Iron drives anabolic metabolism through active histone demethylation and mTORC1.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

DNA Damage and Nuclear Morphological Changes in Cardiac Hypertrophy Are Mediated by SNRK Through Actin Depolymerization.

BACKGROUND: Proper nuclear organization is critical for cardiomyocyte function, because global structural remodeling of nuclear morphology and chromatin structure underpins the development and progression of cardiovascular disease. Previous reports have implicated a role for DNA damage in cardiac hypertrophy; however, the mechanism for this process is not well delineated. AMPK (AMP-activated protein kinase) family of proteins regulates metabolism and DNA damage response (DDR). Here, we examine whether a member of this family, SNRK (SNF1-related kinase), which plays a role in cardiac metabolism, is also involved in hypertrophic remodeling through changes in DDR and structural properties of the nucleus. METHODS: We subjected cardiac-specific Snrk-/- mice to transaortic banding to assess the effect on cardiac function and DDR. In parallel, we modulated SNRK in vitro and assessed its effects on DDR and nuclear parameters. We also used phosphoproteomics to identify novel proteins that are phosphorylated by SNRK. Last, coimmunoprecipitation was used to verify Destrin (DSTN) as the binding partner of SNRK that modulates its effects on the nucleus and DDR. RESULTS: Cardiac-specific Snrk-/- mice display worse cardiac function and cardiac hypertrophy in response to transaortic banding, and an increase in DDR marker pH2AX (phospho-histone 2AX) in their hearts. In addition, in vitro Snrk knockdown results in increased DNA damage and chromatin compaction, along with alterations in nuclear flatness and 3-dimensional volume. Phosphoproteomic studies identified a novel SNRK target, DSTN, a member of F-actin depolymerizing factor proteins that directly bind to and depolymerize F-actin. SNRK binds to DSTN, and DSTN downregulation reverses excess DNA damage and changes in nuclear parameters, in addition to cellular hypertrophy, with SNRK knockdown. We also demonstrate that SNRK knockdown promotes excessive actin depolymerization, measured by the increased ratio of G-actin to F-actin. Last, jasplakinolide, a pharmacological stabilizer of F-actin, rescues the increased DNA damage and aberrant nuclear morphology in SNRK-downregulated cells. CONCLUSIONS: These results indicate that SNRK is a key player in cardiac hypertrophy and DNA damage through its interaction with DSTN. This interaction fine-tunes actin polymerization to reduce DDR and maintain proper cardiomyocyte nuclear shape and morphology.

DNA damage and nuclear morphological changes in cardiac hypertrophy are mediated by SNRK through actin depolymerization.

BACKGROUND: Proper nuclear organization is critical for cardiomyocyte (CM) function, as global structural remodeling of nuclear morphology and chromatin structure underpins the development and progression of cardiovascular disease. Previous reports have implicated a role for DNA damage in cardiac hypertrophy, however, the mechanism for this process is not well delineated. AMPK family of proteins regulate metabolism and DNA damage response (DDR). Here, we examine whether a member of this family, SNF1-related kinase (SNRK), which plays a role in cardiac metabolism, is also involved in hypertrophic remodeling through changes in DDR and structural properties of the nucleus. METHODS: We subjected cardiac specific (cs)- Snrk -/- mice to trans-aortic banding (TAC) to assess the effect on cardiac function and DDR. In parallel, we modulated SNRK in vitro and assessed its effects on DDR and nuclear parameters. We also used phospho-proteomics to identify novel proteins that are phosphorylated by SNRK. Finally, co-immunoprecipitation (co-IP) was used to verify Destrin (DSTN) as the binding partner of SNRK that modulates its effects on the nucleus and DDR. RESULTS: cs- Snrk -/- mice display worse cardiac function and cardiac hypertrophy in response to TAC, and an increase in DDR marker pH2AX in their hearts. Additionally, in vitro Snrk knockdown results in increased DNA damage and chromatin compaction, along with alterations in nuclear flatness and 3D volume. Phospho-proteomic studies identified a novel SNRK target, DSTN, a member of F-actin depolymerizing factor (ADF) proteins that directly binds to and depolymerize F-actin. SNRK binds to DSTN, and DSTN downregulation reverses excess DNA damage and changes in nuclear parameters, in addition to cellular hypertrophy, with SNRK knockdown. We also demonstrate that SNRK knockdown promotes excessive actin depolymerization, measured by the increased ratio of globular (G-) actin to F-actin. Finally, Jasplakinolide, a pharmacological stabilizer of F-actin, rescues the increased DNA damage and aberrant nuclear morphology in SNRK downregulated cells. CONCLUSIONS: These results indicate that SNRK is a key player in cardiac hypertrophy and DNA damage through its interaction with DSTN. This interaction fine-tunes actin polymerization to reduce DDR and maintain proper CM nuclear shape and morphology. Clinical Perspective: What is new? Animal hearts subjected to pressure overload display increased SNF1-related kinase (SNRK) protein expression levels and cardiomyocyte specific SNRK deletion leads to aggravated myocardial hypertrophy and heart failure.We have found that downregulation of SNRK impairs DSTN-mediated actin polymerization, leading to maladaptive changes in nuclear morphology, higher DNA damage response (DDR) and increased hypertrophy. What are the clinical implications? Our results suggest that disruption of DDR through genetic loss of SNRK results in an exaggerated pressure overload-induced cardiomyocyte hypertrophy.Targeting DDR, actin polymerization or SNRK/DSTN interaction represent promising therapeutic targets in pressure overload cardiac hypertrophy.

