Science Note: Cellular metabolism

Scientists have discovered that in both basal and activated states, neuronal somata have higher levels of aerobic glycolysis and lower levels of OXPHOS than terminals. Their findings update the conventional view that neurons use OXPHOS uniformly under basal conditions and highlight the important role of somatic aerobic glycolysis in maintaining antioxidant capacity.

Aerobic glycolysis is the predominant means of glucose metabolism in neuronal somata, which protects against oxidative damage
Click here for the original article: Yao Wei, et. al., Nature Neuroscience, 2023.

Point of Interest
- Neuronal somata engage more in aerobic glycolysis and less in OXPHOS compared to axon terminals, both at rest and when activated.
- The enzyme pyruvate kinase 2 (PKM2) is primarily found in neuronal somata, not terminals.
- Deleting Pkm2 in mice shifts somatic metabolism from aerobic glycolysis to OXPHOS.
- This shift causes oxidative damage and loss of dopaminergic neurons.

Comparison of metabolic pathway in two types of cancer cells

The dependence of OXPHOS and Glycolysis in two types of cancer cells, HeLa and HepG2, were compared based on Lactate production, ATP levels, and OCR values.

Many cancer cells produce ATP through the glycolytic pathway. On the other hand, it has been recently reported that cancer cells whose glycolytic pathway is suppressed survive by shifting their energy metabolism to OXPHOS by enhancing mitochondrial function, and the dependency of metabolic pathways differs depending on cell lines.

＜Evaluation by Lactate production and ATP levels＞

We confirmed the changes in ATP and Lactate production when ATP synthesis by OXPHOS was inhibited by Oligomycin stimulation and by 2-Deoxy-D-glucose (2-DG) in the glycolytic pathway. The results showed that HeLa cells depend on Glycolysis and HepG2 cells depend on OXPHOS to synthesize ATP.

＜Evaluation by Lactate production and ATP levels＞
When OXPHOS was inhibited in HeLa cells, ATP levels remained unchanged (①), and lactate production increased (②). This suggests that even when OXPHOS is inhibited, glycolysis can be further activated. Conversely, when glycolysis is inhibited, ATP levels decrease significantly (③), indicating that energy production depends on glycolysis. On the other hand, when OXPHOS was inhibited in HepG2 cells, lactate production increased (④), indicating that the cells attempt to compensate for energy production by enhancing glycolysis, but ATP levels still decrease (⑤). This means that even with increased glycolysis, ATP production is not sufficiently compensated. Furthermore, ATP levels decrease more when glycolysis is inhibited (⑥), suggesting that energy production in HepG2 cells depends more on OXPHOS than glycolysis.

Products in Use
   - Glycolysis/OXPHOS Assay Kit

＜Evaluation by OCR value＞

Using the same number of cells, we measured the OCR value when cellular oxygen consumption was promoted by stimulating the cells with FCCP, a mitochondrial uncoupling agent. The results showed that HepG2 cells had higher OCR values than HeLa cells, suggesting a greater dependence on OXPHOS, correlating with the results obtained from ATP level and Lactate production.

Products in Use
   - Extracellular OCR Plate Assay Kit

Scientists have found that deficiency of two key glycolytic enzymes, Glut1 (glucose transporter 1) and Gpi1 (glucose-6-phosphate isomerase 1), enhanced tumor cell killing by cytotoxic T cells. Genetic and pharmacologic inactivation of Glut1 sensitizes tumors to anti-tumor immunity and synergizes with anti-PD-1 therapy through the TNF-α pathway.

Tumor aerobic glycolysis confers immune evasion through modulating sensitivity to T cell-mediated bystander killing via TNF-α
Click here for the original article: Lijian Wu, et. al., Cell Metabolism, 2023.

Point of Interest
- The intrinsic glycolysis pathway within tumors confers resistance to killing by T cells.
- Depletion of Glut1 sensitizes tumor cells to cell death induced by TNF-α through the augmentation of reactive oxygen species (ROS).
- Glut1 and Glut3 exhibit differential expression in tumor cells compared to immune cells.
- Inhibiting Glut1 enhances the anti-tumor immune response in murine models.

