Overexpression of protein kinase STK25 in mice exacerbates ectopic lipid accumulation, mitochondrial dysfunction and insulin resistance in skeletal muscle
Understanding the molecular networks controlling ectopic lipid deposition and insulin responsiveness in skeletal muscle is essential for developing new strategies to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator of liver steatosis, hepatic lipid metabolism and whole body glucose and insulin homeostasis. Here, we assessed the role of STK25 in control of ectopic fat storage and insulin responsiveness in skeletal muscle.
Skeletal muscle morphology was studied by histological examination, exercise performance and insulin sensitivity were assessed by treadmill running and euglycaemic–hyperinsulinaemic clamp, respectively, and muscle lipid metabolism was analysed by ex vivo assays in Stk25 transgenic and wild-type mice fed a high-fat diet. Lipid accumulation and mitochondrial function were also studied in rodent myoblasts overexpressing STK25. Global quantitative phosphoproteomics was performed in skeletal muscle of Stk25 transgenic and wild-type mice fed a high-fat diet to identify potential downstream mediators of STK25 action.
We found that overexpression of STK25 in transgenic mice fed a high-fat diet increases intramyocellular lipid accumulation, impairs skeletal muscle mitochondrial function and sarcomeric ultrastructure, and induces perimysial and endomysial fibrosis, thereby reducing endurance exercise capacity and muscle insulin sensitivity. Furthermore, we observed enhanced lipid accumulation and impaired mitochondrial function in rodent myoblasts overexpressing STK25, demonstrating an autonomous action for STK25 within cells. Global phosphoproteomic analysis revealed alterations in the total abundance and phosphorylation status of different target proteins located predominantly to mitochondria and sarcomeric contractile elements in Stk25 transgenic vs wild-type muscle, respectively, providing a possible molecular mechanism for the observed phenotype.
STK25 emerges as a new regulator of the complex interplay between lipid storage, mitochondrial energetics and insulin action in skeletal muscle, highlighting the potential of STK25 antagonists for type 2 diabetes treatment.
KeywordsEctopic lipid storage Insulin resistance Mitochondrial dysfunction Skeletal muscle
Cytochrome c oxidase
Extensor digitorum longus
Microtubule-associated protein 1S
Mitochondrial fission factor
Myosin heavy chain
Serine/threonine protein kinase 25
Transmission electron microscopy
Type 2 diabetes is strongly associated with ectopic lipid deposition within non-adipose tissue, which actively contributes to the development of insulin resistance [1, 2, 3]. Skeletal muscle plays an important role in the pathophysiology of type 2 diabetes accounting for more than 70% of whole body glucose use . Thus, approaches that can suppress ectopic lipid deposition within the skeletal muscle, and increase the responsiveness of muscle to insulin, offer a potential for the development of new therapies for diabetes.
In the search for novel targets that contribute to the pathogenesis of insulin resistance and type 2 diabetes, we recently described serine/threonine protein kinase 25 (STK25; also referred to as YSK1 or SOK1), a member of the sterile 20 (STE20) kinase superfamily , as a central regulator of ectopic lipid accumulation, and whole body glucose and insulin homeostasis [6, 7, 8, 9, 10, 11]. STK25 is broadly expressed in mouse, rat and human tissues [10, 11, 12, 13]. Previous studies have shown that STK25, present in the Golgi complex, regulates cell polarisation and migration in different cell types [14, 15, 16, 17]. It is also reported that in cells subjected to extreme stresses, STK25 enters the nucleus and induces cell death [18, 19]. We found that in mice fed on a high-fat diet, transgenic mice overexpressing STK25 display hyperinsulinaemia and impaired whole body glucose and insulin homeostasis compared with wild-type littermates . Reciprocally, our studies showed that, compared with wild-type littermates, Stk25 knockout mice are protected against systemic glucose intolerance and insulin resistance induced by a high-fat diet . Notably, we found that in both mouse and human liver cells, STK25 is localised on the surface of cytosolic lipid droplets [7, 8]. We observed that increased STK25 abundance in mouse liver and human hepatocytes enhances fat deposition in intrahepatocellular lipid droplets by suppressing lipolytic activity and thereby fatty acid release for β-oxidation and triacylglycerol secretion; the reciprocal effect was seen with STK25 knockdown [7, 8]. Furthermore, we found a significant positive correlation between STK25 mRNA expression and fat content in human liver biopsies [8, 9]. Moreover, STK25 mRNA levels were higher in the skeletal muscle of individuals with type 2 diabetes than in healthy volunteers .
On the basis of our previous findings, which reveal a central role of STK25 in control of hepatic fat deposition and systemic insulin sensitivity [6, 7, 8, 9, 10], we hypothesised that STK25 is also involved in regulation of ectopic lipid storage and insulin responsiveness in skeletal muscle. Here, we provide the first evidence to support the key cell-specific role of STK25 in the excessive accumulation of intramyocellular lipids in the context of chronic exposure to dietary lipids, which is associated with suppressed mitochondrial function, reduced endurance exercise capacity and exacerbated insulin resistance in skeletal muscle.
