Atlas of fetal metabolism during mid-to-late gestation and diabetic pregnancy.

Mounting evidence suggests metabolism instructs stem cell fate decisions. However, how fetal metabolism changes during development and how altered maternal metabolism shapes fetal metabolism remain unexplored. We present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and liquid chromatography-mass spectrometry (LC-MS), we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Our analysis revealed metabolic features specific to a hyperglycemic environment and signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from hyperglycemic dams. Tracing 13C-glucose revealed disparate fetal nutrient sourcing depending on maternal glycemic states. Regardless of glycemic state, histidine-derived metabolites accumulated in late-stage fetal tissues. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations due to maternal hyperglycemia.

Acute activation of adipocyte lipolysis reveals dynamic lipid remodeling of the hepatic lipidome.

Adipose tissue is the site of long-term energy storage. During the fasting state, exercise, and cold exposure, the white adipose tissue mobilizes energy for peripheral tissues through lipolysis. The mobilization of lipids from white adipose tissue to the liver can lead to excess triglyceride accumulation and fatty liver disease. Although the white adipose tissue is known to release free fatty acids, a comprehensive analysis of lipids mobilized from white adipocytes in vivo has not been completed. In these studies, we provide a comprehensive quantitative analysis of the adipocyte-secreted lipidome and show that there is interorgan crosstalk with liver. Our analysis identifies multiple lipid classes released by adipocytes in response to activation of lipolysis. Time-dependent analysis of the serum lipidome showed that free fatty acids increase within 30 min of ß3-adrenergic receptor activation and subsequently decrease, followed by a rise in serum triglycerides, liver triglycerides, and several ceramide species. The triglyceride composition of liver is enriched for linoleic acid despite higher concentrations of palmitate in the blood. To further validate that these findings were a specific consequence of lipolysis, we generated mice with conditional deletion of adipose tissue triglyceride lipase exclusively in adipocytes. This loss of in vivo adipocyte lipolysis prevented the rise in serum free fatty acids and hepatic triglycerides. Furthermore, conditioned media from adipocytes promotes lipid remodeling in hepatocytes with concomitant changes in genes/pathways mediating lipid utilization. Together, these data highlight critical role of adipocyte lipolysis in interorgan crosstalk between adipocytes and liver.

Endothelial Jagged1 levels and distribution are post-transcriptionally controlled by ZFP36 decay proteins.

Vascular morphogenesis requires a delicate gradient of Notch signaling controlled, in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (JAG1) expression is compartmentalized in the vascular plexus remains unclear. Here, we show that Jag1 mRNA is a direct target of zinc-finger protein 36 (ZFP36), an RNA-binding protein involved in mRNA decay that we find robustly induced by vascular endothelial growth factor (VEGF). Endothelial cells lacking ZFP36 display high levels of JAG1 and increase angiogenic sprouting in vitro. Furthermore, mice lacking Zfp36 in endothelial cells display mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front alters NOTCH1 signaling, increasing the number of tip cells, a phenotype that is rescued by imposing haploinsufficiency of Jag1. Our findings reveal an important feedforward loop whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.

MYC is a regulator of androgen receptor inhibition-induced metabolic requirements in prostate cancer.

Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effects of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR-blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.

Vitamin B6 is governed by the local compartmentalization of metabolic enzymes during growth.

Vitamin B6 is a vital micronutrient across cell types and tissues, and dysregulated B6 levels contribute to human disease. Despite its importance, how B6 vitamer levels are regulated is not well understood. Here, we provide evidence that B6 dynamics are rapidly tuned by precise compartmentation of pyridoxal kinase (PDXK), the rate-limiting B6 enzyme. We show that canonical Wnt rapidly led to the accumulation of inactive B6 by shunting cytosolic PDXK into lysosomes. PDXK was modified with methyl-arginine Degron (MrDegron), a protein tag for lysosomes, which enabled delivery via microautophagy. Hyperactive lysosomes resulted in the continuous degradation of PDXK and B6 deficiency that promoted proliferation in Wnt-driven colorectal cancer (CRC) cells. Pharmacological or genetic disruption of the coordinated MrDegron proteolytic pathway was sufficient to reduce CRC survival in cells and organoid models. In sum, this work contributes to the repertoire of micronutrient-regulated processes that enable cancer cell growth and provides insight into the functional impact of B6 deficiencies for survival.