Metabolic shift to glycolysis in senescenct cells

＜Evaluation by Lactate production and ATP levels＞

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX
② Cellular Senescence Detection Kit - SPiDER-βGal
③ NAD/NADH Assay Kit-WST
④ JC-1 MitoMP Detection Kit
⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Scientists have found that neuroinflammatory lesions in the mouse spinal cord cause widespread and persistent axonal ATP deficiency that precedes mitochondrial oxidation and calcium overload. Notably, viral overexpression of individual TCA enzymes can ameliorate axonal energy deficits in neuroinflammatory lesions. This suggests that TCA cycle dysfunction in the common neuroinflammatory disease multiple sclerosis may be amenable to therapy.

Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions
Click here for the original article: Yi-Heng Tai, et. al., Nature Metabolism, 2023.

Point of Interest
- Neuroinflammatory lesions in mice cause long-lasting axonal ATP deficiency.
- This deficiency precedes mitochondrial oxidation and calcium overload.
- It's linked to tricarboxylic acid (TCA) cycle enzyme imbalances.
- Correcting TCA enzyme levels can potentially treat these energy deficits.

Comparison of metabolic pathway in two types of cancer cells

The dependence of OXPHOS and Glycolysis in two types of cancer cells, HeLa and HepG2, were compared based on Lactate production, ATP levels, and OCR values.

Many cancer cells produce ATP through the glycolytic pathway. On the other hand, it has been recently reported that cancer cells whose glycolytic pathway is suppressed survive by shifting their energy metabolism to OXPHOS by enhancing mitochondrial function, and the dependency of metabolic pathways differs depending on cell lines.

＜Evaluation by Lactate production and ATP levels＞

We confirmed the changes in ATP and Lactate production when ATP synthesis by OXPHOS was inhibited by Oligomycin stimulation and by 2-Deoxy-D-glucose (2-DG) in the glycolytic pathway. The results showed that HeLa cells depend on Glycolysis and HepG2 cells depend on OXPHOS to synthesize ATP.

＜Evaluation by Lactate production and ATP levels＞
When OXPHOS was inhibited in HeLa cells, ATP levels remained unchanged (①), and lactate production increased (②). This suggests that even when OXPHOS is inhibited, glycolysis can be further activated. Conversely, when glycolysis is inhibited, ATP levels decrease significantly (③), indicating that energy production depends on glycolysis. On the other hand, when OXPHOS was inhibited in HepG2 cells, lactate production increased (④), indicating that the cells attempt to compensate for energy production by enhancing glycolysis, but ATP levels still decrease (⑤). This means that even with increased glycolysis, ATP production is not sufficiently compensated. Furthermore, ATP levels decrease more when glycolysis is inhibited (⑥), suggesting that energy production in HepG2 cells depends more on OXPHOS than glycolysis.

Products in Use
   - Glycolysis/OXPHOS Assay Kit

＜Evaluation by OCR value＞

Using the same number of cells, we measured the OCR value when cellular oxygen consumption was promoted by stimulating the cells with FCCP, a mitochondrial uncoupling agent. The results showed that HepG2 cells had higher OCR values than HeLa cells, suggesting a greater dependence on OXPHOS, correlating with the results obtained from ATP level and Lactate production.

Products in Use
   - Extracellular OCR Plate Assay Kit

Scientists have found that lysosomal cystine deprivation, unlike general amino acid starvation, triggers ATF4 induction at the transcriptional level, but cytosolic cysteine deprivation does not. Blocking lysosomal cystine efflux diminishes ATF4 induction and enhances sensitivity to ferroptosis. Therefore, intracellular nutrient reprogramming could potentially induce selective ferroptosis in cancer cells without causing systemic starvation.

Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response
Click here for the original article: Robert V. Swanda, et. al., Molecular Cell, 2023.

Point of Interest
- Depletion of cysteine induces adaptive ATF4 expression at the transcriptional level.
- A shortage of cystine in lysosomes stimulates ATF4 expression through the AhR signaling pathway.
- A weakened cysteine stress response increases sensitivity to ferroptosis during cysteine deprivation.
- CysRx promotes cancer cell ferroptosis through intracellular nutrient reprogramming..