The generation of Stk25 transgenic mice, where mouse Stk25 expression in the targeting construct is controlled by chicken β-actin promoter, and the subsequent breeding with C57BL/6NCrl mice (Charles River, Sulzfeld, Germany), have been described previously . From the age of 6 weeks, male transgenic mice and wild-type littermates were fed a pelleted high-fat diet (45% kilocalories from fat; D12451; Research Diets, New Brunswick, NJ, USA). At the age of 24 weeks, the mice were killed after 4 h of food withdrawal. Gastrocnemius skeletal muscle samples were collected for histological analysis (see Histology and immunofluorescence) or snap frozen in liquid nitrogen and stored at −80°C for analysis of protein and gene expression (see ESM Fig. 1 for a schematic overview). All animal experiments were performed after approval from the local Ethics Committee for Animal Studies at the Administrative Court of Appeals in Gothenburg, Sweden, and followed appropriate guidelines.
Histology and immunofluorescence
Gastrocnemius muscle samples were embedded in optimal cutting temperature mounting medium (Histolab Products, Gothenburg, Sweden) and frozen in liquid nitrogen followed by cryosectioning and staining with haematoxylin-eosin (H-E; Histolab Products), Nile Red (Sigma-Aldrich, St Louis, MO, USA) or MitoTracker Red (Thermo Fisher Scientific, Waltham, MA, USA). Enzymatic stainings were performed as previously described . For immunofluorescence, sections were incubated with primary antibodies followed by incubation with secondary antibodies (see ESM Table 1). Gastrocnemius muscle samples were also fixed with 4% formaldehyde in phosphate buffer (Histolab Products), embedded in paraffin, sectioned and stained with Picrosirius Red (Histolab Products) or Periodic acid–Schiff (PAS; Sigma-Aldrich). Ultrastructural analysis of gastrocnemius muscle was performed by transmission electron microscopy (TEM; LEO 912AB; Omega; Carl Zeiss NTS, Oberkochen, Germany) as previously described . Gastrocnemius muscle homogenates were analysed using a Hydroxyproline Colorimetric Assay Kit (Sigma-Aldrich) and a Triglyceride Calorimetric Assay Kit (Biovision, Mountain View, CA, USA).
Cell culture and transient overexpression
L6 myoblasts (Rattus norvegicus, American Type Culture Collection, Manassas, VA, USA) were maintained as described  and transfected with pFLAG-Stk25 (cytomegalovirus promoter; GeneCopoeia, Rockville, MD, USA) or an empty control plasmid using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). Cells were incubated with 50 μmol/l oleic acid for 24 h and stained with Oil Red O or MitoTracker Red as described . Palmitate oxidation was measured as previously described . Cells have been demonstrated to be free of mycoplasma infection by use of the MycoAlert Mycoplasma Detection kit (Lonza, Basel, Switzerland).
Western blot and quantitative real-time PCR
Western blotting was performed as previously described  in gastrocnemius muscle of Stk25 transgenic and wild-type mice and/or transfected L6 myoblasts using anti-STK25, anti-adipose triacylglycerol lipase (ATGL), anti-hormone-sensitive lipase (HSL), anti-PLIN2 and anti-actin primary antibodies (see ESM Table 1). The anti-STK25 antibody has been validated by using Stk25-knockout mice . Quantitative real-time PCR was performed in gastrocnemius muscle of Stk25 transgenic and wild-type mice and transfected L6 myoblasts using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) as described  (see ESM Table 2).
Ex vivo measurement of lipid metabolism
The oxidation rate of palmitate was measured in quadriceps muscle homogenates as described previously . Oleic acid uptake and triacylglycerol synthesis from [14C]-oleic acid were measured in isolated extensor digitorum longus (EDL) and soleus muscles as described [22, 23].
In vivo assessment of exercise performance and insulin sensitivity
Endurance exercise was assessed by treadmill running until the mice reached fatigue (see ESM Methods). Insulin sensitivity was measured by euglycaemic–hyperinsulinaemic clamp as previously described  using an insulin infusion rate of 7 mU/min/kg (see ESM Methods).
Liquid chromatography mass spectrometry analysis
Gastrocnemius muscle samples were heat stabilised and prepared, including tryptic digestion, chemical labelling for relative quantification, enrichment of phosphopeptides and pre-fractionation, as described in ESM Methods. Liquid chromatography mass spectrometry (LC-MS)/MS of these combined tandem mass tagged labelled samples was performed on an Orbitrap Fusion Tribrid MS interfaced to an Easy-nLC 1000 (Thermo Fisher Scientific).
Statistical significance between groups was calculated with an unpaired two-tailed Student’s t test or by two-way ANOVA followed by Tukey post hoc test. A p < 0.05 was considered statistically significant.
