Transcription factors KLF15 and PPARδ cooperatively orchestrate genome-wide regulation of lipid metabolism in skeletal muscle.

Skeletal muscle dynamically regulates systemic nutrient homeostasis through transcriptional adaptations to physiological cues. In response to changes in the metabolic environment (e.g., alterations in circulating glucose or lipid levels), networks of transcription factors and coregulators are recruited to specific genomic loci to fine-tune homeostatic gene regulation. Elucidating these mechanisms is of particular interest as these gene regulatory pathways can serve as potential targets to treat metabolic disease. The zinc-finger transcription factor Krüppel-like factor 15 (KLF15) is a critical regulator of metabolic homeostasis; however, its genome-wide distribution in skeletal muscle has not been previously identified. Here, we characterize the KLF15 cistrome in vivo in skeletal muscle and find that the majority of KLF15 binding is localized to distal intergenic regions and associated with genes related to circadian rhythmicity and lipid metabolism. We also identify critical interdependence between KLF15 and the nuclear receptor PPARδ in the regulation of lipid metabolic gene programs. We further demonstrate that KLF15 and PPARδ colocalize genome-wide, physically interact, and are dependent on one another to exert their transcriptional effects on target genes. These findings reveal that skeletal muscle KLF15 plays a critical role in metabolic adaptation through its direct actions on target genes and interactions with other nodal transcription factors such as PPARδ.

CCN3 Regulates Macrophage Foam Cell Formation and Atherosclerosis.

Recent studies implicate the Cyr61, CTGF, Nov (CCN) matricellular signaling protein family as emerging players in vascular biology, with NOV (alias CCN3) as an important regulator of vascular homeostasis. Herein, we examined the role of CCN3 in the pathogenesis of atherosclerosis. In response to a 15-week high-fat diet feeding, CCN3-deficient mice on the atherosclerosis-prone Apoe-/- background developed increased aortic lipid-rich plaques compared to control Apoe-/- mice, a result that was observed in the absence of alterations in plasma lipid content. To address the cellular contributor(s) responsible for the atherosclerotic phenotype, we performed bone marrow transplantation experiments. Transplantation of Apoe; Ccn3 double-knockout bone marrow into Apoe-/- mice resulted in an increase of atherosclerotic plaque burden, whereas transplantation of Apoe-/- marrow to Apoe; Ccn3 double-knockout mice caused a reduction of atherosclerosis. These results indicate that CCN3 deficiency, specifically in the bone marrow, plays a major role in the development of atherosclerosis. Mechanistically, cell-based studies in isolated peritoneal macrophages demonstrated that CCN3 deficiency leads to an increase of lipid uptake and foam cell formation, an effect potentially attributed to the increased expression of scavenger receptors CD36 and SRA1, key factors involved in lipoprotein uptake. These results suggest that bone marrow-derived CCN3 is an essential regulator of atherosclerosis and point to a novel role of CCN3 in modulating lipid accumulation within macrophages.

Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program.

Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

BACKGROUND: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. METHODS AND RESULTS: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. CONCLUSIONS: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Kruppel-like factor 15 is a critical regulator of cardiac lipid metabolism.

The mammalian heart, the body's largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.

Kruppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation.

The ability of skeletal muscle to enhance lipid utilization during exercise is a form of metabolic plasticity essential for survival. Conversely, metabolic inflexibility in muscle can cause organ dysfunction and disease. Although the transcription factor Kruppel-like factor 15 (KLF15) is an important regulator of glucose and amino acid metabolism, its endogenous role in lipid homeostasis and muscle physiology is unknown. Here we demonstrate that KLF15 is essential for skeletal muscle lipid utilization and physiologic performance. KLF15 directly regulates a broad transcriptional program spanning all major segments of the lipid-flux pathway in muscle. Consequently, Klf15-deficient mice have abnormal lipid and energy flux, excessive reliance on carbohydrate fuels, exaggerated muscle fatigue, and impaired endurance exercise capacity. Elucidation of this heretofore unrecognized role for KLF15 now implicates this factor as a central component of the transcriptional circuitry that coordinates physiologic flux of all three basic cellular nutrients: glucose, amino acids, and lipids.