Lysosomal Acidic pH Detection Kit-Green/Red 
Lysosomal Acidic pH Detection Kit-Green/Deep Red 

Lysosomal Function and Mitochondrial ROS

CCCP and Antimycin are recognized inducers of mitochondrial ROS, linked to the loss of mitochondrial membrane potential. Recent studies have shown that CCCP induces not only mitochondrial ROS but also lysosomal dysfunction. To observe mitochondrial ROS, HeLa cells were labeled with MitoBright ROS Deep Red for Mitochondrial Superoxide Detection, and the lysosomal mass and pH were independently detected with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes revealed that CCCP, unlike Antimycin, triggers concurrent lysosomal neutralization and mitochondrial ROS induction.

Reference: Benjamin S Padman, et. al., Autophagy (2013)

Products in Use
   - LysoPrime Green
   - pHLys Red
   - Lysosomal Acidic pH Detection Kit
   - MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

Lysosomal Function and Iron Homeostasis

Recent reports suggest that lysosomal neutralization can result in iron depletion, consequently leading to the disruption of cell viability. To verify this, HeLa cells were labeled with FerroOrange for Fe²⁺ detection, and the lysosomal mass and pH were separately detected with LysoPrime DeepRed and pHLys Green (a product currently under development). Co-staining with FerroOrange and Lysosomal dyes demonstrated that Bafilomycin A1 (Baf. A1), an inhibitor of lysosomal acidification, causes iron depletion consistent with the findings reported in the article. Interestingly, the iron chelator, Deferiprone (DFP), did not impact lysosomal pH, suggesting that lysosomal function plays a key role in managing iron homeostasis.

Reference: Ross A Weber, et. al., Mol Cell (2020)

Products in Use
   - FerroOrange
   - pHLys Green*
   - LysoPrime Deep Red

*pHLys Green is a component of "Lysosomal Acidic pH Detection Kit-Green/Deep Red".

Scientists have discovered that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia–reperfusion injury. This process involves epigenetic inhibition of cardiomyocyte maturation via activation of the alpha-ketoglutarate-dependent lysine demethylase KDM5.

Inhibition of fatty acid oxidation enables heart regeneration in adult mice
Click here for the original article: Xiang Li, et. al., Nature, 2023.

Point of Interest
- Cardiomyocytes undergo postnatal maturation which limits heart regeneration.
- Disabling fatty acid oxidation in these cells promotes their proliferation and aids heart repair after injury.  
- This process is linked to changes in energy metabolism and the activation of KDM5, altering gene expression.  
- Reversing metabolic maturation can rejuvenate cardiomyocytes and facilitate heart healing.

Fatty acid starvation induced by uptake inhibitor evoke reprogramming of cellular metabolism 

Mitochondrial fatty acid β-oxidation and oxidative phosphorylation (OXPHOS) are crucial biochemical processes that metabolize fats and sugars to produce ATP, the cell's primary energy source. In this section, we underscored the significance of fatty acid starvation and energy pathways, with an emphasis on the fatty acid uptake inhibitor, FATP2. Here are the key findings from our experiments conducted on HeLa cells:

・Inhibition of fatty acid uptake results in reduced cell proliferation, though it does not lead to cell death. This was determined through the use of a Cell Counting Kit-8 and Fatty Acid Uptake Kit (Image 1).

・Fatty acid starvation shifts cellular metabolism from OXPHOS to glycolysis, as indicated by the Glycolysis/JC-1 MitoMP Assay Kit. (Image 2)

・When fatty acid uptake is inhibited, a compensatory increase in glucose and glutamine uptake occurs to preserve cell viability, as observed using the Glucose Assay Kit and Glutamine Assay Kit. (Image 3)

Products in Use
for Fatty Acid Uptake Assay
  ① Fatty Acid Uptake Assay Kit

for Cell Proliferation/Cytotoxicity Assay
  ② Cell Counting Kit-8

for Glycolysis Assay and Mitochondrial Membrane Potential Detection
  ③ Glycolysis/JC-1 MitoMP Assay Kit

for Glucose and Glutamine Consumption Assay
  ④ Glucose Assay Kit

  ⑤ Glutamine Assay Kit

In recent years, the discovery of several novel lipid-mitochondria axis in metabolism has attracted much attention. A link between acylcarnitine accumulation and lipid intolerance in skeletal muscle mitochondria is tightly linked to poor health. In other breakthroughs, decreased expression in fatty acid transporters and β-oxidation enzymes causes fatty acid buildup in lipid droplets. Research also uncovers that knocking out a vital enzyme in mitochondrial fatty acid oxidation astrocytes leads to cognitive impairment.

