Thrombospondin-1 promotes liver fibrosis by enhancing TGF-ß action in hepatic stellate cells.

Insulin resistance in adipose tissue is thought to be a key contributor to the pathogenesis of various metabolic disorders including metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis (MASLD/MASH), but the mechanism underlying this contribution to MASLD/MASH has remained unknown. We previously showed that dysregulation of the PDK1-FoxO1 signaling axis in adipocytes plays a role in the development of MASLD/MASH by analysis of adipocyte-specific PDK1 knockout (A-PDK1KO) and adipocyte-specific PDK1/FoxO1 double-knockout (A-PDK1/FoxO1DKO) mice. We here focused on the role of the extracellular matrix protein thrombospondin-1 (TSP-1) as a secreted factor whose expression in adipose tissue is increased in A-PDK1KO mice and normalized in A-PDK1/FoxO1DKO mice. Genetic ablation of TSP-1 markedly ameliorated liver fibrosis in A-PDK1KO mice fed a high-fat diet. With regard to the potential mechanism of this effect, TSP-1 augmented the expression of fibrosis-related genes induced by TGF-ß in LX-2 human hepatic stellate cells. We also showed that TSP-1 expression and secretion were negatively regulated by insulin signaling via the PDK1-FoxO1 axis in cultured adipocytes. Our results thus indicate that TSP-1 plays a key role in the pathogenesis of liver fibrosis in MASH. Regulation of TSP-1 expression by PDK1-FoxO1 axis in adipocytes may provide a basis for targeted therapy of hepatic fibrosis in individuals with MASH.

Krüppel-like factor 15 deficiency exacerbates osteoarthritis through reduced expression of peroxisome proliferator-activated receptor gamma signaling in mice.

OBJECTIVE: Krüppel-like zinc finger transcription factors (KLFs) play diverse roles in mammalian cell differentiation and development. In this study, we investigated the function of KLF15 in the progression of osteoarthritis (OA). METHODS: 0Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male wild-type control (WT) mice and cartilage-specific KLF15 knockout (KO) mice. Histological analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining were performed. Morphological changes were measured using microcomputed tomography. Six mice from each group were analyzed (total number of mice analyzed: 60). In vitro, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western blot analyses were performed. RESULTS: KLF15 KO DMM mice exhibited significant cartilage degradation compared to WT mice. According to the Osteoarthritis Research Society International cartilage OA-histopathology scoring system, the mean sum score in KLF15 KO mice was significantly higher than that in WT mice at 8 weeks after surgery. Immunohistochemistry results revealed KLF15 KO mice exhibited reduced peroxisome proliferator-activated receptor gamma (PPARγ) expression, increased pIKKα/ß, a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) 5, and Matrix metalloproteinases (MMP13) expression, and reduced Forkhead box O (FOXO1) and Light chain 3B (LC3B) expression. Inhibition of PPARγ phosphorylation accelerated the effects of interleukin (IL) 1ß-treatment in both KLF15 KO and WT chondrocytes, and activation of PPARγ expression canceled the IL1ß-induced catabolic effects. CONCLUSION: Our results indicated that the OA phenotype of KLF15 KO DMM mice was influenced by reduced PPARγ expression, including enhanced pIKKα/ß, ADAMTS5, and MMP13 expression, reduced autophagy, and increased apoptosis. KLF15 regulation may constitute a possible therapeutic strategy for the treating OA.

Preferences for drinking water temperature in mice vary with ambient temperature.

Temperature detection and temperature preference are critical for the maintenance of thermal homeostasis and protection from noxious temperature. Methods to measure the time spent on plates of different temperatures such as the two-plate test and thermal-gradient test are generally used to evaluate temperature preference. In this study, we established a temperature preference test that assesses water drinking behavior by modifying the two-bottle taste preference test. We found that mice in a common-temperature environment of 23 °C avoid drinking water more than 40 °C. While mice in a common-temperature environment preferred 10 °C and 30 °C water equally, mice in a hot environment at 35 °C preferred to 10 °C water compared to 30 °C water. Moreover, mice in a cold environment of 10 °C did not avoid drinking 40 °C water compared to 10 °C water. From these results, the temperature preference test that we developed could be used to evaluate temperature preference owing to the ambient temperature changes.