Muscle Krüppel-like factor 15 regulates lipid flux and systemic metabolic homeostasis.

Skeletal muscle is a major determinant of systemic metabolic homeostasis that plays a critical role in glucose metabolism and insulin sensitivity. By contrast, despite being a major user of fatty acids, and evidence that muscular disorders can lead to abnormal lipid deposition (e.g., nonalcoholic fatty liver disease in myopathies), our understanding of skeletal muscle regulation of systemic lipid homeostasis is not well understood. Here we show that skeletal muscle Krüppel-like factor 15 (KLF15) coordinates pathways central to systemic lipid homeostasis under basal conditions and in response to nutrient overload. Mice with skeletal muscle-specific KLF15 deletion demonstrated (a) reduced expression of key targets involved in lipid uptake, mitochondrial transport, and utilization, (b) elevated circulating lipids, (c) insulin resistance/glucose intolerance, and (d) increased lipid deposition in white adipose tissue and liver. Strikingly, a diet rich in short-chain fatty acids bypassed these defects in lipid flux and ameliorated aspects of metabolic dysregulation. Together, these findings establish skeletal muscle control of lipid flux as critical to systemic lipid homeostasis and metabolic health.

Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia.

Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.

Leukocyte integrin signaling regulates FOXP1 gene expression via FOXP1-IT1 long non-coding RNA-mediated IRAK1 pathway.

Leukocyte integrin-dependent downregulation of the transcription factor FOXP1 is required for monocyte differentiation and macrophage functions, but the precise gene regulatory mechanism is unknown. Here, we identify multi-promoter structure (P1, P2, and P3) of the human FOXP1 gene. Clustering of the ß2-leukocyte integrin Mac-1 downregulated transcription from these promoters. We extend our prior observation that IL-1 receptor-associated kinase 1 (IRAK1) is physically associated with Mac-1 and provide evidence that IRAK1 is a potent suppressor of human FOXP1 promoter. IRAK1 reduced phosphorylation of histone deacetylase 4 (HDAC4) via inhibiting phosphorylation of calcium/calmodulin dependent protein kinase II delta (CaMKIIδ), thereby promoting recruitment of HDAC4 to P1 chromatin. A novel human FOXP1 intronic transcript 1 (FOXP1-IT1) long non-coding RNA (lncRNA), whose gene is embedded within that of FOXP1, has been cloned and found to bind directly to HDAC4 and regulate FOXP1 in cis manner. Overexpression of FOXP1-IT1 counteracted Mac-1 clustering-dependent downregulation of FOXP1, reduced IRAK1 downregulation of HDAC4 phosphorylation, and attenuated differentiation of THP-1 monocytic cells. In contrast, Mac-1 clustering inhibited FOXP1-IT1 expression with reduced binding to HDAC4 as well as phosphorylation of CaMKIIδ to activate the IRAK1 signaling pathway. Importantly, both IRAK1 and HDAC4 inhibitors significantly reduced integrin clustering-triggered downregulation of FOXP1 expression in purified human blood monocytes. Identification of this Mac-1/IRAK-1/FOXP1-IT1/HDAC4 signaling network featuring crosstalk between lncRNA and epigenetic factor for the regulation of FOXP1 expression provides new targets for anti-inflammatory therapeutics.

ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects.

