Imbalance in glucose metabolism regulates the transition of microglia from homeostasis to Disease Associated Microglia stage 1.

Microglia undergo two-stage activation in neurodegenerative diseases, known as disease-associated microglia (DAM). TREM2 mediates the DAM2 stage transition, but what regulates the first DAM1 stage transition is unknown. We report that glucose dyshomeostasis inhibits DAM1 activation, and PKM2 plays a role. As in tumors, PKM2 was aberrantly elevated in both male and female human AD brains, but unlike in tumors, it is expressed as active tetramers, as well as among TREM2+ microglia surrounding plaques in 5XFAD male and female mice. snRNAseq analyses of microglia without Pkm2 in 5XFAD mice revealed significant increases in DAM1 markers in a distinct 'metabolic' cluster, which is enriched in genes for glucose metabolism, DAM1, and AD risk. 5XFAD mice incidentally exhibited a significant reduction in amyloid pathology without microglial Pkm2 Surprisingly, microglia in 5XFAD without Pkm2 exhibited increases in glycolysis and spare respiratory capacity, which correlated with restoration of mitochondrial cristae alterations. In addition, in situ spatial metabolomics of plaque-bearing microglia revealed an increase in respiratory activity. These results together suggest that it is not only glycolytic but also respiratory inputs that are critical to the development of DAM signatures in 5XFAD mice.Significance Statement Although reduced glucose uptake in the brain has been recognized as one of the earliest pathological events that accompany Alzheimer's disease (AD), it has not been clear whether this was due to a brain-wide defect in glucose metabolism or a dysfunction in a particular cell type. Our data suggest that dysregulation of metabolic homeostasis in microglia is critical for global AD pathology in a mouse model. Upon restoring glucose metabolism in microglia genetically, AD pathology is attenuated with a concomitant increase in DAM genes, especially DAM1 genes. These results suggest that this increase in DAM gene expression is protective against AD pathology, and glucose dyshomeostasis is a trigger to inhibit expression of protective DAM genes in AD.

Small-molecule inhibition of glycogen synthase 1 for the treatment of Pompe disease and other glycogen storage disorders.

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.

Voltage-gated potassium channels control extended access cocaine seeking: a role for nucleus accumbens astrocytes.

Dopaminergic signaling in the nucleus accumbens shell (NAc) regulates neuronal activity relevant to reward-related learning, including cocaine-associated behaviors. Although astrocytes respond to dopamine and cocaine with structural changes, the impact of dopamine and cocaine on astrocyte functional plasticity has not been widely studied. Specifically, behavioral implications of voltage-gated channel activity in the canonically non-excitable astrocytes are not known. We characterized potassium channel function in NAc astrocytes following exposure to exogenous dopamine or cocaine self-administration training under short (2 h/day) and extended (6 h/day) access schedules. Electrophysiological, Ca2+ imaging, mRNA, and mass spectrometry tools were used for molecular characterization. Behavioral effects were examined after NAc-targeted microinjections of channel antagonists and astroglial toxins. Exogenous dopamine increased activity of currents mediated by voltage-gated (Kv7) channels in NAc astrocytes. This was associated with a ~5-fold increase in expression of Kcnq2 transcript level in homogenized NAc micropunches. Matrix-assisted laser desorption/ionization mass spectrometry revealed increased NAc dopamine levels in extended access, relative to short access, rats. Kv7 inhibition selectively increased frequency and amplitude of astrocyte intracellular Ca2+ transients in NAc of extended access rats. Inhibition of Kv7 channels in the NAc attenuated cocaine-seeking in extended access rats only, an effect that was occluded by microinjection of the astrocyte metabolic poison, fluorocitrate. These results suggest that voltage-gated K+ channel signaling in NAc astrocytes is behaviorally relevant, support Kv7-mediated regulation of astrocyte Ca2+ signals, and propose novel mechanisms of neuroglial interactions relevant to drug use.

MetaVision3D: Automated Framework for the Generation of Spatial Metabolome Atlas in 3D.

High-resolution spatial imaging is transforming our understanding of foundational biology. Spatial metabolomics is an emerging field that enables the dissection of the complex metabolic landscape and heterogeneity from a thin tissue section. Currently, spatial metabolism highlights the remarkable complexity in two-dimensional space and is poised to be extended into the three-dimensional world of biology. Here, we introduce MetaVision3D, a novel pipeline driven by computer vision techniques for the transformation of serial 2D MALDI mass spectrometry imaging sections into a high-resolution 3D spatial metabolome. Our framework employs advanced algorithms for image registration, normalization, and interpolation to enable the integration of serial 2D tissue sections, thereby generating a comprehensive 3D model of unique diverse metabolites across host tissues at mesoscale. As a proof of principle, MetaVision3D was utilized to generate the mouse brain 3D metabolome atlas (available at https://metavision3d.rc.ufl.edu/ ) as an interactive online database and web server to further advance brain metabolism and related research.