Evidence for muscle damage in Stk25 transgenic mice, with fibre type composition remaining unaltered
We previously showed that STK25 is highly expressed in mouse, rat and human skeletal muscle . Here we found that endogenous STK25 protein was 2.1 ± 0.3-fold higher in white portions of the gastrocnemius muscle (predominantly type IIb fibres) compared with red portions (predominantly type IIa fibres) in wild-type mice (ESM Fig. 2a). Nonetheless, the endogenous STK25 protein was detected by immunofluorescence in all fibre types (ESM Fig. 2b). STK25 was markedly increased in both red and white portions of Stk25 transgenic vs wild-type muscle (ESM Fig. 2a, b).
Picrosirius Red staining for collagen revealed that perimysial fibrosis was increased in Stk25 transgenic vs wild-type muscle and endomysial fibrosis, not present in wild-type muscle, was readily observed in transgenic muscle (Fig. 1h). Consistently, hydroxylated proline, a main constituent of collagens, was 1.6 ± 0.2-fold higher in Stk25 transgenic muscle homogenates (Fig. 1i).
STK25 overexpression in mice augments fat storage and impairs mitochondrial function in skeletal muscle
TEM demonstrated that a significant fraction of mitochondria in Stk25 transgenic, but not wild-type, muscle were structurally distorted and appeared swollen, and displayed disarrayed cristae, reduced electron density of the matrix and/or internal vesicles (Fig. 2d, ESM Fig. 3). TEM also revealed the presence of large lipid droplets in transgenic but not wild-type muscle (Fig. 2d). Mitochondrial DNA (mtDNA) copy number, and the expression of key transcriptional activators mediating mitochondrial biogenesis—PGC1α (Ppargc1a), PGC1β (Ppargc1b) and nuclear respiratory factor 1 (Nrf1)—were similar in Stk25 transgenic vs wild-type muscle (ESM Figs 4, 5).
Our previous studies have shown that the glycogen content was similar in gastrocnemius muscle homogenates of Stk25 transgenic and wild-type mice . Consistent with these findings, PAS staining in glycogen-rich type IIa and IIx fibres was similar between the genotypes (Fig. 2e).
Overexpression of STK25 induces lipid accumulation and represses mitochondrial function in myoblasts
As with the results obtained in Stk25 transgenic vs wild-type muscle (see above), mtDNA copy number and the expression of key transcriptional activators mediating mitochondrial biogenesis (PGC1α, PGC1β and Nrf1) were largely similar in cells transfected with the STK25 expression plasmid and vector control (ESM Fig. 6b, c). Our previous studies have shown that in skeletal muscle of Stk25 transgenic mice fed a high-fat diet, the mRNA expression of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid oxidation in mitochondria, was significantly reduced . Surprisingly, Cpt1 mRNA levels were markedly increased in STK25-overexpressing L6 cells (ESM Fig. 6c); while the significance of this observation remains unclear, in view of other data suggesting repressed mitochondrial function.
Overexpression of STK25 in mice reduces skeletal muscle β-oxidation, while lipid uptake and synthesis remain unaltered
In addition, we incubated isolated skeletal muscle from Stk25 transgenic and wild-type mice with radiolabelled oleate and observed that cell-associated radioactivity was not significantly altered between the genotypes, indicating similar fatty acid influx (Fig. 4b). Furthermore, skeletal muscles isolated from both genotypes displayed similar incorporation of oleate into triacylglycerol (Fig. 4c).
Stk25 transgenic mice have reduced running performance
Stk25 transgenic mice display reduced in vivo insulin-stimulated glucose uptake in skeletal muscle
The observation that STK25 overexpression increased intramyocellular lipid levels and favoured fat storage rather than oxidation in the muscle prompted us to investigate whether these changes would affect skeletal muscle insulin sensitivity. To this end, euglycaemic–hyperinsulinaemic clamp experiments with a glucose tracer were performed in Stk25 transgenic and wild-type mice fed a high-fat diet. Insulin infusion significantly increased plasma insulin concentration at the end of the clamp for both genotypes (ESM Fig. 8a). There was no difference in glucose infusion rate or blood glucose level at steady state of the clamp comparing the two genotypes (ESM Fig. 8b). Insulin-stimulated glucose uptake was 1.5 ± 0.1-fold and 1.6 ± 0.1-fold lower in gastrocnemius and quadriceps muscles of Stk25 transgenic mice, respectively, with a similar tendency seen in EDL and soleus muscles (Fig. 5d).
