Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.

Histone lactylation in macrophages is predictive for gene expression changes during ischemia induced-muscle regeneration.

OBJECTIVES: We have previously shown that lactate is an essential metabolite for macrophage polarisation during ischemia-induced muscle regeneration. Recent in vitro work has implicated histone lactylation, a direct derivative of lactate, in macrophage polarisation. Here, we explore the in vivo relevance of histone lactylation for macrophage polarisation after muscle injury. METHODS: To evaluate macrophage dynamics during muscle regeneration, we subjected mice to ischemia-induced muscle damage by ligating the femoral artery. Muscle samples were harvested at 1, 2, 4, and 7 days post injury (dpi). CD45+CD11b+F4/80+CD64+ macrophages were isolated and processed for RNA sequencing, Western Blotting, and CUT&Tag-sequencing to investigate gene expression, histone lactylation levels, and histone lactylation genomic localisation and enrichment, respectively. RESULTS: We show that, over time, macrophages in the injured muscle undergo extensive gene expression changes, which are similar in nature and in timing to those seen after other types of muscle-injuries. We find that the macrophage histone lactylome is modified between 2 and 4 dpi, which is a crucial window for macrophage polarisation. Absolute histone lactylation levels increase, and, although subtly, the genomic enrichment of H3K18la changes. Overall, we find that histone lactylation is important at both promoter and enhancer elements. Lastly, H3K18la genomic profile changes from 2 to 4 dpi were predictive for gene expression changes later in time, rather than being a reflection of prior gene expression changes. CONCLUSIONS: Our results suggest that histone lactylation dynamics are functionally important for the function of macrophages during muscle regeneration.

Combinatorial Treatment with PARP and MAPK Inhibitors Overcomes Phenotype Switch-Driven Drug Resistance in Advanced Melanoma.

Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors (MAPKi). A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of patients with melanoma. Using a drug screen targeting chromatin regulators in patient-derived three-dimensional MAPKi-resistant melanoma cell cultures, we discovered that PARP inhibitors (PARPi) restore sensitivity to MAPKis, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARPis induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed epithelial-mesenchymal transition-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPKi-sensitive state. The combination of PARP and MAPKis synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating patients with melanoma with PARPis in combination with MAPKis to abrogate acquired therapy resistance. SIGNIFICANCE: PARP inhibitors can overcome resistance to MAPK inhibitors by activating autophagic cell death and reversing phenotype switching, suggesting that this synergistic combination could help improve the prognosis of patients with melanoma.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Shaping the brain vasculature in development and disease in the single-cell era.

The CNS critically relies on the formation and proper function of its vasculature during development, adult homeostasis and disease. Angiogenesis - the formation of new blood vessels - is highly active during brain development, enters almost complete quiescence in the healthy adult brain and is reactivated in vascular-dependent brain pathologies such as brain vascular malformations and brain tumours. Despite major advances in the understanding of the cellular and molecular mechanisms driving angiogenesis in peripheral tissues, developmental signalling pathways orchestrating angiogenic processes in the healthy and the diseased CNS remain incompletely understood. Molecular signalling pathways of the 'neurovascular link' defining common mechanisms of nerve and vessel wiring have emerged as crucial regulators of peripheral vascular growth, but their relevance for angiogenesis in brain development and disease remains largely unexplored. Here we review the current knowledge of general and CNS-specific mechanisms of angiogenesis during brain development and in brain vascular malformations and brain tumours, including how key molecular signalling pathways are reactivated in vascular-dependent diseases. We also discuss how these topics can be studied in the single-cell multi-omics era.

Exercise-induced angiogenesis is dependent on metabolically primed ATF3/4+ endothelial cells.

Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4+ mECs are enriched in red oxidative muscle areas while ATF3/4low ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4+ mECs are more angiogenic when compared with white ATF3/4low mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4+) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs.

Fat Induces Glucose Metabolism in Nontransformed Liver Cells and Promotes Liver Tumorigenesis.