RIT1 regulation of CNS lipids RIT1 deficiency Alters cerebral lipid metabolism and reduces white matter tract oligodendrocytes and conduction velocities.

Oligodendrocytes (OLs) generate lipid-rich myelin membranes that wrap axons to enable efficient transmission of electrical impulses. Using a RIT1 knockout mouse model and in situ high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) coupled with MS-based lipidomic analysis to determine the contribution of RIT1 to lipid homeostasis. Here, we report that RIT1 loss is associated with altered lipid levels in the central nervous system (CNS), including myelin-associated lipids within the corpus callosum (CC). Perturbed lipid metabolism was correlated with reduced numbers of OLs, but increased numbers of GFAP+ glia, in the CC, but not in grey matter. This was accompanied by reduced myelin protein expression and axonal conduction deficits. Behavioral analyses revealed significant changes in voluntary locomotor activity and anxiety-like behavior in RIT1KO mice. Together, these data reveal an unexpected role for RIT1 in the regulation of cerebral lipid metabolism, which coincide with altered white matter tract oligodendrocyte levels, reduced axonal conduction velocity, and behavioral abnormalities in the CNS.

Loss of carnitine palmitoyltransferase 1a reduces docosahexaenoic acid-containing phospholipids and drives sexually dimorphic liver disease in mice.

BACKGROUND AND AIMS: Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism. APPROACH AND RESULTS: Male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (60% kcal fat) for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis (Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism (Elovl5, Fads1, Elovl2), while only female LKO mice increased genes involved in inflammation (Ly6d, Mmp12, Cxcl2). Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. CONCLUSIONS: Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Inhibition of mitochondrial fission activates glycogen synthesis to support cell survival in colon cancer.

Metabolic reprogramming has been recognized as one of the major mechanisms that fuel tumor initiation and progression. Our previous studies demonstrate that activation of Drp1 promotes fatty acid oxidation and downstream Wnt signaling. Here we investigate the role of Drp1 in regulating glycogen metabolism in colon cancer. Knockdown of Drp1 decreases mitochondrial respiration without increasing glycolysis. Analysis of cellular metabolites reveals that the levels of glucose-6-phosphate, a precursor for glycogenesis, are significantly elevated whereas pyruvate and other TCA cycle metabolites remain unchanged in Drp1 knockdown cells. Additionally, silencing Drp1 activates AMPK to stimulate the expression glycogen synthase 1 (GYS1) mRNA and promote glycogen storage. Using 3D organoids from Apcf/f/Villin-CreERT2 models, we show that glycogen levels are elevated in tumor organoids upon genetic deletion of Drp1. Similarly, increased GYS1 expression and glycogen accumulation are detected in xenograft tumors derived from Drp1 knockdown colon cancer cells. Functionally, increased glycogen storage provides survival advantage to Drp1 knockdown cells. Co-targeting glycogen phosphorylase-mediated glycogenolysis sensitizes Drp1 knockdown cells to chemotherapy drug treatment. Taken together, our results suggest that Drp1-loss activates glucose uptake and glycogenesis as compensative metabolic pathways to promote cell survival. Combined inhibition of glycogen metabolism may enhance the efficacy of chemotherapeutic agents for colon cancer treatment.

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides in Toxoplasma gondii tachyzoites and bradyzoites.

The asexual stages of Toxoplasma gondii are defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature of T. gondii bradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs) that are believed to play a role in reactivation, but their functions during the chronic infection remain largely unexplored. More recently, the presence of storage glucans has been recognized in tachyzoites where their precise function and architecture have yet to be fully defined. Importantly, the T. gondii genome encodes activities needed for glucan turnover: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. The expression of these enzymes in tachyzoites supports the existence of a storage glucan, evidence that is corroborated by specific labeling with the anti-glycogen antibody IV58B6. Disruption of reversible glucan phosphorylation via a CRISPR/Cas9 knockout (KO) of TgLaforin revealed no growth defects under nutrient-replete conditions in tachyzoites. However, the growth of TgLaforin-KO tachyzoites was severely stunted when starved of glutamine, even under glucose replete conditions. The loss of TgLaforin also resulted in the attenuation of acute virulence in mice accompanied by a lower cyst burden. Defective cyst formation due to profound changes in AG morphology was also observed in TgLaforin-KO parasites, both in vitro and in vivo. Together, these data demonstrate the importance of glucan turnover across the T. gondii asexual cycle. These findings, alongside our previously identified class of small molecules that inhibit TgLaforin, implicate reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronic T. gondii infections.