Global phosphoproteomic analysis of skeletal muscle in Stk25 transgenic and wild-type mice
Differential regulation of the total protein abundance in gastrocnemius skeletal muscle of Stk25 transgenic and wild-type mice fed a high-fat diet
Serine/threonine protein kinase 25 (EC 188.8.131.52)
STK25 belongs to the STE20 serine/threonine protein kinase superfamily. It has been shown to be involved in regulation of cell migration, modulation of cell death and control of glucose tolerance and insulin sensitivity
The taxilin family is composed of three members in mammals, where taxilin beta is abundantly expressed in the skeletal muscle and heart. Available evidence suggests that taxilin proteins interact with several syntaxin family members and are involved in intracellular vesicle traffic, especially transport of the vesicles delivered to the plasma membrane
Homer homologue 1
Homer proteins belong to a family of adaptor proteins, which play different roles in cell function, including the regulation of GPCRs and Ca2+ homeostasis. HOMER1 associates both with STIM1 and the Cav1.2 alpha1 subunit upon Ca2+ store depletion, which indicates a functional role in supporting the interaction between STIM1 and Cav1.2 channels
Carbonyl reductase 4
CRB4 and HSD17B8 are the two subunits that make up KAR (EC 184.108.40.206), which catalyses the second step of the mitochondrial fatty acid synthesis pathway
Rbmx (alias Hnrnpg)
RNA binding motif protein, X chromosome (alias heterogeneous nuclear ribonucleoprotein G)
RBMX is a ubiquitously expressed nuclear glycoprotein implicated in pre-mRNA splicing and in regulation of cellular splicing preferences
Fhod3 (alias Fhos2)
Formin homology 2 domain containing 3 (alias formin homologue overexpressed in spleen 2)
FHOD3 is a formin family protein, which has actin-organising activity and has been shown to associate with the nestin intermediate filaments
Galactose-1-phosphate uridyl transferase (EC 220.127.116.11)
GALT is the second enzyme in the evolutionarily conserved galactose metabolic pathway, and facilitates the simultaneous conversion of uridine diphosphoglucose and galactose-1 phosphate to uridine diphosphogalactose and glucose-1 phosphate It has been shown that GALT-deficient mice accumulate galactitol and galactonate in heart and skeletal muscle
Chromodomain helicase DNA binding protein 4
CHD4 is a chromatin-remodelling enzyme that has been reported to regulate DNA damage responses
Mitochondrial fission factor
MFF is anchored to the mitochondrial outer membrane through a C-terminal transmembrane domain. Depletion of MFF promotes mitochondrial fusion, resulting in an interconnected tubular network of mitochondria; in contrast, exogenous expression of MFF induces extensive mitochondrial fragmentation
Prkaca (alias Pkaca)
Protein kinase, cAMP dependent, catalytic, alpha (alias protein kinase A catalytic subunit alpha)
Most of the effects of cAMP in the eukaryotic cell are mediated through the phosphorylation of target proteins on Ser or Thr residues by PKA (EC 18.104.22.168). Inactive PKA is a tetramer composed of 2 regulatory and 2 catalytic subunits. The cooperative binding of 4 molecules of cAMP dissociates the enzyme in a regulatory subunit dimer and 2 free active catalytic subunits. In the human, 3 catalytic subunits (PRKACA, PRKACB and PRKACG) have been identified. PKA anchoring proteins (AKAPs) modulate PKA-dependent phosphorylation by tethering this kinase to a specific subcellular location
Cathepsins are the ubiquitously expressed major lysosomal proteases and they primarily determine the proteolytic capacity of lysosomes. Cathepsin B has been implicated in signalling pathways of apoptosis, liver fibrosis and muscle wasting
Von Willebrand factor A domain-containing protein 8
VWA8 is a protein of unknown function with a high mitochondrial localisation prediction
Ywhaz (alias 14-3-3ζ)
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (alias 14-3-3 protein zeta)
YWHAZ is involved in the import of precursor proteins into mitochondria. The expression of YWHAZ appears to be upregulated in a number of human tumours, suggesting that this protein may exhibit oncogenic properties. YWHAZ is reported to interact with several targets such as with IRS1 and PKB (Akt1), although the functional significance of these interactions remains unclear
Gps1 (alias Csn1)
G protein pathway suppressor 1 (alias COP9/signalosome complex subunit)
GPS1 is one of the 8 subunits of the COP9 signalosome (CSN). The cullin deneddylation activity of CSN requires all its subunits and regulates cullin-RING ligases, thereby controlling ubiquitination of a large number of proteins
Epilepsy, progressive myoclonic epilepsy, type 2 gene alpha (alias laforin)
EPM2A is, by sequence, a member of the atypical dual specificity protein phosphatase subfamily, but it has been shown to dephosphorylate polysaccharides including glycogen and amylopectin. Loss-of-function mutations in the EPM2A gene cause the fatal neurodegenerative disorder in humans, Lafora disease, characterised by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies
Rtn4ip1 (alias Nimp)
Reticulon 4 interacting protein 1 (alias NOGO-interacting mitochondrial protein)
RTN4IP1 is a highly conserved and ubiquitously expressed novel mitochondrial ubiquinol oxydo-reductase. Mutations in RTN4IP1 have been associated with a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light
N-ethylmaleimide sensitive fusion protein
NSF was the first protein found to play a key role in eukaryotic trafficking. In concert with the adaptor protein SNAP, NSF disassembles the SNARE complex into individual proteins upon ATP hydrolysis. By disassembling post-fusion SNARE complexes, NSF is essential for maintaining pools of fusion-ready individual SNARE proteins that mediate membrane fusion in a variety of cellular processes
Mitochondrial ribosomal protein L2
Mitochondria have their own translation system for production of proteins essential for oxidative phosphorylation. MRPL2 is one of the protein components of mitochondrial ribosomes that are encoded by the nuclear genome
Leucine-rich PPR-motif containing
LRPPRC regulates stability of mitochondrial DNA-encoded mRNAs. Inactivation of LRPPRC impairs mitochondrial respiration and reduces ATP production caused mainly by an ATP synthase deficiency
Rras2 (alias Tc21)
Related RAS viral (R-Ras) oncogene homologue 2
RRAS2 is the ubiquitously expressed member of the R-Ras family of Ras-related proteins. Deregulated RRAS2 activity has been suggested to contribute to human oncogenesis
Glyoxalase domain containing 4
GLOD4 is a mitochondrial protein implicated in metabolic detoxification; however, the exact function of GLOD4 is not known
Stromal interaction molecule 1
STIM1 is a transmembrane protein, mainly located to the membrane of the endoplasmic reticulum. STIM1 is considered a key element in the activation of store-operated Ca2+ (SOC) entry by mediating the communication of the filling state of the Ca2+ stores to the plasma membrane channels
Proteasome (prosome, macropain) 26S subunit, non-ATPase, 6
In eukaryotes, protein turnover is almost entirely accomplished by a single enzyme, the 26S proteasome. PSMD6 acts as a regulatory subunit of the 26S proteasome, and is probably involved in the ATP-dependent degradation of ubiquitinated proteins
Anxa7 (alias Anx7)
Annexin A7 (alias annexin-7, synexin)
ANXA7 is a calcium-dependent membrane-binding protein that regulates membrane fusion and acts as a voltage-dependent calcium channel. ANXA7 participates in cellular Ca2+ signalling and has been implicated if different cellular processes such as spherocytosis, inflammatory myopathies, cardiac remodelling, regulation of cell survival and tumour growth as well as in excitation–contraction coupling in skeletal muscle
Aldehyde dehydrogenase 2, mitochondrial (EC 22.214.171.124)
Aldehyde dehydrogenases catalyse the conversion of reactive aldehydes to carboxylates. Mitochondrial ALDH2 is known to oxidise acetaldehyde produced from ethanol into acetate
Aldh6a1 (alias Mmsdh)
Aldehyde dehydrogenase family 6, subfamily A1 [alias methylmalonate-semialdehyde dehydrogenase (acylating), mitochondrial] (EC 126.96.36.199)
The Aldh6a1 gene encodes mitochondrial methylmalonate semialdehyde dehydrogenase, an enzyme that catalyses the irreversible oxidative decarboxylation of malonate and methylmalonate semialdehydes to acetyl- and propionyl-CoA, respectively. This activity is part of the valine and pyrimidine catabolic pathways
Echs1 (alias Sceh)
Enoyl coenzyme A hydratase, short chain, 1, mitochondrial (alias enoyl-CoA hydratase 1)
ECHS1 catalyses the second step in mitochondrial fatty acid oxidation
Map1s (alias Map8, C19ORF5)
Microtubule-associated protein 1S (alias microtubule-associated protein 8)
The ubiquitously distributed MAP1S has been implicated in microtubule dynamics and mitotic abnormalities, mitotic cell death and autophagy regulation. MAP1S is an interactive partner of mitochondrion-associated LRPPRC and RASSF1 as well as ND1 and COX-I. MAP1S deficiency in mice causes an accumulation of large swollen mitochondria indicative of a potential defect in mitophagy, while accumulation of MAP1S was associated with irreversible aggregation of mitochondria in mammalian cells
NADH dehydrogenase (ubiquinone) 1 beta subcomplex 7
NDUFB7 is a eukaryotic complex I subunit that resides in the mitochondrial intermembrane space. NADH:ubiquinone oxidoreductase (complex I) catalyses the first step in oxidative phosphorylation. It couples the oxidation of NADH to the reduction of ubiquinone and the translocation of protons across the inner membrane
Differential regulation of the phosphorylation pattern in gastrocnemius skeletal muscle of Stk25 transgenic and wild-type mice fed a high-fat diet
Titin is a giant muscle protein that spans from Z-disk to M-disk and plays a key role in muscle assembly, force transmission at the Z-disk, and maintenance of resting tension in the I-band region
Mylpf (alias Mlc2)
Myosin light chain, phosphorylatable, fast skeletal muscle