Hepatic fat accumulation is associated with diabetes and hepatocellular carcinoma (HCC). Here, we characterize the metabolic response that high-fat availability elicits in livers before disease development. After a short term on a high-fat diet (HFD), otherwise healthy mice showed elevated hepatic glucose uptake and increased glucose contribution to serine and pyruvate carboxylase activity compared with control diet (CD) mice. This glucose phenotype occurred independently from transcriptional or proteomic programming, which identifies increased peroxisomal and lipid metabolism pathways. HFD-fed mice exhibited increased lactate production when challenged with glucose. Consistently, administration of an oral glucose bolus to healthy individuals revealed a correlation between waist circumference and lactate secretion in a human cohort. In vitro, palmitate exposure stimulated production of reactive oxygen species and subsequent glucose uptake and lactate secretion in hepatocytes and liver cancer cells. Furthermore, HFD enhanced the formation of HCC compared with CD in mice exposed to a hepatic carcinogen. Regardless of the dietary background, all murine tumors showed similar alterations in glucose metabolism to those identified in fat exposed nontransformed mouse livers, however, particular lipid species were elevated in HFD tumor and nontumor-bearing HFD liver tissue. These findings suggest that fat can induce glucose-mediated metabolic changes in nontransformed liver cells similar to those found in HCC. SIGNIFICANCE: With obesity-induced hepatocellular carcinoma on a rising trend, this study shows in normal, nontransformed livers that fat induces glucose metabolism similar to an oncogenic transformation.

Macrophage-derived glutamine boosts satellite cells and muscle regeneration.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1-4. Macrophages and satellite cells communicate in different ways1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.

Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization.

Endothelial cell (EC)-derived signals contribute to organ regeneration, but angiocrine metabolic communication is not described. We found that EC-specific loss of the glycolytic regulator pfkfb3 reduced ischemic hindlimb revascularization and impaired muscle regeneration. This was caused by the reduced ability of macrophages to adopt a proangiogenic and proregenerative M2-like phenotype. Mechanistically, loss of pfkfb3 reduced lactate secretion by ECs and lowered lactate levels in the ischemic muscle. Addition of lactate to pfkfb3-deficient ECs restored M2-like polarization in an MCT1-dependent fashion. Lactate shuttling by ECs enabled macrophages to promote proliferation and fusion of muscle progenitors. Moreover, VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis. Finally, increasing lactate levels during ischemia rescued macrophage polarization and improved muscle reperfusion and regeneration, whereas macrophage-specific mct1 deletion prevented M2-like polarization. In summary, ECs exploit glycolysis for angiocrine lactate shuttling to steer muscle regeneration from ischemia.

Hallmarks of frailty and osteosarcopenia in prematurely aged PolgA(D257A/D257A) mice.

BACKGROUND: Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed. METHODS: To evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild-type littermates (PolgA(+/+) , WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed. RESULTS: PolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (-14%, P < 0.05, -23%, P < 0.0001) and fibre area (-20%, P < 0.05, -22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro-computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load-induced bone adaptation, suggesting reduced mechanosensitivity with age. CONCLUSIONS: PolgA(D257A/D257A) mutation leads to hallmarks of age-related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.

Differentiation but not ALS mutations in FUS rewires motor neuron metabolism.

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.

Restoration of histone acetylation ameliorates disease and metabolic abnormalities in a FUS mouse model.

Dysregulation of epigenetic mechanisms is emerging as a central event in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). In many models of neurodegeneration, global histone acetylation is decreased in the affected neuronal tissues. Histone acetylation is controlled by the antagonistic actions of two protein families -the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). Drugs inhibiting HDAC activity are already used in the clinic as anti-cancer agents. The aim of this study was to explore the therapeutic potential of HDAC inhibition in the context of ALS. We discovered that transgenic mice overexpressing wild-type FUS ("Tg FUS+/+"), which recapitulate many aspects of human ALS, showed reduced global histone acetylation and alterations in metabolic gene expression, resulting in a dysregulated metabolic homeostasis. Chronic treatment of Tg FUS+/+ mice with ACY-738, a potent HDAC inhibitor that can cross the blood-brain barrier, ameliorated the motor phenotype and substantially extended the life span of the Tg FUS+/+ mice. At the molecular level, ACY-738 restored global histone acetylation and metabolic gene expression, thereby re-establishing metabolite levels in the spinal cord. Taken together, our findings link epigenetic alterations to metabolic dysregulation in ALS pathology, and highlight ACY-738 as a potential therapeutic strategy to treat this devastating disease.

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis.

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.

Metabolic regulation of exercise-induced angiogenesis.