Nutrient regulation of development and cell fate decisions.

Diet contributes to health at all stages of life, from embryonic development to old age. Nutrients, including vitamins, amino acids, lipids and sugars, have instructive roles in directing cell fate and function, maintaining stem cell populations, tissue homeostasis and alleviating the consequences of aging. This Review highlights recent findings that illuminate how common diets and specific nutrients impact cell fate decisions in healthy and disease contexts. We also draw attention to new models, technologies and resources that help to address outstanding questions in this emerging field and may lead to dietary approaches that promote healthy development and improve disease treatments.

FH Variant Pathogenicity Promotes Purine Salvage Pathway Dependence in Kidney Cancer.

Fumarate accumulation due to loss of fumarate hydratase (FH) drives cellular transformation. Germline FH alterations lead to hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to an aggressive form of kidney cancer. There is an unmet need to classify FH variants by cancer-associated risk. We quantified catalytic efficiencies of 74 variants of uncertain significance. Over half were enzymatically inactive, which is strong evidence of pathogenicity. We next generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks de novo purine biosynthesis, rendering FH-deficient cells reliant on purine salvage for proliferation. Genetic or pharmacologic inhibition of the purine salvage pathway reduced HLRCC tumor growth in vivo. These findings suggest the pathogenicity of patient-associated FH variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors. SIGNIFICANCE: This study functionally characterizes patient-associated FH variants with unknown significance for pathogenicity. This study also reveals nucleotide salvage pathways as a targetable feature of FH-deficient cancers, which are shown to be sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. This presents a new rapidly translatable treatment strategy for FH-deficient cancers. This article is featured in Selected Articles from This Issue, p. 1949.

ZFP36-mediated mRNA decay regulates metabolism.

Cellular metabolism is tightly regulated by growth factor signaling, which promotes metabolic rewiring to support growth and proliferation. While growth factor-induced transcriptional and post-translational modes of metabolic regulation have been well defined, whether post-transcriptional mechanisms impacting mRNA stability regulate this process is less clear. Here, we present the ZFP36/L1/L2 family of RNA-binding proteins and mRNA decay factors as key drivers of metabolic regulation downstream of acute growth factor signaling. We quantitatively catalog metabolic enzyme and nutrient transporter mRNAs directly bound by ZFP36 following growth factor stimulation-many of which encode rate-limiting steps in metabolic pathways. Further, we show that ZFP36 directly promotes the mRNA decay of Enolase 2 (Eno2), altering Eno2 protein expression and enzymatic activity, and provide evidence of a ZFP36/Eno2 axis during VEGF-stimulated developmental retinal angiogenesis. Thus, ZFP36-mediated mRNA decay serves as an important mode of metabolic regulation downstream of growth factor signaling within dynamic cell and tissue states.

Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells.

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

TGFß superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor ß (TGFß) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFß-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Fumarate hydratase variant prevalence and manifestations among individuals receiving germline testing.