The PDK1-FoxO1 signaling in adipocytes controls systemic insulin sensitivity through the 5-lipoxygenase-leukotriene B4 axis.

Although adipocytes are major targets of insulin, the influence of impaired insulin action in adipocytes on metabolic homeostasis remains unclear. We here show that adipocyte-specific PDK1 (3'-phosphoinositide-dependent kinase 1)-deficient (A-PDK1KO) mice manifest impaired metabolic actions of insulin in adipose tissue and reduction of adipose tissue mass. A-PDK1KO mice developed insulin resistance, glucose intolerance, and hepatic steatosis, and this phenotype was suppressed by additional ablation of FoxO1 specifically in adipocytes (A-PDK1/FoxO1KO mice) without an effect on adipose tissue mass. Neither circulating levels of adiponectin and leptin nor inflammatory markers in adipose tissue differed between A-PDK1KO and A-PDK1/FoxO1KO mice. Lipidomics and microarray analyses revealed that leukotriene B4 (LTB4) levels in plasma and in adipose tissue as well as the expression of 5-lipoxygenase (5-LO) in adipose tissue were increased and restored in A-PDK1KO mice and A-PDK1/FoxO1KO mice, respectively. Genetic deletion of the LTB4 receptor BLT1 as well as pharmacological intervention to 5-LO or BLT1 ameliorated insulin resistance in A-PDK1KO mice. Furthermore, insulin was found to inhibit LTB4 production through down-regulation of 5-LO expression via the PDK1-FoxO1 pathway in isolated adipocytes. Our results indicate that insulin signaling in adipocytes negatively regulates the production of LTB4 via the PDK1-FoxO1 pathway and thereby maintains systemic insulin sensitivity.

Canagliflozin ameliorates hepatic fat deposition in obese diabetic mice: Role of prostaglandin E2.

Clinical and animal studies have suggested a possible beneficial effect of sodium-glucose cotransporter 2 (SGLT2) inhibitors on nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH). Although SGLT2 inhibitors have been shown to reduce hepatic fat deposition in association with loss of body weight, the mechanism of this action has remained unknown. We here show that the SGLT2 inhibitor canagliflozin ameliorated fatty liver and hyperglycemia without affecting body weight or epididymal fat weight in obese diabetic KKAy mice. Lipidomics analysis based on liquid chromatography and tandem mass spectrometry revealed that canagliflozin treatment increased the amounts of prostaglandin E2 (PGE2) and resolvin E3 in the liver of these mice. We also found that PGE2 attenuated fat deposition in mouse primary hepatocytes exposed to palmitic acid. Our results thus suggest that PGE2 may play an important role in the amelioration of hepatic fat deposition by canagliflozin, with elucidation of its mechanism of action potentially providing a basis for the development of new therapeutics for NAFLD-NASH.

Modulation of lipid mediator profile may contribute to amelioration of chronic inflammation in adipose tissue of obese mice by pioglitazone.

Thiazolidinediones exert their antidiabetic effect in part by ameliorating chronic inflammation in adipose tissue. However, the precise mechanism of this anti-inflammatory action has remained unclear. We here investigated the effects of the TZD pioglitazone on the lipid mediator profile of adipose tissue in obese diabetic KKAy mice by metabololipidomics analysis based on liquid chromatography and tandem mass spectrometry. Pioglitazone treatment increased the amounts of pro-resolving lipid mediators including lipoxin B4 (LXB4), resolvin E2, and eicosapentaenoic acid as well as reduced those of prostaglandin E2 and 4-hydroxydocosahexaenoic acid in epididymal adipose tissue of KKAy mice. These effects were accompanied by increased expression of genes for the anti-inflammatory proteins arginase 1, interleukin (IL)-13, and IL-10 in this tissue. Pioglitazone also increased LXB4 production in cultured 3T3-L1 adipocytes. Finally, LXB4 increased IL-10 gene expression in adipose tissue explants from KKAy mice. Together, our results suggest that up-regulation of LXB4 may contribute to the anti-inflammatory effect of pioglitazone in obese adipose tissue.