Liver-specific overexpression of HKDC1 increases hepatocyte size and proliferative capacity.

A primary role of the liver is to regulate whole body glucose homeostasis. Glucokinase (GCK) is the main hexokinase (HK) expressed in hepatocytes and functions to phosphorylate the glucose that enters via GLUT transporters to become glucose-6-phosphate (G6P), which subsequently commits glucose to enter downstream anabolic and catabolic pathways. In the recent years, hexokinase domain-containing-1 (HKDC1), a novel 5th HK, has been characterized by our group and others. Its expression profile varies but has been identified to have low basal expression in normal liver but increases during states of stress including pregnancy, nonalcoholic fatty liver disease (NAFLD), and liver cancer. Here, we have developed a stable overexpression model of hepatic HKDC1 in mice to examine its effect on metabolic regulation. We found that HKDC1 overexpression, over time, causes impaired glucose homeostasis in male mice and shifts glucose metabolism towards anabolic pathways with an increase in nucleotide synthesis. Furthermore, we observed these mice to have larger liver sizes due to greater hepatocyte proliferative potential and cell size, which in part, is mediated via yes-associated protein (YAP) signaling.

Iron Metabolism in Cardiovascular Disease: Physiology, Mechanisms, and Therapeutic Targets.

The cardiovascular system requires iron to maintain its high energy demands and metabolic activity. Iron plays a critical role in oxygen transport and storage, mitochondrial function, and enzyme activity. However, excess iron is also cardiotoxic due to its ability to catalyze the formation of reactive oxygen species and promote oxidative damage. While mammalian cells have several redundant iron import mechanisms, they are equipped with a single iron-exporting protein, which makes the cardiovascular system particularly sensitive to iron overload. As a result, iron levels are tightly regulated at many levels to maintain homeostasis. Iron dysregulation ranges from iron deficiency to iron overload and is seen in many types of cardiovascular disease, including heart failure, myocardial infarction, anthracycline-induced cardiotoxicity, and Friedreich's ataxia. Recently, the use of intravenous iron therapy has been advocated in patients with heart failure and certain criteria for iron deficiency. Here, we provide an overview of systemic and cellular iron homeostasis in the context of cardiovascular physiology, iron deficiency, and iron overload in cardiovascular disease, current therapeutic strategies, and future perspectives.

[WITHDRAWN] Hexokinase-1 mitochondrial dissociation and protein O-GlcNAcylation drive heart failure with preserved ejection fraction.

The authors have requested that this preprint be removed from Research Square.

Optimized protocol to isolate primary mouse peritoneal macrophage metabolites.

Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches. For complete details on the use and execution of this protocol, please refer to De Jesus et al., (2022).

ZFP36L2 suppresses mTORc1 through a P53-dependent pathway to prevent peripartum cardiomyopathy in mice.

