A Metabolic Basis for Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor ß (TGF-ß) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.

Shear stress and oxygen availability drive differential changes in opossum kidney proximal tubule cell metabolism and endocytosis.

Kidney proximal tubule (PT) cells have high-metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT functions are impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96 hours showed significant time- and culture-dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis vs development of ion transport and endocytic capacities in our culture system that highlight the complexity of O2 -dependent and mechanosensitive crosstalk to regulate PT cell function.

Adropin treatment restores cardiac glucose oxidation in pre-diabetic obese mice.

Exposure to a high fat (HF) diet promotes increased fatty acid uptake, fatty acid oxidation and lipid accumulation in the heart. These maladaptive changes impact cellular energy metabolism and may promote the development of cardiac dysfunction. Attempts to increase cardiac glucose utilization have been proposed as a way to reverse cardiomyopathy in obese and diabetic individuals. Adropin is a nutrient-regulated metabolic hormone shown to promote glucose oxidation over fatty acid oxidation in skeletal muscle homogenates in vitro. The focus of the current study was to investigate whether adropin can regulate substrate metabolism in the heart following prolonged exposure to a HF diet in vivo. Mice on a long-term HF diet received serial intraperitoneal injections of vehicle or adropin over three days. Cardiac glucose oxidation was significantly reduced in HF animals, which was rescued by acute adropin treatment. Significant decreases in cardiac pyruvate dehydrogenase activity were observed in HF animals, which were also reversed by adropin treatment. In contrast to previous studies, this change was unrelated to Pdk4 expression, which remained elevated in both vehicle- and adropin-treated HF mice. Instead, we show that adropin modulated the expression of the mitochondrial acetyltransferase enzyme GCN5L1, which altered the acetylation status and activity of fuel metabolism enzymes to favor glucose utilization. Our findings indicate that adropin exposure leads to increased cardiac glucose oxidation under HF conditions, and may provide a future therapeutic avenue in the treatment of diabetic cardiomyopathy.

Coordinated Activities of Multiple Myc-dependent and Myc-independent Biosynthetic Pathways in Hepatoblastoma.

Hepatoblastoma (HB) is associated with aberrant activation of the ß-catenin and Hippo/YAP signaling pathways. Overexpression of mutant ß-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc+/+ (WT) and myc-/- (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid ß-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid ß-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetyl-CoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

Reduced intestinal lipid absorption and body weight-independent improvements in insulin sensitivity in high-fat diet-fed Park2 knockout mice.

Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. PARK2 is a ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of PARK2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed a high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after 1-wk HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of PARK2 during metabolic stress.

c-Myc programs fatty acid metabolism and dictates acetyl-CoA abundance and fate.

myc(-/-) rat fibroblasts (KO cells) differ from myc(+/+) (WT) cells and KO cells with enforced Myc re-expression (KO-Myc cells) with respect to mitochondrial structure and function, utilization of glucose and glutamine as energy-generating substrates, and ATP levels. Specifically, KO cells demonstrate low levels of glycolysis and oxidative phosphorylation, dysfunctional mitochondria and electron transport chain complexes, and depleted ATP stores. We examined here how these cells adapt to their energy-deficient state and how they differ in their uptake and utilization of long- and medium-chain fatty acids such as palmitate and octanoate, respectively. Metabolic tracing of these molecules showed that KO cells preferentially utilize them as ß-oxidation substrates and that, rather than directing them into phospholipids, preferentially store them as neutral lipids. KO cell transcriptional profiling and functional assays revealed a generalized up-regulation of pathways involved in fatty acid transport and catabolism as well as evidence that these cells attempt to direct acetyl-CoA into the tricarboxylic acid (TCA) cycle for ATP production rather than utilizing it for anabolic purposes. Additional evidence to support this idea included the finding that AMP-dependent protein kinase was constitutively activated in KO cells. The complex control of pyruvate dehydrogenase, which links glycolysis to the TCA cycle, was also maximized to ensure the conversion of pyruvate to acetyl-CoA. Despite these efforts to maximize acetyl-CoA for energy-generating purposes, its levels remained chronically low in KO cells. This suggests that tumor cells with Myc deregulation might be susceptible to novel therapies that limit acetyl-CoA availability.

Reduced hepatocyte mitophagy is an early feature of NAFLD pathogenesis and hastens the onset of steatosis, inflammation and fibrosis.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN hastens the onset but not severity of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.

GCN5L1 impairs diastolic function in mice exposed to a high fat diet by restricting cardiac pyruvate oxidation.

Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.

Liver-specific Prkn knockout mice are more susceptible to diet-induced hepatic steatosis and insulin resistance.