Paternal allelic mutation at the Kcnq1 locus reduces pancreatic ß-cell mass by epigenetic modification of Cdkn1c.

Genetic factors are important determinants of the onset and progression of diabetes mellitus. Numerous susceptibility genes for type 2 diabetes, including potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1), have been identified in humans by genome-wide analyses and other studies. Experiments with genetically modified mice have also implicated various genes in the pathogenesis of diabetes. However, the possible effects of the parent of origin for diabetes susceptibility alleles on disease onset have remained unclear. Here, we show that a mutation at the Kcnq1 locus reduces pancreatic ß-cell mass in mice by epigenetic modulation only when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus and regulates the expression of neighboring genes on the paternal allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 expression as well as the increased expression of cyclin-dependent kinase inhibitor 1C (Cdkn1c), an imprinted gene that encodes a cell cycle inhibitor, only when the mutation is on the paternal allele. Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic ß-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.

Regulation of insulin signaling in skeletal muscle by PIP3 phosphatase, SKIP, and endoplasmic reticulum molecular chaperone glucose-regulated protein 78.

Insulin resistance is characterized as a pathogenic factor in type 2 diabetes. Despite skeletal muscle being primarily responsible for systemic glucose disposal, the mechanisms underlying the induction of insulin resistance in skeletal muscle have not been fully elucidated. A number of studies have shown that it is characterized by the inhibition of the phosphatidylinositol (PI) 3-kinase signaling pathway. Here, we show that skeletal muscle- and kidney-enriched inositol polyphosphate phosphatase (SKIP), a phosphatidylinositol-3,4,5-trisphosphate (PIP3) phosphatase, and glucose-regulated protein 78 (GRP78) are implicated in the inhibition of insulin-dependent PI 3-kinase signaling in skeletal muscle. Mechanistically, under resting conditions, SKIP forms a complex with GRP78 at the endoplasmic reticulum (ER). Insulin stimulation facilitates the dissociation of SKIP from GRP78 and its binding to the activated form of Pak1. GRP78 is necessary for membrane localization and Pak1-binding of SKIP, which facilitates inactivation of the insulin signaling pathway. These findings underscore the specific and prominent role of SKIP and GRP78 in the regulation of insulin-dependent PI 3-kinase signaling in skeletal muscle.

Novel role for retinol-binding protein 4 in the regulation of blood pressure.

Elevated levels of serum retinol-binding protein 4 (RBP4) contribute to insulin resistance and correlate with increased prevalence of hypertension and myocardial infarction. We sought to determine whether lowering RBP4 would improve blood pressure (BP) and protect against obesity- or angiotensin (Ang)-II-induced hypertension. Systolic and diastolic BP were lower in the RBP4-knockout (RBP4-KO) mice and higher in the RBP4-overexpressing (RBP4-Tg) mice compared with BP in the wild-type (WT) littermates. Carbachol-induced vasodilatation was increased in arteries from the RBP4-KO compared with the WT mice and was impaired in the RBP4-Tg mice. Aortic eNOS(Ser1177) phosphorylation was enhanced ∼50% in the RBP4-KO mice, with no change in total eNOS protein. Feeding a high-fat diet increased BP in the RBP4-KO mice only to the level in the WT mice fed chow and had no effect on aortic eNOS(Ser1177) phosphorylation. Ang-II infusion resulted in 22 mmHg lower systolic BP in the RBP4-KO than in the WT mice, although the relative BP increase over saline infusion was ∼30% in both. Ang-II treatment decreased aortic eNOS(Ser1177) phosphorylation in the WT and RBP4-KO mice, but phosphorylation remained higher in the RBP4-KO mice. Cardiac hypertrophy with Ang-II treatment was diminished by 56% in the RBP4-KO mice. Thus, elevated serum RBP4 raises BP and lack of RBP4 reduces it, with commensurate changes in aortic eNOS(Ser1177) phosphorylation. Lowering RBP4 may reduce BP through enhanced eNOS-mediated vasodilatation and may be a novel therapeutic approach for hypertension.