MYLPF is controlled by phosphorylation and modulates muscle contraction properties
Sarcalumenin is a Ca2+ binding protein localised to the sarcoplasmic reticulum that is considered to be important in the excitation–contraction–relaxation cycle in skeletal muscle cells
Ckm (alias M-ck)
Creatine kinase, muscle (EC 188.8.131.52)
Creatine kinase catalyses the reversible transfer of a phosphate from phosphocreatine to ADP, maintaining intracellular ATP levels
Aldolase A, fructose-bisphosphate (EC 184.108.40.206)
ALDOA is a glycolytic enzyme that catalyses the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate
SMTNL2 is a functionally uncharacterised protein from the SMTN family with notably high expression in skeletal muscle
Tropomyosin 1, alpha
Tropomyosin 1 represents a critical myofilament protein in the Ca2+ regulation of actin–myosin interaction, striated muscle contraction, and relaxation. TPM1 is the predominant isoform in the mammalian heart and fast skeletal muscle
Enolase 3, beta muscle (EC 220.127.116.11)
Muscle-specific ENO3 is a glycolytic enzyme that resides at the M-disk and converts the glycolytic intermediate 2-phospho-d-glycerate to phosphoenolpyruvate
Myoz2 (alias Cs-1)
Myozenin 2 (alias calsarcin-1)
Myozenins are calcineurin-interacting proteins that also bind to ACTN2 and LDB3 at the Z-disk, suggesting a role in modulating calcium–calcineurin-dependent signalling
Cardiomyopathy associated 5 (alias myospryn)
CMYA5 is a multifunctional desmin-binding protein that also associates with PKA. CMYA5 is suggested to participate in the subcellular targeting of PKA activity in striated muscle
SPEG complex locus
SPEG is a protein localised to the sarcoplasmic reticulum that interacts with myotubularin; SPEG deficiency causes CNM in humans
Eukaryotic translation initiation factor 3, subunit B
EIF3B is part of the EIF3 complex
Ybx3 (alias Csda)
Y box protein 3 (alias cold-shock domain protein A)
YBX3 is a transcriptional repressor, which is highly expressed in skeletal muscle, where it specifically regulates myogenin expression
Myoz1 (alias Cs-2)
Myozenin 1 (alias calsarcin-2)
Myozenins are calcineurin-interacting proteins that also bind to ACTN2 and LDB3 at the Z-disk, suggesting a role in modulating calcium–calcineurin-dependent signalling
Actinin alpha 3
ACTN3 is localised to the Z-disk, where it helps to anchor the actin filaments and regulates contractile properties of the muscle
Myh4 (alias MhcIIb)
Myosin, heavy polypeptide 4, skeletal muscle (alias myosin heavy chain IIb)
Skeletal muscle fibres are classified by speed of contraction, where expression of MYH4 defines the fast-twitch glycolytic type IIb fibres
Glyceraldehyde-3-phosphate dehydrogenase (EC 18.104.22.168)
GAPDH catalyses an important energy-yielding step in carbohydrate metabolism, the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and NAD
Bin1 (alias Amph2)
Bridging integrator 1 (alias amphiphysin II)
BIN1 is localised in skeletal muscle at deep sarcolemmal invaginations, the T-tubules, implicated in excitation–contraction coupling; mutations in BIN1 have been described in CNM in humans
Junctional sarcoplasmic reticulum protein 1
JSRP1 is an integral protein constituent of the skeletal muscle sarcoplasmic reticulum membrane, involved in the development and maintenance of skeletal muscle strength
Nascent polypeptide-associated complex alpha polypeptide
NACA, exclusively found in skeletal and heart muscle, is suggested to be involved in skeletal muscle development, homeostasis and regeneration, although the underlying mechanisms remain unclear
Ldb3 (alias Zasp)
LIM domain binding 3 (alias Z-band alternatively spliced PDZ motif-containing protein)
LDB3 is a PDZ-LIM domain binding factor that plays an important role in maintaining the structural integrity of the striated muscle Z-disk. LDB3 binds to myozenin and ACTN2
Assessment of the known cellular localisation of the differentially expressed proteins revealed a marked enrichment of the targets located to mitochondria (Fig. 6d, Table 1). Among these candidates, carbonyl reductase 4 (CBR4), an enzyme in the mitochondrial fatty acid synthesis pathway , reticulon 4 interacting protein 1 (RTN4IP1), enoyl coenzyme A hydratase, short chain, 1 (ECHS1), and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 7 (NDUFB7)—all involved in mitochondrial oxidation [25, 26, 27]—as well as two mitochondrial aldehyde dehydrogenases (ALDH2 and ALDH6A1 [28, 29]) were upregulated in transgenic muscle. In addition, mitochondrial fission factor (MFF) and microtubule-associated protein 1S (MAP1S), which control mitochondrial morphology by regulating dynamic fission and fusion process [30, 31], were differentially expressed comparing the genotypes. Apart from these candidates with relatively well-known functions, several additional proteins with poorly described roles in mitochondria were differentially expressed in transgenic muscle, including von Willebrand factor A domain-containing protein 8 (VWA8) and glyoxalase domain-containing 4 (GLOD4).