Mitochondrial fragmentation and bioenergetic failure manifest in Huntington's disease (HD), a fatal neurodegenerative disease. The factors that couple mitochondrial fusion/fission with bioenergetics and their impacts on neurodegeneration however remain poorly understood. Our proteomic analysis identifies mitochondrial protein ATAD3A as an interactor of mitochondrial fission GTPase, Drp1, in HD. Here we show that, in HD, ATAD3A dimerization due to deacetylation at K135 residue is required for Drp1-mediated mitochondrial fragmentation. Disturbance of ATAD3A steady state impairs mtDNA maintenance by disrupting TFAM/mtDNA binding. Blocking Drp1/ATAD3A interaction with a peptide, DA1, abolishes ATAD3A oligomerization, suppresses mitochondrial fragmentation and mtDNA lesion, and reduces bioenergetic deficits and cell death in HD mouse- and patient-derived cells. DA1 treatment reduces behavioral and neuropathological phenotypes in HD transgenic mice. Our findings demonstrate that ATAD3A plays a key role in neurodegeneration by linking Drp1-induced mitochondrial fragmentation to defective mtDNA maintenance, suggesting that DA1 might be useful for developing HD therapeutics.

Kruppel-like factor 15 is required for the cardiac adaptive response to fasting.

Cardiac metabolism is highly adaptive in response to changes in substrate availability, as occur during fasting. This metabolic flexibility is essential to the maintenance of contractile function and is under the control of a group of select transcriptional regulators, notably the nuclear receptor family of factors member PPARα. However, the diversity of physiologic and pathologic states through which the heart must sustain function suggests the possible existence of additional transcriptional regulators that play a role in matching cardiac metabolism to energetic demand. Here we show that cardiac KLF15 is required for the normal cardiac response to fasting. Specifically, we find that cardiac function is impaired upon fasting in systemic and cardiac specific Klf15-null mice. Further, cardiac specific Klf15-null mice display a fasting-dependent accumulation of long chain acylcarnitine species along with a decrease in expression of the carnitine translocase Slc25a20. Treatment with a diet high in short chain fatty acids relieves the KLF15-dependent long chain acylcarnitine accumulation and impaired cardiac function in response to fasting. Our observations establish KLF15 as a critical mediator of the cardiac adaptive response to fasting through its regulation of myocardial lipid utilization.

Interventions Targeting Glucocorticoid-Krüppel-like Factor 15-Branched-Chain Amino Acid Signaling Improve Disease Phenotypes in Spinal Muscular Atrophy Mice.

The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn-/-;SMN2 and Smn2B/- mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling.

Adipose KLF15 Controls Lipid Handling to Adapt to Nutrient Availability.

Adipose tissue stores energy in the form of triglycerides. The ability to regulate triglyceride synthesis and breakdown based on nutrient status (e.g., fed versus fasted) is critical for physiological homeostasis and dysregulation of this process can contribute to metabolic disease. Whereas much is known about hormonal control of this cycle, transcriptional regulation is not well understood. Here, we show that the transcription factor Kruppel-like factor 15 (KLF15) is critical for the control of adipocyte lipid turnover. Mice lacking Klf15 in adipose tissue (AK15KO) display decreased adiposity and are protected from diet-induced obesity. Mechanistic studies suggest that adipose KLF15 regulates key genes of triglyceride synthesis and inhibits lipolytic action, thereby promoting lipid storage in an insulin-dependent manner. Finally, AK15KO mice demonstrate accelerated lipolysis and altered systemic energetics (e.g., locomotion, ketogenesis) during fasting conditions. Our study identifies adipose KLF15 as an essential regulator of adipocyte lipid metabolism and systemic energy balance.

A conserved KLF-autophagy pathway modulates nematode lifespan and mammalian age-associated vascular dysfunction.