Skeletal muscle relies on an ingenious network of blood vessels, which ensures optimal oxygen and nutrient supply. An increase in muscle vascularization is an early adaptive event to exercise training, but the cellular and molecular mechanisms underlying exercise-induced blood vessel formation are not completely clear. In this review, we provide a concise overview on how exercise-induced alterations in muscle metabolism can evoke metabolic changes in endothelial cells (ECs) that drive muscle angiogenesis. In skeletal muscle, angiogenesis can occur via sprouting and splitting angiogenesis and is dependent on vascular endothelial growth factor (VEGF) signaling. In the resting muscle, VEGF levels are controlled by the estrogen-related receptor γ (ERRγ). Upon exercise, the transcriptional coactivator peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC1α) orchestrates several adaptations to endurance exercise within muscle fibers and simultaneously promotes transcriptional activation of Vegf expression and increased muscle capillary density. While ECs are highly glycolytic and change their metabolism during sprouting angiogenesis in development and disease, a similar role for EC metabolism in exercise-induced angiogenesis in skeletal muscle remains to be elucidated. Nonetheless, recent studies have illustrated the importance of endothelial hydrogen sulfide and sirtuin 1 (SIRT1) activity for exercise-induced angiogenesis, suggesting that EC metabolic reprogramming may be fundamental in this process. We hypothesize that the exercise-induced angiogenic response can also be modulated by metabolic crosstalk between muscle and the endothelium. Defining the underlying molecular mechanisms responsible for skeletal muscle angiogenesis in response to exercise will yield valuable insight into metabolic regulation as well as the determinants of exercise performance.

The Warburg Effect in Endothelial Cells and its Potential as an Anti-angiogenic Target in Cancer.

Endothelial cells (ECs) make up the lining of our blood vessels and they ensure optimal nutrient and oxygen delivery to the parenchymal tissue. In response to oxygen and/or nutrient deprivation, ECs become activated and sprout into hypo-vascularized tissues forming new vascular networks in a process termed angiogenesis. New sprouts are led by migratory tip cells and extended through the proliferation of trailing stalk cells. Activated ECs rewire their metabolism to cope with the increased energetic and biosynthetic demands associated with migration and proliferation. Moreover, metabolic signaling pathways interact and integrate with angiogenic signaling events. These metabolic adaptations play essential roles in determining EC fate and function, and are perturbed during pathological angiogenesis, as occurs in cancer. The angiogenic switch, or the growth of new blood vessels into an expanding tumor, increases tumor growth and malignancy. Limiting tumor angiogenesis has therefore long been a goal for anticancer therapy but the traditional growth factor targeted anti-angiogenic treatments have met with limited success. In recent years however, it has become increasingly recognized that focusing on altered tumor EC metabolism provides an attractive alternative anti-angiogenic strategy. In this review, we will describe the EC metabolic signature and how changes in EC metabolism affect EC fate during physiological sprouting, as well as in the cancer setting. Then, we will discuss the potential of targeting EC metabolism as a promising approach to develop new anti-cancer therapies.

Endothelial Cell Metabolism in Health and Disease.

The metabolism of endothelial cells (ECs) has only recently been recognized as a driving force of angiogenesis. Metabolic pathways, such as glycolysis, fatty acid oxidation, and glutamine metabolism, have distinct, essential roles during vessel formation. Moreover, EC metabolism is markedly perturbed in pathologies such as cancer and diabetes. For instance, because tumor ECs increase glycolysis, lowering hyperglycolysis in tumor ECs induces therapeutic benefits in preclinical tumor models. Expanding our knowledge of how ECs alter their metabolism in disease could pave the way for novel therapeutic opportunities. In this review, we discuss the most recent insights into EC metabolism in health and disease, with emphasis on the changes in metabolism in the tumor endothelium.

Premorbid obesity, but not nutrition, prevents critical illness-induced muscle wasting and weakness.

BACKGROUND: The 'obesity paradox' of critical illness refers to better survival with a higher body mass index. We hypothesized that fat mobilized from excess adipose tissue during critical illness provides energy more efficiently than exogenous macronutrients and could prevent lean tissue wasting. METHODS: In lean and premorbidly obese mice, the effect of 5 days of sepsis-induced critical illness on body weight and composition, muscle wasting, and weakness was assessed, each with fasting and parenteral feeding. Also, in lean and overweight/obese prolonged critically ill patients, markers of muscle wasting and weakness were compared. RESULTS: In mice, sepsis reduced body weight similarly in the lean and obese, but in the obese with more fat loss and less loss of muscle mass, better preservation of myofibre size and muscle force, and less loss of ectopic lipids, irrespective of administered feeding. These differences between lean and obese septic mice coincided with signs of more effective hepatic fatty acid and glycerol metabolism, and ketogenesis in the obese. Also in humans, better preservation of myofibre size and muscle strength was observed in overweight/obese compared with lean prolonged critically ill patients. CONCLUSIONS: During critical illness premorbid obesity, but not nutrition, optimized utilization of stored lipids and attenuated muscle wasting and weakness.

Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization, Impairs Metastasis, and Improves Chemotherapy.

Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.