BACKGROUND: Germline variants in fumarate hydratase (FH) are associated with autosomal dominant (AD) hereditary leiomyomatosis and renal cell cancer (HLRCC) and autosomal recessive (AR) fumarase deficiency (FMRD). The prevalence and cancer penetrance across different FH variants remain unclear. METHODS: A database containing 120,061 records from individuals undergoing cancer germline testing was obtained. FH variants were classified into 3 categories: AD HLRCC variants, AR FMRD variants, and variants of unknown significance (VUSs). Individuals with variants from these categories were compared with those with negative genetic testing. RESULTS: FH variants were detected in 1.3% of individuals (AD HLRCC, 0.3%; AR FMRD, 0.4%; VUS, 0.6%). The rate of AD HLRCC variants discovered among reportedly asymptomatic individuals without a clear indication for HLRCC testing was 1 in 2668 (0.04%). In comparison with those with negative genetic testing, the renal cell carcinoma (RCC) prevalence was elevated with AD HLRCC variants (17.0% vs 4.5%; P < .01) and VUSs (6.4% vs 4.5%; P = .02) but not with AR FMRD variants. CONCLUSIONS: The prevalence of HLRCC discovered incidentally on germline testing is similar to recent population carrier estimates, and this suggests that this is a relatively common cancer syndrome. Compared with those with negative genetic testing, those with VUSs had an elevated risk of RCC, whereas those with AR FMRD variants did not.

A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich's ataxia.

Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. A source of damage to Fe-S clusters is cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ for copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3­mediated Cu1+ toxicity is a major determinant of cellular functional pool of Fe-S clusters. Inadequate Fe-S cluster supply, due to diminished assembly as occurs in Friedreich's ataxia or defective distribution, causes severe metabolic and growth defects in Saccharomyces cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster­dependent metabolism and growth. Our findings reveal an interplay between chromatin and mitochondria in Fe-S cluster homeostasis and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.

Targeting nucleotide metabolism as the nexus of viral infections, cancer, and the immune response.

Virus-infected cells and cancers share metabolic commonalities that stem from their insatiable need to replicate while evading the host immune system. These similarities include hijacking signaling mechanisms that induce metabolic rewiring in the host to up-regulate nucleotide metabolism and, in parallel, suppress the immune response. In both cancer and viral infections, the host immune cells and, specifically, lymphocytes augment nucleotide synthesis to support their own proliferation and effector functions. Consequently, established treatment modalities targeting nucleotide metabolism against cancers and virally infected cells may result in restricted immune response. Encouragingly, following the introduction of immunotherapy against cancers, multiple studies improved our understanding for improving antigen presentation to the immune system. We propose here that understanding the immune consequences of targeting nucleotide metabolism against cancers may be harnessed to optimize therapy against viral infections.

Monocarboxylate transporter antagonism reveals metabolic vulnerabilities of viral-driven lymphomas.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the "Warburg effect"- and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6-glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.

SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Challenges in Studying Stem Cell Metabolism.

The expanding field of stem cell metabolism has been supported by technical advances in metabolite profiling and novel functional analyses. While use of these methodologies has been fruitful, many challenges are posed by the intricacies of culturing stem cells in vitro, along with the distinctive scarcity of adult tissue stem cells and the complexities of their niches in vivo. This review provides an examination of the methodologies used to characterize stem cell metabolism, highlighting their utility while placing a sharper focus on their limitations and hurdles the field needs to overcome for the optimal study of stem cell metabolic networks.

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

Development of Novel Mitochondrial Pyruvate Carrier Inhibitors to Treat Hair Loss.

Herein, we report the synthesis and evaluation of novel analogues of UK-5099 both in vitro and in vivo for the development of mitochondrial pyruvate carrier (MPC) inhibitors to treat hair loss. A comprehensive understanding of the structure-activity relationship was obtained by varying four positions of the hit compound, namely, the alkyl group on the N1 position, substituents on the indole core, various aromatic and heteroaromatic core structures, and various Michael acceptors. The major discovery was that the inhibitors with a 3,5-bis(trifluoromethyl)benzyl group at the N1 position were shown to have much better activity than JXL001 (UK-5099) to increase cellular lactate production. Additionally, analogue JXL069, possessing a 7-azaindole heterocycle, was also shown to have significant MPC inhibition activity, which further increases the chemical space for drug design. Finally, more than 10 analogues were tested on shaved mice by topical treatment and promoted obvious hair growth on mice.

O-Mg! Lactate Drives Mg2+ Mobilization.

Lactate initiates Mg2+ release from the ER and subsequent uptake by the mitochondria.

GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the ß-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, ß-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.