Pregnancy is associated with substantial physiological changes of the heart, and disruptions in these processes can lead to peripartum cardiomyopathy (PPCM). The molecular processes that cause physiological and pathological changes in the heart during pregnancy are not well characterized. Here, we show that mTORc1 was activated in pregnancy to facilitate cardiac enlargement that was reversed after delivery in mice. mTORc1 activation in pregnancy was negatively regulated by the mRNA-destabilizing protein ZFP36L2 through its degradation of Mdm2 mRNA and P53 stabilization, leading to increased SESN2 and REDD1 expression. This pathway impeded uncontrolled cardiomyocyte hypertrophy during pregnancy, and mice with cardiac-specific Zfp36l2 deletion developed rapid cardiac dysfunction after delivery, while prenatal treatment of these mice with rapamycin improved postpartum cardiac function. Collectively, these data provide what we believe to be a novel pathway for the regulation of mTORc1 through mRNA stabilization of a P53 ubiquitin ligase. This pathway was critical for normal cardiac growth during pregnancy, and its reduction led to PPCM-like adverse remodeling in mice.

Hexokinase 1 cellular localization regulates the metabolic fate of glucose.

The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH.

Hepatocyte-Specific Loss of PPARγ Protects Mice From NASH and Increases the Therapeutic Effects of Rosiglitazone in the Liver.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is commonly observed in patients with type 2 diabetes, and thiazolidinediones (TZD) are considered a potential therapy for NASH. Although TZD increase insulin sensitivity and partially reduce steatosis and alanine aminotransferase, the efficacy of TZD on resolving liver pathology is limited. In fact, TZD may activate peroxisome proliferator-activated receptor gamma (PPARγ) in hepatocytes and promote steatosis. Therefore, we assessed the role that hepatocyte-specific PPARγ plays in the development of NASH, and how it alters the therapeutic effects of TZD on the liver of mice with diet-induced NASH. METHODS: Hepatocyte-specific PPARγ expression was knocked out in adult mice before and after the development of NASH induced with a high fat, cholesterol, and fructose (HFCF) diet. RESULTS: HFCF diet increased PPARγ expression in hepatocytes, and rosiglitazone further activated PPARγ in hepatocytes of HFCF-fed mice in vivo and in vitro. Hepatocyte-specific loss of PPARγ reduced the progression of HFCF-induced NASH in male mice and increased the benefits derived from the effects of TZD on extrahepatic tissues and non-parenchymal cells. RNAseq and metabolomics indicated that HFCF diet promoted inflammation and fibrogenesis in a hepatocyte PPARγ-dependent manner and was associated with dysregulation of hepatic metabolism. Specifically, hepatocyte-specific loss of PPARγ plays a positive role in the regulation of methionine metabolism, and that could reduce the progression of NASH. CONCLUSIONS: Because of the negative effect of hepatocyte PPARγ in NASH, inhibition of mechanisms promoted by endogenous PPARγ in hepatocytes may represent a novel strategy that increases the efficiency of therapies for NAFLD.

Hepatic HKDC1 Expression Contributes to Liver Metabolism.

Glucokinase (GCK) is the principal hexokinase (HK) in the liver, operating as a glucose sensor to regulate glucose metabolism and lipid homeostasis. Recently, we proposed HK domain-containing 1 (HKDC1) to be a fifth HK with expression in the liver. Here, we reveal HKDC1 to have low glucose-phosphorylating ability and demonstrate its association with the mitochondria in hepatocytes. As we have shown previously that genetic deletion of HKDC1 leads to altered hepatic triglyceride levels, we also explored the influence of overexpression of HKDC1 in hepatocytes on cellular metabolism, observing reduced glycolytic capacity and maximal mitochondrial respiration with concurrent reductions in glucose oxidation and mitochondrial membrane potential. Furthermore, we found that acute in vivo overexpression of HKDC1 in the liver induced substantial changes in mitochondrial dynamics. Altogether, these findings suggest that overexpression of HKDC1 causes mitochondrial dysfunction in hepatocytes. However, its overexpression was not enough to alter energy storage in the liver but led to mild improvement in glucose tolerance. We next investigated the conditions necessary to induce HKDC1 expression, observing HKDC1 expression to be elevated in human patients whose livers were at more advanced stages of nonalcoholic fatty liver disease (NAFLD) and similarly, found high liver expression in mice on diets causing high levels of liver inflammation and fibrosis. Overall, our data suggest that HKDC1 expression in hepatocytes results in defective mitochondrial function and altered hepatocellular metabolism and speculate that its expression in the liver may play a role in the development of NAFLD.

Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21.

The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.