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles
Click here for the original article: Timothy R. Koves, et. al., Cell Metabolism, 2023.

Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis
Click here for the original article: Ludovic Enkler, et. al., Nature Cell Biology, 2023.

Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition
Click here for the original article: Brenda Morant-Ferrando , et. al., Nature Metabolism, 2023.

Point of Interest
- The metabolic pathway of long-chain fatty acid oxidation is susceptible to bottlenecks.
- Bottlenecks in the oxidation of fat can lead to CoA trapping and a decrease in respiratory efficiency.
- The reverse flux of pyruvate-derived acetyl CoA through the medium-chain ketothiolase pathway can regenerate free CoA.
- High levels of medium-chain ketothiolase in the heart and red muscles enhance mitochondrial tolerance to lipids.

Point of Interest
- Expression of fatty acid transporters and the key enzyme in β-oxidation decreased in cells with a hyperactive Arf1 mutant, causing fatty acids to accumulate in lipid droplets.
- As a result, mitochondria fragmented and ATP synthesis declined.
- Genetic and drug-induced reduction of fatty acids mimicked the mitochondrial effects of the arf1 mutant.
- Although β-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism remains conserved.

Point of Interest
- Knockout of CPT1A, a key enzyme of mitochondrial fatty acid oxidation, leads to cognitive impairment in adult mouse astrocytes.
- Decreased fatty acid oxidation alters astrocytic pyruvate metabolism and reduces reactive oxygen species formation.
- Astrocytes naturally metabolize fatty acids to maintain the mitochondrial respiratory chain in an energetically inefficient, disassembled conformation.
- This disassembled conformation helps in preserving the signaling of reactive oxygen species and supports cognitive performance.

Fatty acid starvation induced by uptake inhibitor evoke reprogramming of cellular metabolism 

Mitochondrial fatty acid β-oxidation and oxidative phosphorylation (OXPHOS) are crucial biochemical processes that metabolize fats and sugars to produce ATP, the cell's primary energy source. In this section, we underscored the significance of fatty acid starvation and energy pathways, with an emphasis on the fatty acid uptake inhibitor, FATP2. Here are the key findings from our experiments conducted on HeLa cells:

・Inhibition of fatty acid uptake results in reduced cell proliferation, though it does not lead to cell death. This was determined through the use of a Cell Counting Kit-8 and Fatty Acid Uptake Kit (Image 1).

・Fatty acid starvation shifts cellular metabolism from OXPHOS to glycolysis, as indicated by the Glycolysis/JC-1 MitoMP Assay Kit. (Image 2)

・When fatty acid uptake is inhibited, a compensatory increase in glucose and glutamine uptake occurs to preserve cell viability, as observed using the Glucose Assay Kit and Glutamine Assay Kit. (Image 3)

Products in Use
for Fatty Acid Uptake Assay
  ① Fatty Acid Uptake Assay Kit

for Cell Proliferation/Cytotoxicity Assay
  ② Cell Counting Kit-8

for Glycolysis Assay and Mitochondrial Membrane Potential Detection
  ③ Glycolysis/JC-1 MitoMP Assay Kit NEW

for Glucose and Glutamine Consumption Assay
  ④ Glucose Assay Kit

  ⑤ Glutamine Assay Kit

Loss of UCHL1 rescues the defects related to Parkinson’s disease by suppressing glycolysis
Su Jin Ham, et. al., Sci Adv, 7 (2021)

   Point of Interest
   - The loss of UCHL1 regulates glucose metabolism and rescues the defects related to PD. 
   - Specific glycolytic metabolites are decreased and an energy-dependent mitophagy pathway is induced by inhibition of UCHL1.
   - UCHL1 plays a critical role in regulating mitophagy independent of the PINK1-Parkin pathway and PKM is the responsible target of UCHL1 in controlling mitophagy and PD pathogenesis.