Analysis of proteins containing differentially regulated phosphosites, on the other hand, revealed an enrichment of the targets with known location in the Z-disk, where actin-containing thin filaments from neighbouring sarcomeres overlap cross-linked by alpha-actinin, and in the myosin-containing A-band of the sarcomere (Fig. 6e, Table 2). These myofibril-associated candidates included proteins with well-characterised roles in regulation of contractile properties of the skeletal muscle, such as titin (TTN), myosin regulatory light chain (MYLPF), creatine kinase (CKM), tropomyosin 1, alpha (TPM1), actinin alpha 3 (ACTN3) and myosin heavy chain II beta (MYH4 or MHCIIB), as well as proteins with less defined roles, such as myozenin 1 and 2 (MYOZ1 and 2) and LIM domain binding 3 (LDB3). Interestingly, the phosphorylation of the three enzymes in the glycolytic pathway—aldolase A, fructose-bisphosphate (ALDOA), enolase 3, beta muscle (ENO3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—was altered in Stk25 transgenic compared with wild-type muscle. Of the 26 differentially regulated phosphosites, 24 were annotated in PhosphoSitePlus . However, the functional implication of the phosphorylation at these sites has only been described for TPM1, where phosphorylation at a single Ser-283 residue has been associated with increased Ca2+ activated ATPase activity  and regulation of Ca2+ sensitivity .
Notably, our previous studies have shown that overexpression of STK25 repressed lipolysis in mouse and human hepatocytes, which probably contributed to the increased lipid storage observed in these cells [7, 8]. The global phosphoproteomic analysis by MS/MS did not detect any lipases above the level of quantification in the skeletal muscle. However, we also measured the skeletal muscle mRNA and protein levels of two lipases (ATGL and HSL) by quantitative real-time PCR and western blot, respectively, and observed no differences between the genotypes (ESM Fig. 9). Recently, the levels of the lipid droplet binding proteins Perilipin 2 (PLIN2 also known as adipose differentiation-related protein [ADRP]) and Perilipin 3 (PLIN3), as well as the activity of AMP-activated protein kinase (AMPK), have been implicated in the regulation of lipid content in muscle cells [35, 36]. PLIN3 was quantified by MS/MS analysis without any alterations in total protein abundance observed between the genotypes, while PLIN2 and AMPK were below the level of quantification. However, we found that the skeletal muscle mRNA of Plin2 and Plin3, and protein abundance of PLIN2 measured by western blot, were not changed when comparing the genotypes (ESM Fig. 9). Furthermore, our previous studies have shown that AMPK activity, measured by AMPKα Thr172 phosphorylation, was similar when comparing the skeletal muscle of Stk25 transgenic and wild-type mice .
The main finding of this study is the substantially increased presence of ultrastructural abnormalities of both subsarcolemmal and intermyofibrillar mitochondria in Stk25 transgenic skeletal muscle, which was associated with reduced MitoTracker Red staining, lower abundance of oxidative metabolism markers and suppressed β-oxidation. Previous studies using TEM have demonstrated alterations in mitochondrial morphology in skeletal muscle of humans and rodent models with insulin resistance and type 2 diabetes [38, 39, 40]. In addition, reduced in vivo mitochondrial oxidative capacity has been reported in the skeletal muscle of patients with type 2 diabetes [41, 42, 43]. Furthermore, studies in healthy elderly individuals and insulin-resistant offspring of parents with type 2 diabetes have demonstrated that repressed mitochondrial function may predispose these individuals to intramyocellular lipid accumulation and insulin resistance . Moreover, coordinated reduction in the expression of genes involved in mitochondrial function and oxidative phosphorylation was reported in skeletal muscle from type 2 diabetes patients, but also from individuals with insulin resistance but normal glucose tolerance [45, 46]. Based on this evidence, impaired mitochondrial function observed in Stk25 transgenic muscle probably contributed to increased myocellular lipid storage and development of skeletal muscle insulin resistance.
We further explored the underlying mechanisms involved in the regulation of the mitochondrial structure and activity by STK25. We failed to detect any accumulation of STK25 protein in skeletal muscle mitochondria (ESM Fig. 2c), and therefore, STK25 is likely to control mitochondrial function indirectly through the regulation of abundance and/or phosphorylation pattern of, as yet, unknown mitochondrial target proteins. Interestingly, global phosphoproteomic analysis revealed that the two key regulators of mitochondrial fusion and fission process were differentially expressed, with MFF protein levels decreased and MIP1S protein levels increased, in Stk25 transgenic vs wild-type muscle. MFF knockdown in mammalian cells resulted in an interconnected tubular network of mitochondria, whereas MFF overexpression stimulated mitochondrial fission [31, 47]. Accumulation of MAP1S, on the other hand, was associated with irreversible aggregation of mitochondria . Based on this evidence, we speculate that altered protein abundance of MFF and MAP1S may be one underlying mechanism for impaired mitochondrial morphology in Stk25 transgenic muscle.
