KLF15 and PPAR α

The metabolic myocardium is an omnivore and utilizes various carbon substrates to meet its energetic demand. While the adult heart preferentially consumes fatty acids (FAs) over carbohydrates, myocardial fuel plasticity is essential for organismal survival. This metabolic plasticity governing fuel utilization is under robust transcriptional control and studies over the past decade have illuminated members of the nuclear receptor family of factors (e.g., PPARα) as important regulators of myocardial lipid metabolism. However, given the complexity of myocardial metabolism in health and disease, it is likely that other molecular pathways are likely operative and elucidation of such pathways may provide the foundation for novel therapeutic approaches. We previously demonstrated that Kruppel-like factor 15 (KLF15) is an independent regulator of cardiac lipid metabolism thus raising the possibility that KLF15 and PPARα operate in a coordinated fashion to regulate myocardial gene expression requisite for lipid oxidation. In the current study, we show that KLF15 binds to, cooperates with, and is required for the induction of canonical PPARα-mediated gene expression and lipid oxidation in cardiomyocytes. As such, this study establishes a molecular module involving KLF15 and PPARα and provides fundamental insights into the molecular regulation of cardiac lipid metabolism

KLF15 Is a Molecular Link between Endoplasmic Reticulum Stress and Insulin Resistance

Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15-/- mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15-/- liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action

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

Kruppel-like Factor 15 Is a Critical Regulator of Cardiac Lipid Metabolism

Background: Metabolic homeostasis is central to normal cardiac function. The molecular mechanisms underlying metabolic plasticity in the heart remain poorly understood. Results: Kruppel-like factor 15 (KLF15) is a direct and independent regulator of myocardial lipid flux. Conclusion: KLF15 is a core component of the transcriptional circuitry that governs cardiac metabolism. Significance: This work is the first to implicate the KLF transcription factor family in cardiac metabolism. The mammalian heart, the body\u27s 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

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

Impact of anaerobic glycolysis and oxidative substrate selection on contractile function and mechanical efficiency during moderate severity ischemia

The role of anaerobic glycolysis and oxidative substrate selection on contractile function and mechanical efficiency during moderate severity myocardial ischemia is unclear. We hypothesize that 1) preventing anaerobic glycolysis worsens contractile function and mechanical efficiency and 2) increasing glycolysis and glucose oxidation while inhibiting free fatty acid oxidation improves contractile function during ischemia. Experiments were performed in anesthetized pigs, with regional ischemia induced by a 60% decrease in left anterior descending coronary artery blood flow for 40 min. Three groups were studied: 1) no treatment, 2) inhibition of glycolysis with iodoacetate (IAA), or 3) hyperinsulinemia and hyperglycemia (HI + HG). Glucose and free fatty acid oxidation were measured using radioisotopes and anaerobic glycolysis from net lactate efflux and myocardial lactate content. Regional contractile power was assessed from left ventricular pressure and segment length in the anterior wall. We found that preventing anaerobic glycolysis with IAA during ischemia in the absence of alterations in free fatty acid and glucose oxidation did not adversely affect contractile function or mechanical efficiency during myocardial ischemia, suggesting that anaerobic glycolysis is not essential for maintaining residual contractile function. Increasing glycolysis and glucose oxidation with HI + HG inhibited free fatty acid oxidation and improved contractile function and mechanical efficiency. In conclusion, these results show a dissociation between myocardial function and anaerobic glycolysis during moderate severity ischemia in vivo, suggesting that metabolic therapies should not be aimed at inhibiting anaerobic glycolysis per se, but rather activating insulin signaling and/or enhancing carbohydrate oxidation and/or decreasing fatty acid oxidation

Autocrine ATP release coupled to extracellular pyrophosphate accumulation in vascular smooth muscle cells

Extracellular inorganic pyrophosphate (PPi) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PPi via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PPi via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog β,γ-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PPi to accumulate ≥600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PPi accumulation while increasing ATP accumulation. Probenecid, which inhibits PPi efflux dependent on ANK, a putative PPi transporter or transport regulator, reduced extracellular PPi accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises ≥50% of the extracellular PPi-generating capacity of VSMC. The accumulation of extracellular PPi and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PPi accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PPi generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix

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

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