Fatty acid starvation induced by uptake inhibitor evoke reprogramming of cellular metabolism 

Mitochondrial fatty acid β-oxidation and oxidative phosphorylation (OXPHOS) are crucial biochemical processes that metabolize fats and sugars to produce ATP, the cell's primary energy source. In this section, we underscored the significance of fatty acid starvation and energy pathways, with an emphasis on the fatty acid uptake inhibitor, FATP2. Here are the key findings from our experiments conducted on HeLa cells:

・Inhibition of fatty acid uptake results in reduced cell proliferation, though it does not lead to cell death. This was determined through the use of a Cell Counting Kit-8 and Fatty Acid Uptake Kit (Image 1).

・Fatty acid starvation shifts cellular metabolism from OXPHOS to glycolysis, as indicated by the Glycolysis/JC-1 MitoMP Assay Kit. (Image 2)

・When fatty acid uptake is inhibited, a compensatory increase in glucose and glutamine uptake occurs to preserve cell viability, as observed using the Glucose Assay Kit and Glutamine Assay Kit. (Image 3)

Products in Use
for Fatty Acid Uptake Assay
  ① Fatty Acid Uptake Assay Kit

for Cell Proliferation/Cytotoxicity Assay
  ② Cell Counting Kit-8

for Glycolysis Assay and Mitochondrial Membrane Potential Detection
  ③ Glycolysis/JC-1 MitoMP Assay Kit NEW

for Glucose and Glutamine Consumption Assay
  ④ Glucose Assay Kit

  ⑤ Glutamine Assay Kit

Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma
Zijian Yang, et. al., Nature Metabolism, 5, 61-79 (2023)

   Point of Interest
   - Lactate drives the posttranslational modification, lysine lactylation (Kla), on core histones but other targets remain unclear.
   - Lactylome and proteome analysis of the tumors and adjacent livers reveals 99.8% of Kla sites are on non-histone proteins. 
   - Kla affects enzymes involved in metabolic pathways, including nucleotide metabolism.
   - Lactylation at K28 inhibits adenylate kinase 2, facilitating the proliferation and metastasis of HCC cells.

Metabolic shift to glycolysis in senescenct cells

	NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products. *S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Products in Use
① DNA Damage Detection Kit - γH2AX

② Cellular Senescence Detection Kit - SPiDER-βGal

③ NAD/NADH Assay Kit-WST

④ JC-1 MitoMP Detection Kit

⑤ Glycolysis/OXPHOS Assay Kit、Lactate Assay Kit-WST

Large neutral amino acid levels tune perinatal neuronal excitability and survival
Lisa Knaus, et. al., Cell, 186, 1-18 (2023)

   Point of Interest
   - Metabolic reprogramming of the cerebral cortex throughout development
   - Essential role of large neutral amino acids in perinatal neuronal metabolic state
   - Link between Slc7a5 deletion in neurons, branched-chain amino acids, and lipid metabolism
   - Impact of reduced large neutral amino acid levels on neuronal excitability and survival

The metabolic shift to glycolysis under the treatment of fatty acid transporter inhibitors

Products in Use
for Fatty Acid Uptake Assay
  ① Fatty Acid Uptake Assay Kit

for Cell Proliferation/Cytotoxicity Assay
  ② Cell Counting Kit-8

for Glycolysis Assay
  ③ Glucose Assay Kit-WST
  ④ Lactate Assay Kit-WST

Related Products
for Glycolysis and OXPHOS Assay
  - Glycolysis/OXPHOS Assay Kit

for OXPHOS Assay
  - Extracellular OCR Plate Assay Kit

• - Using ¹³C-tracer analysis, the activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes was identified in the nuclei.
• - Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins.
• - 2-oxoglutarate dehydrogenase, which produces succinyl-CoA, also localizes at the nucleus in mouse embryonic stem cells.
• - The nuclear localization of this enzyme changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation.

• - iNs from patients with AD express cancer-associated PKM2
• - PKM2 facilitates Warburg-effect-like glycolytic reprogramming of old neurons
• - Nuclear PKM2 associates with STAT3 and HIF1α to promote neuronal fate loss in AD iNs
• - Modulation of PKM2 with shikonin restores healthy neuronal features

• - Adipocytes transfer mitochondria to tissue-specific networks of cells in fat
• - Diet-induced obesity, but not aging, impairs mitochondria transfer to macrophages
• - Dietary long-chain fatty acids inhibit mitochondria uptake by macrophages
• - Macrophages limit the release of adipocyte mitochondria into blood