Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during Drosophila melanogaster larval development.

The dramatic growth that occurs during Drosophila larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis. To explore the potential role of Drosophila LDH in promoting biosynthesis, we examined how Ldh mutations influence larval development. Our studies unexpectedly found that Ldh mutants grow at a normal rate, indicating that LDH is dispensable for larval biomass production. However, subsequent metabolomic analyses suggested that Ldh mutants compensate for the inability to produce lactate by generating excess glycerol-3-phosphate (G3P), the production of which also influences larval redox balance. Consistent with this possibility, larvae lacking both LDH and G3P dehydrogenase (GPDH1) exhibit growth defects, synthetic lethality and decreased glycolytic flux. Considering that human cells also generate G3P upon inhibition of lactate dehydrogenase A (LDHA), our findings hint at a conserved mechanism in which the coordinate regulation of lactate and G3P synthesis imparts metabolic robustness to growing animal tissues.

l-2-Hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule l-2-hydroxyglutarate (l-2HG) is elevated in the most common histology of renal cancer. Similarly to other oncometabolites, l-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that l-2HG remodels amino acid metabolism in renal cancer cells through combined effects on histone methylation and RNA N6-methyladenosine. The combined effects of l-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high-l-2HG kidney cancers demonstrate reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high-l-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies.

The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. Here we genetically manipulated expression of Drosophila melanogaster lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. We also assessed survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. We provide evidence that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, our findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner.

Metabolomic analysis of Drosophila melanogaster larvae lacking Pyruvate kinase.

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster , however, are complicated by the fact that the fly genome encodes six Pyk paralogs whose functions remain poorly defined. To address this issue, we used sequence distance and phylogenetic approaches to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other five Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of two different Pyk mutant backgrounds revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectedly reveals that steady state pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and peptidase activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans.

Metabolomic analysis of Drosophila melanogaster larvae lacking pyruvate kinase.

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster are complicated by the fact that the fly genome encodes 6 Pyk paralogs whose functions remain poorly defined. To address this issue, we used sequence distance and phylogenetic approaches to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other 5 Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of 2 different Pyk mutant strains revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectedly reveals that pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and protease activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans.

Renal oncometabolite L-2-hydroxyglutarate imposes a block in kidney tubulogenesis: Evidence for an epigenetic basis for the L-2HG-induced impairment of differentiation.

2-Hydroxyglutarate (2HG) overproducing tumors arise in a number of tissues, including the kidney. The tumorigenesis resulting from overproduced 2HG has been attributed to the ability of 2HG alter gene expression by inhibiting α-ketoglutarate (αKG)-dependent dioxygenases, including Ten-eleven-Translocation (TET) enzymes. Genes that regulate cellular differentiation are reportedly repressed, blocking differentiation of mesenchymal cells into myocytes, and adipocytes. In this report, the expression of the enzyme responsible for L2HG degradation, L-2HG dehydrogenase (L2HGDH), is knocked down, using lentiviral shRNA, as well as siRNA, in primary cultures of normal Renal Proximal Tubule (RPT) cells. The knockdown (KD) results in increased L-2HG levels, decreased demethylation of 5mC in genomic DNA, and increased methylation of H3 Histones. Consequences include reduced tubulogenesis by RPT cells in matrigel, and reduced expression of molecular markers of differentiation, including membrane transporters as well as HNF1α and HNF1ß, which regulate their transcription. These results are consistent with the hypothesis that oncometabolite 2HG blocks RPT differentiation by altering the methylation status of chromatin in a manner that impedes the transcriptional events required for normal differentiation. Presumably, similar alterations are responsible for promoting the expansion of renal cancer stem-cells, increasing their propensity for malignant transformation.

The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis.

As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH1) has been the focus of biochemical genetics studies for several decades and, as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. Here, we expand upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. Our findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, our findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive the loss of this key enzyme.

The oncometabolite L-2-hydroxyglutarate is a common product of dipteran larval development.

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.