Gys1 Antisense Therapy Prevents Disease-Driving Aggregates and Epileptiform Discharges in a Lafora Disease Mouse Model.

Patients with Lafora disease have a mutation in EPM2A or EPM2B, resulting in dysregulation of glycogen metabolism throughout the body and aberrant glycogen molecules that aggregate into Lafora bodies. Lafora bodies are particularly damaging in the brain, where the aggregation drives seizures with increasing severity and frequency, coupled with neurodegeneration. Previous work employed mouse genetic models to reduce glycogen synthesis by approximately 50%, and this strategy significantly reduced Lafora body formation and disease phenotypes. Therefore, an antisense oligonucleotide (ASO) was developed to reduce glycogen synthesis in the brain by targeting glycogen synthase 1 (Gys1). To test the distribution and efficacy of this drug, the Gys1-ASO was administered to Epm2b-/- mice via intracerebroventricular administration at 4, 7, and 10 months. The mice were then sacrificed at 13 months and their brains analyzed for Gys1 expression, glycogen aggregation, and neuronal excitability. The mice treated with Gys1-ASO exhibited decreased Gys1 protein levels, decreased glycogen aggregation, and reduced epileptiform discharges compared to untreated Epm2b-/- mice. This work provides proof of concept that a Gys1-ASO halts disease progression of EPM2B mutations of Lafora disease.

Spatial Metabolome Lipidome and Glycome from a Single brain Section.

Metabolites, lipids, and glycans are fundamental biomolecules involved in complex biological systems. They are metabolically channeled through a myriad of pathways and molecular processes that define the physiology and pathology of an organism. Here, we present a blueprint for the simultaneous analysis of spatial metabolome, lipidome, and glycome from a single tissue section using mass spectrometry imaging. Complimenting an original experimental protocol, our workflow includes a computational framework called Spatial Augmented Multiomics Interface (Sami) that offers multiomics integration, high dimensionality clustering, spatial anatomical mapping with matched multiomics features, and metabolic pathway enrichment to providing unprecedented insights into the spatial distribution and interaction of these biomolecules in mammalian tissue biology.

Loss of Carnitine Palmitoyltransferase 1a Reduces Docosahexaenoic Acid-Containing Phospholipids and Drives Sexually Dimorphic Liver Disease in Mice.

Background and Aims: Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the impact by which liver-specific CPT1a deletion impacts hepatic lipid metabolism. Approach and Results: Six-to-eight-week old male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (HFD; 60% kcal fat) for 15 weeks. Mice were necropsied after a 16 hour fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase (ALT) levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in both whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis ( Plin2 , Cidec , G0S2 ) and in polyunsaturated fatty acid (PUFA) metabolism ( Elovl5, Fads1, Elovl2 ), while only female LKO mice increased genes involved in inflammation ( Ly6d, Mmp12, Cxcl2 ). Kinase profiling showed decreased protein kinase A (PKA) activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. Conclusions: Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Current avenues of gene therapy in Pompe disease.

PURPOSE OF REVIEW: Pompe disease is a rare, inherited, devastating condition that causes progressive weakness, cardiomyopathy and neuromotor disease due to the accumulation of glycogen in striated and smooth muscle, as well as neurons. While enzyme replacement therapy has dramatically changed the outcome of patients with the disease, this strategy has several limitations. Gene therapy in Pompe disease constitutes an attractive approach due to the multisystem aspects of the disease and need to address the central nervous system manifestations. This review highlights the recent work in this field, including methods, progress, shortcomings, and future directions. RECENT FINDINGS: Recombinant adeno-associated virus (rAAV) and lentiviral vectors (LV) are well studied platforms for gene therapy in Pompe disease. These products can be further adapted for safe and efficient administration with concomitant immunosuppression, with the modification of specific receptors or codon optimization. rAAV has been studied in multiple clinical trials demonstrating safety and tolerability. SUMMARY: Gene therapy for the treatment of patients with Pompe disease is feasible and offers an opportunity to fully correct the principal pathology leading to cellular glycogen accumulation. Further work is needed to overcome the limitations related to vector production, immunologic reactions and redosing.

The multifaceted roles of the brain glycogen.

