesis and proteomic data indicated that upregulation of specific proteins in response to denervation is mainly achieved by protein stabilization. This study provides the first integrated analysis of protein expression, synthesis and ubiquitin signatures during muscular atrophy in a living animal.