We also found disorganised myofibril architecture and pronounced fibrosis in Stk25 transgenic muscle, which probably contributed to reduced endurance exercise capacity. This was not observed in wild-type muscle. Notably, global phosphoproteomic analysis revealed a marked enrichment of the differentially phosphorylated proteins located in the myofibril, in particular to the Z-disk and A-band of the sarcomere, in Stk25 transgenic vs wild-type muscle. However, at present it is not known whether these changes have contributed to the disrupted sarcomere organisation in transgenic muscle. Interestingly, recent evidence shows that perturbations in skeletal muscle sarcomere ultrastructure in individuals with heart failure and type 2 diabetes can be improved by stimulating mitochondrial function , which supports a close functional connection between mitochondrial alterations and muscle damage, and suggests that mitochondrial abnormalities observed in Stk25 transgenic muscle may have contributed to altered myofibril architecture.
The metabolic changes in the skeletal muscle of Stk25 transgenic mice are consistent with our previous observations in mouse and human liver cells, where STK25 overexpression in conditions of excess dietary fuels increased intrahepatocellular lipid deposition while repressing mitochondrial function and insulin sensitivity [7, 8, 9]. Taken together, these findings suggest that STK25 may regulate the shift in the metabolic balance from lipid use to lipid storage in several tissues prone to diabetic damage, contributing to the pathogenesis of whole body insulin resistance and type 2 diabetes.
In this study, we characterised the skeletal muscle phenotype of Stk25 transgenic and wild-type mice challenged with a high-fat diet in order to mimic conditions in high-risk individuals. Notably, our previous investigations have shown that liver lipid deposition and whole body insulin sensitivity were not significantly altered comparing Stk25 transgenic vs wild-type mice fed regular chow [9, 10]. Moreover, we found that skeletal muscle triacylglycerol content and fibrosis were not increased in Stk25 transgenic mice fed regular chow compared with corresponding wild-type littermates (ESM Fig. 10). These data suggest that overexpression of STK25 leads to significant metabolic alterations in mice only after a dietary challenge.
To date, it remains unknown whether any physiological situations occur in which the STK25 protein abundance is enhanced to a level observed in the Stk25 transgenic muscle, which is a limitation of the animal model. Notably, our findings in Stk25 transgenic muscle are reciprocal to our previous observations of increased β-oxidation and improved insulin action in STK25-deficient myoblasts  as well as reduced lipid accumulation and enhanced insulin sensitivity in the skeletal muscle of Stk25-/- mice fed a high-fat diet , reinforcing the physiological validity of the results.
In light of the current epidemic of type 2 diabetes, research aimed at understanding the interplay between intramyocellular lipid storage, mitochondrial energetics, and insulin action in skeletal muscle is of utmost importance for the development of new therapeutic strategies. This study provides several layers of evidence that STK25 is an interesting new mediator in the interconnected metabolic network controlling skeletal muscle insulin sensitivity, and that the development of STK25 antagonists for therapeutic applications in type 2 diabetes and related metabolic disease is warranted.
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.
This work was supported by grants from the Swedish Research Council, the Novo Nordisk Foundation, the Swedish Heart and Lung Foundation, the Diabetes Wellness Network Sweden, the Estonian Research Council, the Swedish Diabetes Foundation, the Royal Society of Arts and Sciences in Gothenburg, the M. Bergvalls Foundation, the Wiberg Foundation, the Adlerbert Research Foundation, the I. Hultman Foundation, the S. and E. Goljes Foundation, the West Sweden ALF Program, the F. Neubergh Foundation, the I.-B. and A. Lundbergs Research Foundation, W. and M. Lundgrens Foundation and the European Foundation for the Study of Diabetes and Novo Nordisk Partnership for Diabetes Research in Europe. The study sponsor was not involved in the design of the study; the collection, analysis, and interpretation of data; writing the report; or the decision to submit the report for publication.
Duality of interest
FB is co-founder and shareholder in Metabogen AB. FB has participated on advisory boards and received honoraria from Unilever, Boehringer and Merck. The authors declare that there is no other duality of interest associated with this article.
All the authors made substantial contributions to conception and design, and/or analysis and interpretation of data. UC and EN-D generated the bulk of the results. EC, MA, SS, MEJ, FB and MM contributed to the research data. MS and JB performed the analysis of acylcarnitines. B-MO and CS conducted the primary global phosphoproteomic analysis. BRJ conducted the TEM studies. All the authors revised the article critically for important intellectual content and approved the final version of the article to be published. MM directed the project, designed the study, interpreted the data and wrote the manuscript. MM is the guarantor of this work.
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