Glycogen is a biologically essential macromolecule that is directly involved in multiple human diseases. While its primary role in carbohydrate storage and energy metabolism in the liver and muscle is well characterized, recent research has highlighted critical metabolic and non-metabolic roles for glycogen in the brain. In this review, the emerging roles of glycogen homeostasis in the healthy and diseased brain are discussed with a focus on advancing our understanding of the role of glycogen in the brain. Innovative technologies that have led to novel insights into glycogen functions are detailed. Key insights into how cellular localization impacts neuronal and glial function are discussed. Perturbed glycogen functions are observed in multiple disorders of the brain, including where it serves as a disease driver in the emerging category of neurological glycogen storage diseases (n-GSDs). n-GSDs include Lafora disease (LD), adult polyglucosan body disease (APBD), Cori disease, Glucose transporter type 1 deficiency syndrome (G1D), GSD0b, and late-onset Pompe disease (PD). They are neurogenetic disorders characterized by aberrant glycogen which results in devastating neurological and systemic symptoms. In the most severe cases, rapid neurodegeneration coupled with dementia results in death soon after diagnosis. Finally, we discuss current treatment strategies that are currently being developed and have the potential to be of great benefit to patients with n-GSD. Taken together, novel technologies and biological insights have resulted in a renaissance in brain glycogen that dramatically advanced our understanding of both biology and disease. Future studies are needed to expand our understanding and the multifaceted roles of glycogen and effectively apply these insights to human disease.

In situ microwave fixation provides an instantaneous snapshot of the brain metabolome.

Brain glucose metabolism is highly heterogeneous among brain regions and continues postmortem. In particular, we demonstrate exhaustion of glycogen and glucose and an increase in lactate production during conventional rapid brain resection and preservation by liquid nitrogen. In contrast, we show that these postmortem changes are not observed with simultaneous animal sacrifice and in situ fixation with focused, high-power microwave. We further employ microwave fixation to define brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple brain regions, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the tricarboxylic acid (TCA) cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation for more accurate studies of brain metabolism in rodent models.

Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis.

Matrix assisted laser desorption/ionization imaging has greatly improved our understanding of spatial biology, however a robust bioinformatic pipeline for data analysis is lacking. Here, we demonstrate the application of high-dimensionality reduction/spatial clustering and histopathological annotation of matrix assisted laser desorption/ionization imaging datasets to assess tissue metabolic heterogeneity in human lung diseases. Using metabolic features identified from this pipeline, we hypothesize that metabolic channeling between glycogen and N-linked glycans is a critical metabolic process favoring pulmonary fibrosis progression. To test our hypothesis, we induced pulmonary fibrosis in two different mouse models with lysosomal glycogen utilization deficiency. Both mouse models displayed blunted N-linked glycan levels and nearly 90% reduction in endpoint fibrosis when compared to WT animals. Collectively, we provide conclusive evidence that lysosomal utilization of glycogen is required for pulmonary fibrosis progression. In summary, our study provides a roadmap to leverage spatial metabolomics to understand foundational biology in pulmonary diseases.

Cervical spinal cord injury leads to injury and altered metabolism in the lungs.

High-cervical spinal cord injury often disrupts respiratory motor pathways and disables breathing in the affected population. Moreover, cervically injured individuals are at risk for developing acute lung injury, which predicts substantial mortality rates. While the correlation between acute lung injury and spinal cord injury has been found in the clinical setting, the field lacks an animal model to interrogate the fundamental biology of this relationship. To begin to address this gap in knowledge, we performed an experimental cervical spinal cord injury (N = 18) alongside sham injury (N = 3) and naïve animals (N = 15) to assess lung injury in adult rats. We demonstrate that animals display some early signs of lung injury two weeks post-spinal cord injury. While no obvious histological signs of injury were observed, the spinal cord injured cohort displayed significant signs of metabolic dysregulation in multiple pathways that include amino acid metabolism, lipid metabolism, and N-linked glycosylation. Collectively, we establish for the first time a model of lung injury after spinal cord injury at an acute time point that can be used to monitor the progression of lung damage, as well as identify potential targets to ameliorate acute lung injury.

PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence.

Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge.

The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response: two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses in mice expressing human APOE to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNA sequencing (RNA-seq) highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression and a disrupted tricarboxylic acid (TCA) cycle and are inherently pro-glycolytic, while spatial transcriptomics and mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism and provide valuable, interactive resources for discovery and validation research.

Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidation.

BACKGROUND & AIMS: The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. METHODS: Six-week-old, male, C57Bl/6 J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with in vivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. RESULTS: Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein in vivo. In vitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C in vitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. CONCLUSIONS: KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. IMPACT AND IMPLICATIONS: Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.