OBJECTIVE: PARKIN is an E3 ubiquitin ligase that regulates mitochondrial quality control through a process called mitophagy. Recent human and rodent studies suggest that loss of hepatic mitophagy may occur during the pathogenesis of obesity-associated fatty liver and contribute to changes in mitochondrial metabolism associated with this disease. Whole-body Prkn knockout mice are paradoxically protected against diet-induced hepatic steatosis; however, liver-specific effects of Prkn deficiency cannot be discerned in this model due to pleotropic effects of germline Prkn deletion on energy balance and subsequent protection against diet-induced obesity. We therefore generated the first liver-specific Prkn knockout mouse strain (LKO) to directly address the role of hepatic Prkn. METHODS: Littermate control (WT) and LKO mice were fed regular chow (RC) or high-fat diet (HFD) and changes in body weight and composition were measured over time. Liver mitochondrial content was assessed using multiple, complementary techniques, and mitochondrial respiratory capacity was assessed using Oroboros O2K platform. Liver fat was measured biochemically and assessed histologically, while global changes in hepatic gene expression were measured by RNA-seq. Whole-body and tissue-specific insulin resistance were assessed by hyperinsulinemic-euglycemic clamp with isotopic tracers. RESULTS: Liver-specific deletion of Prkn had no effect on body weight or adiposity during RC or HFD feeding; however, hepatic steatosis was increased by 45% in HFD-fed LKO compared with WT mice (P < 0.05). While there were no differences in mitochondrial content between genotypes on either diet, mitochondrial respiratory capacity and efficiency in the liver were significantly reduced in LKO mice. Gene enrichment analyses from liver RNA-seq results suggested significant changes in pathways related to lipid metabolism and fibrosis in HFD-fed Prkn knockout mice. Finally, whole-body insulin sensitivity was reduced by 35% in HFD-fed LKO mice (P < 0.05), which was primarily due to increased hepatic insulin resistance (60% of whole-body effect; P = 0.11). CONCLUSIONS: These data demonstrate that PARKIN contributes to mitochondrial homeostasis in the liver and plays a protective role against the pathogenesis of hepatic steatosis and insulin resistance.

Hepatic insulin sensitivity is improved in high-fat diet-fed Park2 knockout mice in association with increased hepatic AMPK activation and reduced steatosis.

Park2 is an E3 ubiquitin ligase known for its role in mitochondrial quality control via the mitophagy pathway. Park2 KO mice are protected from diet-induced obesity and hepatic insulin sensitivity is improved in high-fat diet (HFD)-fed Park2 KO mice even under body weight-matched conditions. In order to better understand the cellular mechanism by which Park2 KO mice are protected from diet-induced hepatic insulin resistance, we determined changes in multiple pathways commonly associated with the pathogenesis of insulin resistance, namely levels of bioactive lipid species, activation of the endoplasmic reticulum (ER) stress response and changes in cytokine levels and signaling. We report for the first time that whole-body insulin sensitivity is unchanged in regular chow (RC)-fed Park2 KO mice, and that liver diacylglycerol levels are reduced and very-long-chain ceramides are increased in Park2 KO mice fed HFD for 1 week. Hepatic transcriptional markers of the ER stress response were reduced and plasma tumor necrosis factor-α (TNFα), interleukin-6 and -10 (IL6, IL10) were significantly increased in HFD-fed Park2 KO mice; however, there were no detectable differences in hepatic inflammatory signaling pathways between groups. Interestingly, hepatic adenylate charge was reduced in HFD-fed Park2 KO liver and was associated increased activation of AMPK. These data suggest that negative energy balance that contributed to protection from obesity during chronic HFD manifested at the level of the hepatocyte during short-term HFD feeding and contributed to the improved hepatic insulin sensitivity.

Adropin reduces blood glucose levels in mice by limiting hepatic glucose production.

Adropin is a liver- and brain-secreted peptide hormone with striking effects on fuel metabolism regulation in a number of tissues. Previous studies demonstrated that adropin secretion is decreased in obese mice subjected to a long-term high-fat diet (HFD), and that whole-body loss of adropin expression resulted in systemic insulin resistance. Treatment of obese mice with adropin improves glucose tolerance, which has been linked to increased glucose oxidation and inhibition of fatty acid utilization in isolated skeletal muscle homogenates. In this study, we used in vivo physiological measurements to determine how treatment of obese mice with adropin affects whole-body glucose metabolism. Treatment with adropin reduced fasting blood glucose and, as shown previously, increased glucose tolerance in HFD mice during standard glucose tolerance tests. Under hyperinsulinemic-euglycemic clamp conditions, adropin treatment led to a nonsignificant increase in whole-body insulin sensitivity, and a significant reduction in whole-body glucose uptake. Finally, we show that adropin treatment suppressed hepatic glucose production and improved hepatic insulin sensitivity. This correlated with reduced expression of fatty acid import proteins and gluconeogenic regulatory enzymes in the liver, suggesting that adropin treatment may impact the pathways that drive vital aspects of hepatic glucose metabolism.

Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFß1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFß1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.

Adipocyte JAK2 Regulates Hepatic Insulin Sensitivity Independently of Body Composition, Liver Lipid Content, and Hepatic Insulin Signaling.