Role for RalA downstream of Rac1 in skeletal muscle insulin signalling.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. The small GTPase Rac1 has been implicated in this insulin signalling, but the mechanism whereby Rac1 stimulates GLUT4 translocation remains obscure. In the present study, we examined the role of the small GTPase RalA downstream of Rac1 in skeletal muscle fibres isolated from genetically modified mice. A dominant-negative mutant of RalA, when ectopically overexpressed, significantly reduced GLUT4 translocation in response to insulin or either one of constitutively activated mutants of Rac1 and its upstream regulators, including the guanine-nucleotide-exchange factor FLJ00068, the protein kinase Akt2 and phosphoinositide 3-kinase. Constitutively activated Rac1 also failed to induce GLUT4 translocation in mouse skeletal muscle fibres in which the expression of RalA was abrogated by specific siRNA molecules. Furthermore, we applied a novel approach to detect the activated form of RalA in situ by immunofluorescence microscopy of mouse skeletal muscle fibres, demonstrating that constitutively activated mutants of Rac1 and its upstream regulators as well as insulin indeed cause the activation of RalA. Notably, this RalA activation was remarkably impaired in rac1-deficient skeletal muscle fibres. Taken together, these results provide evidence that RalA is indeed activated and involved in the regulation of GLUT4 translocation in response to insulin downstream of Rac1 in mouse skeletal muscle.

Downregulation of Krüppel-like factor 15 expression delays endochondral bone ossification during fracture healing.

OBJECTIVE: The role of Krüppel-like zinc finger transcription factor 15 (KLF15) in endochondral ossification during fracture healing remains unexplored. In this study, we aimed to elucidate the impact of KLF15 in a mouse model of tibial transverse fracture. METHODS: We created tamoxifen-inducible, cartilage-specific KLF15 knockout mice (KLF15 KO). KLF15 fl/fl Col2-CreERT mice from the same litters as the KLF15 KO mice, but not treated with 4-hydroxytamoxifen, were used as controls (CT). At 10 weeks, male KLF15 KO and CT mice underwent tibial fracture followed by intramedullary nailing. Both groups were administered tamoxifen at days 0, 3, and 7 after surgery. The tibiae were harvested on post-surgery days 7, 10, and 14 for radiological assessment using micro-computed tomography. Histological staining (Safranin-O) and immunohistochemistry for KLF15, SOX9, Indian hedgehog (IHH), RUNX2, and Osterix were performed. Additionally, cartilage from mouse fetus was cultured for qRT-PCR and western blot analyses of KLF15, SOX9, IHH, Col2, RUNX2, Osterix, TGF-ß, SMAD3, and phosphor-SMAD3. RESULTS: The radiological assessment revealed that immature callus formation was delayed in KLF15 KO, compared with that in CT, peaking on day 14 compared with that on day 10 in CT. KLF15 KO mice exhibited delayed fracture healing and reduced Safranin-O staining at days 7 and 10 post-surgery. The ratio of cells positive for KLF15 and SOX9 was significantly lower in KLF15 KO than in CT, whereas the ratios for IHH, RUNX2, and Osterix showed no significant difference. RT-PCR revealed reduced expression of KLF15, SOX9, and COL2, with no significant changes in IHH, Osterix, RUNX2, TGF-ß, and SMAD3. Western blot analysis indicated decreased SMAD3 phosphorylation in KLF15 KO mice. CONCLUSION: KLF15 regulates SOX9 via the TGF-ß-SMAD3-SOX9 pathway, independent of IHH, in endochondral ossification. The KLF15 deficiency decreases SOX9 expression through reduced SMAD3 phosphorylation, subsequently delaying fracture healing.

Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

Insulin resistance in adipose tissue and metabolic diseases.