Loss of protein and organelle quality control secondary to reduced autophagy is a hallmark of aging. However, the physiologic and molecular regulation of autophagy in long-lived organisms remains incompletely understood. Here we show that the Kruppel-like family of transcription factors are important regulators of autophagy and healthspan in C. elegans, and also modulate mammalian vascular age-associated phenotypes. Kruppel-like family of transcription factor deficiency attenuates autophagy and lifespan extension across mechanistically distinct longevity nematode models. Conversely, Kruppel-like family of transcription factor overexpression extends nematode lifespan in an autophagy-dependent manner. Furthermore, we show the mammalian vascular factor Kruppel-like family of transcription factor 4 has a conserved role in augmenting autophagy and improving vessel function in aged mice. Kruppel-like family of transcription factor 4 expression also decreases with age in human vascular endothelium. Thus, Kruppel-like family of transcription factors constitute a transcriptional regulatory point for the modulation of autophagy and longevity in C. elegans with conserved effects in the murine vasculature and potential implications for mammalian vascular aging.KLF family transcription factors (KLFs) regulate many cellular processes, including proliferation, survival and stress responses. Here, the authors position KLFs as important regulators of autophagy and lifespan in C. elegans, a role that may extend to the modulation of age-associated vascular phenotypes in mammals.

Matricellular protein CCN3 mitigates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease.

Kruppel-like factors in muscle health and disease.

Kruppel-like factors (KLF) are zinc-finger DNA-binding transcription factors that are critical regulators of tissue homeostasis. Emerging evidence suggests that KLFs are critical regulators of muscle biology in the context of cardiovascular health and disease. The focus of this review is to provide an overview of the current state of knowledge regarding the physiologic and pathologic roles of KLFs in the three lineages of muscle: cardiac, smooth, and skeletal.

Investigating mechanisms underpinning the detrimental impact of a high-fat diet in the developing and adult hypermuscular myostatin null mouse.

BACKGROUND: Obese adults are prone to develop metabolic and cardiovascular diseases. Furthermore, over-weight expectant mothers give birth to large babies who also have increased likelihood of developing metabolic and cardiovascular diseases. Fundamental advancements to better understand the pathophysiology of obesity are critical in the development of anti-obesity therapies not only for this but also future generations. Skeletal muscle plays a major role in fat metabolism and much work has focused in promoting this activity in order to control the development of obesity. Research has evaluated myostatin inhibition as a strategy to prevent the development of obesity and concluded in some cases that it offers a protective mechanism against a high-fat diet. METHODS: Pregnant as well as virgin myostatin null mice and age matched wild type animals were raised on a high fat diet for up to 10 weeks. The effect of the diet was tested on skeletal muscle, liver and fat. Quantitate PCR, Western blotting, immunohistochemistry, in-vivo and ex-vivo muscle characterisation, metabonomic and lipidomic measurements were from the four major cohorts. RESULTS: We hypothesised that myostatin inhibition should protect not only the mother but also its developing foetus from the detrimental effects of a high-fat diet. Unexpectedly, we found muscle development was attenuated in the foetus of myostatin null mice raised on a high-fat diet. We therefore re-examined the effect of the high-fat diet on adults and found myostatin null mice were more susceptible to diet-induced obesity through a mechanism involving impairment of inter-organ fat utilization. CONCLUSIONS: Loss of myostatin alters fatty acid uptake and oxidation in skeletal muscle and liver. We show that abnormally high metabolic activity of fat in myostatin null mice is decreased by a high-fat diet resulting in excessive adipose deposition and lipotoxicity. Collectively, our genetic loss-of-function studies offer an explanation of the lean phenotype displayed by a host of animals lacking myostatin signalling.

KLF15 Establishes the Landscape of Diurnal Expression in the Heart.

Circadian rhythms offer temporal control of anticipatory physiologic adaptations in animals. In the mammalian cardiovascular system, the importance of these rhythms is underscored by increased cardiovascular disease in shift workers, findings recapitulated in experimental animal models. However, a nodal regulator that allows integration of central and peripheral information and coordinates cardiac rhythmic output has been elusive. Here, we show that kruppel-like factor 15 (KLF15) governs a biphasic transcriptomic oscillation in the heart with a maximum ATP production phase and a remodeling and repair phase corresponding to the active and resting phase of a rodent. Depletion of KLF15 in cardiomyocytes leads to a disorganized oscillatory behavior without phasic partition despite an intact core clock. Thus, KLF15 is a nodal connection between the clock and meaningful rhythmicity in the heart.