Disruption of hepatocyte growth hormone (GH) signaling through disruption of Jak2 (JAK2L) leads to fatty liver. Previously, we demonstrated that development of fatty liver depends on adipocyte GH signaling. We sought to determine the individual roles of hepatocyte and adipocyte Jak2 on whole-body and tissue insulin sensitivity and liver metabolism. On chow, JAK2L mice had hepatic steatosis and severe whole-body and hepatic insulin resistance. However, concomitant deletion of Jak2 in hepatocytes and adipocytes (JAK2LA) completely normalized insulin sensitivity while reducing liver lipid content. On high-fat diet, JAK2L mice had hepatic steatosis and insulin resistance despite protection from diet-induced obesity. JAK2LA mice had higher liver lipid content and no protection from obesity but retained exquisite hepatic insulin sensitivity. AKT activity was selectively attenuated in JAK2L adipose tissue, whereas hepatic insulin signaling remained intact despite profound hepatic insulin resistance. Therefore, JAK2 in adipose tissue is epistatic to liver with regard to insulin sensitivity and responsiveness, despite fatty liver and obesity. However, hepatocyte autonomous JAK2 signaling regulates liver lipid deposition under conditions of excess dietary fat. This work demonstrates how various tissues integrate JAK2 signals to regulate insulin/glucose and lipid metabolism.

Abnormal lipid processing but normal long-term repopulation potential of myc-/- hepatocytes.

Establishing c-Myc's (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc-/- (KO) mice were smaller in young animals and larger in older animals relative to myc+/+ (WT) counterparts. KO mice also consumed more oxygen, produced more CO2 and generated more heat. Although WT and KO hepatocytes showed few mitochondrial structural differences, the latter demonstrated defective electron transport chain function. RNAseq revealed differences in transcripts encoding ribosomal subunits, cytochrome p450 members and enzymes for triglyceride and sterol biosynthesis. KO hepatocytes also accumulated neutral lipids. WT and KO hepatocytes repopulated recipient tyrosinemic livers equally well although the latter were associated with a pro-inflammatory hepatic environment that correlated with worsening lipid accumulation, its extracellular deposition and parenchymal oxidative damage. Our results show Myc to be dispensable for sustained in vivo hepatocyte proliferation but necessary for maintaining normal lipid homeostasis. myc-/- livers resemble those encountered in non-alcoholic fatty liver disease and, under sustained proliferative stress, gradually acquire the features of non-alcoholic steatohepatitis.

c-Myc and AMPK Control Cellular Energy Levels by Cooperatively Regulating Mitochondrial Structure and Function.

The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because Myc over-expression depletes ATP with the resultant activation of AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in ampk+/+ (WT) and ampk-/- (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs had a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox states. Transcriptional profiling of pathways pertinent to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are highly co-dependent and appear to engage in significant cross-talk across numerous pathways which support metabolic and ATP-generating functions.

Vascular pool of releasable soluble VEGF receptor-1 (sFLT1) in women with previous preeclampsia and uncomplicated pregnancy.

CONTEXT: Research examining the source of excess soluble fms-like tyrosine kinase 1 (sFLT1) in preeclampsia has focused on the placenta. The potential contribution of the releasable store of sFLT1 in the systemic vasculature is unknown. OBJECTIVE: We asked whether the nonplacental releasable store of sFLT1 is larger in women with previous preeclampsia than in women with a previous uncomplicated pregnancy. DESIGN: We administered heparin to nulligravid women and to women with previous preeclampsia or a previous uncomplicated pregnancy. We compared post-heparin sFLT1 concentrations with those observed in uncomplicated pregnancy and preeclampsia. SETTING: The study was performed at Magee-Womens Hospital. PATIENTS: Participants included nulligravidas (n = 8), women 6-24 months postpartum (previous uncomplicated pregnancy, n = 16; previous preeclampsia, n = 15), and pregnant women (uncomplicated pregnancy, n = 30; preeclampsia, n = 25). INTERVENTION: Nonpregnant women received an unfractionated heparin bolus. MAIN OUTCOME MEASURES: Pre- and post-heparin plasma sFLT1, placental growth factor, and vascular endothelial growth factor were measured. RESULTS: In nonpregnant women, heparin increased plasma sFLT1 by 250-fold (P < .01), increased placental growth factor by 7-fold (P < .01), and decreased free vascular endothelial growth factor (P < .01). These changes did not differ between nulligravidas, women with previous preeclampsia, and women with a previous uncomplicated pregnancy. Post-heparin sFLT1 in nonpregnant women was higher than sFLT1 in uncomplicated pregnancy, but lower than sFLT1 in preeclampsia. Baseline and post-heparin sFLT1 were positively correlated (r(2) = 0.19; P < .01). Heparin increased the concentration of the 100-kDa sFLT1 isoform. Adding heparin to whole blood or plasma did not increase sFLT1. CONCLUSIONS: Nonpregnant women have a significant vascular store of releasable sFLT1. The size of this store does not differ between women with previous preeclampsia vs women with previous uncomplicated pregnancy.