Adipose tissue regulates systemic energy metabolism through adipokine production as well as energy storage and energy supply to other organs in response to changes in energy status. Adipose tissue dysfunction is therefore thought to be a key contributor to the pathogenesis of a variety of metabolic disorders including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Given that insulin plays a central role in the regulation of many aspects of adipocyte function, insulin resistance in adipose tissue is implicated in the pathogenesis of metabolic disorders as a cause of adipose tissue dysfunction. The concept of metabolic dysfunction-associated fatty liver disease (MAFLD) has recently been proposed for liver disease associated with metabolic disorders in both obese and nonobese individuals, with insulin resistance in adipose tissue likely being an important factor in its pathogenesis. This review outlines the relation between insulin resistance in adipose tissue and metabolic disorders, with a focus on the physiological relevance and mechanism of action of 3'-phosphoinositide-dependent kinase 1 (PDK1), a key kinase in insulin signaling, and its downstream transcription factor FoxO1 in adipocytes.

Adipose tissue insulin resistance exacerbates liver inflammation and fibrosis in a diet-induced NASH model.

BACKGROUND: Insulin regulates various biological processes in adipocytes, and adipose tissue dysfunction due to insulin resistance in this tissue plays a central role in the development of metabolic diseases, including NAFLD and NASH. However, the combined impact of adipose tissue insulin resistance and dietary factors on the pathogenesis of NAFLD-NASH has remained unknown. METHODS AND RESULTS: 3'-phosphoinositide-dependent kinase 1 (PDK1) is a serine-threonine protein kinase that mediates the metabolic actions of insulin. We recently showed that adipocyte-specific PDK1 knockout (A-PDK1KO) mice maintained on normal chow exhibit metabolic disorders, including progressive liver disease leading to NASH, in addition to reduced adipose tissue mass. We here show that maintenance of A-PDK1KO mice on the Gubra amylin NASH (GAN) diet rich in saturated fat, cholesterol, and fructose exacerbates inflammation and fibrosis in the liver. Consistent with these histological findings, RNA-sequencing analysis of the liver showed that the expression of genes related to inflammation and fibrosis was additively upregulated by adipocyte-specific PDK1 ablation and the GAN diet. Of note, the reduced adipose tissue mass of A-PDK1KO mice was not affected by the GAN diet. Our results thus indicate that adipose tissue insulin resistance and the GAN diet additively promote inflammation and fibrosis in the liver of mice. CONCLUSIONS: A-PDK1KO mice fed with the GAN diet, constitute a new mouse model for studies of the pathogenesis of NAFLD-NASH, especially that in lean individuals, as well as for the development of potential therapeutic strategies for this disease.

Nicotinamide mononucleotide induces lipolysis by regulating ATGL expression via the SIRT1-AMPK axis in adipocytes.

Nicotinamide adenine dinucleotide (NAD+) -dependent protein deacetylase SIRT1 plays an important role in the regulation of metabolism. Although the administration of nicotinamide mononucleotide (NMN), a key NAD+ intermediate, has been shown to ameliorate metabolic disorders, such as insulin resistance and glucose intolerance, the direct effect of NMN on the regulation of lipid metabolism in adipocytes remains unclear. We here investigated the effect of NMN on lipid storage in 3T3-L1 differentiated adipocytes. Oil-red O staining showed that NMN treatment reduced lipid accumulation in these cells. NMN was found to enhance lipolysis in adipocytes since the concentration of glycerol in the media was increased by NMN treatment. Western blotting and real-time RT-PCR analysis revealed that adipose triglyceride lipase (ATGL) expression at both protein and mRNA level was increased with NMN treatment in 3T3-L1 adipocytes. Whereas NMN increased SIRT1 expression and AMPK activation, an AMPK inhibitor compound C restored the NMN-dependent upregulation of ATGL expression in these cells, suggesting that NMN upregulates ATGL expression through the SIRT1-AMPK axis. NMN administration significantly decreased subcutaneous fat mass in mice on a high-fat diet. We also found that adipocyte size in subcutaneous fat was decreased with NMN treatment. Consistent with the alteration of fat mass and adipocyte size, the ATGL expression in subcutaneous fat was slightly, albeit significantly, increased with NMN treatment. These results indicate that NMN suppresses subcutaneous fat mass in diet-induced obese mice, potentially in part via the upregulation of ATGL. Unexpectedly, the reduction in fat mass as well as ATGL upregulation with NMN treatment were not observed in epididymal fat, implying that the effects of NMN are site-specific in adipose tissue. Thus, these findings provide important insights into the mechanism of NMN/NAD+ in the regulation of metabolism.

Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a potential therapeutic target for various medical conditions. We here identify a small-molecule compound (RX-375) that activates AMPK and inhibits fatty acid synthesis in cultured human hepatocytes. RX-375 does not bind to AMPK but interacts with prohibitins (PHB1 and PHB2), which were found to form a complex with AMPK. RX-375 induced dissociation of this complex, and PHBs knockdown resulted in AMPK activation, in the cultured cells. Administration of RX-375 to obese mice activated AMPK and ameliorated steatosis in the liver. High-throughput screening based on disruption of the AMPK-PHB interaction identified a second small-molecule compound that activates AMPK, confirming the importance of this interaction in the regulation of AMPK. Our results thus indicate that PHBs are previously unrecognized negative regulators of AMPK, and that compounds that prevent the AMPK-PHB interaction constitute a class of AMPK activator.

A gut microbial metabolite of linoleic acid ameliorates liver fibrosis by inhibiting TGF-ß signaling in hepatic stellate cells.

The antidiabetic drug pioglitazone ameliorates insulin resistance by activating the transcription factor PPARγ. In addition to its blood glucose-lowering action, pioglitazone exerts pleiotropic effects including amelioration of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The mechanism by which pioglitazone achieves this latter effect has remained unclear, however. We here show that pioglitazone administration increases the amount of linoleic acid (LA) metabolites in adipose tissue of KK-Ay mice. These metabolites are produced by lactic acid bacteria in the gut, and pioglitazone also increased the fraction of Lactobacillus in the gut microbiota. Administration of the LA metabolite HYA (10-hydroxy-cis-12-octadecenoic acid) to C57BL/6 J mice fed a high-fat diet improved liver histology including steatosis, inflammatory cell infiltration, and fibrosis. Gene ontology analysis of RNA-sequencing data for the liver revealed that the top category for genes downregulated by HYA treatment was related to extracellular matrix, and the expression of individual genes related to fibrosis was confirmed to be attenuated by HYA treatment. Mechanistically, HYA suppressed TGF-ß-induced Smad3 phosphorylation and fibrosis-related gene expression in human hepatic stellate cells (LX-2). Our results implicate LA metabolites in the mechanism by which pioglitazone ameliorates liver fibrosis, and they suggest that HYA is a potential therapeutic for NAFLD/NASH.

A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy.

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Krüppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca2+ bioimaging revealed that the cytosolic Ca2+ concentration ([Ca2+]i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for Ca2+ signaling in that a decrease in [Ca2+]i from the basal level triggers a defined biological event.

Role of PDK1 in skeletal muscle hypertrophy induced by mechanical load.

Phosphatidylinositol 3-kinase (PI3K) plays an important role in protein metabolism and cell growth. We here show that mice (M-PDK1KO mice) with skeletal muscle-specific deficiency of 3'-phosphoinositide-dependent kinase 1 (PDK1), a key component of PI3K signaling pathway, manifest a reduced skeletal muscle mass under the static condition as well as impairment of mechanical load-induced muscle hypertrophy. Whereas mechanical load-induced changes in gene expression were not affected, the phosphorylation of ribosomal protein S6 kinase (S6K) and S6 induced by mechanical load was attenuated in skeletal muscle of M-PDK1KO mice, suggesting that PDK1 regulates muscle hypertrophy not through changes in gene expression but through stimulation of kinase cascades such as the S6K-S6 axis, which plays a key role in protein synthesis. Administration of the ß2-adrenergic receptor (AR) agonist clenbuterol activated the S6K-S6 axis in skeletal muscle and induced muscle hypertrophy in mice. These effects of clenbuterol were attenuated in M-PDK1KO mice, and mechanical load-induced activation of the S6K-S6 axis and muscle hypertrophy were inhibited in mice with skeletal muscle-specific deficiency of ß2-AR. Our results suggest that PDK1 regulates skeletal muscle mass under the static condition and that it contributes to mechanical load-induced muscle hypertrophy, at least in part by mediating signaling from ß2-AR.