CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology® (CST) where we discuss what to expect from your time at the bench, share tips, tricks, and information.

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How Do I Measure Metabolism?

Several different assays can be used by scientists to measure metabolism in a variety of contexts. These include methods to directly determine metabolic rates as well as to examine key signaling pathways and environmental cues, like hypoxia and oxidative stress, which directly influence metabolism under normal and pathological conditions.

Biomarkers and Assays to Assess Hypoxia and Oxidative Stress

Hypoxia and oxidative stress are both a cause and consequence of alterations in cellular metabolism. Cell Signaling Technology® (CST) is proud to offer antibodies to investigate key biomarkers of hypoxia and oxidative stress. Available products can be accessed directly from the CST Hypoxia Interactive Signaling Pathway.

  • Expression of HIF-1α, a transcription factor and master regulator of cellular hypoxia response pathways, is significantly upregulated in response to a low O2 environment. CST antibodies to HIF1 can be used to examine these changes via an array of assays, including western blotting and ELISA. Elevated HIF-1α expression would inform scientists that cells or tissues are likely exposed to Hypoxia in their model system.
  • As a transcription factor, HIF-1α activates the expression of a broad range of downstream targets. Examination of these target in combination with assessing HIF-1α levels provides valuable confirmation of ongoing cellular processes related to hypoxia and oxidative stress.

Measuring Mitochondrial Respiration

Mitochondria are cellular organelles that serve the primary energy factories to support cellular function.   The conversion of metabolites from the TCA cycle into ATP is achieved by the transfer of electrons from these intermediates through the respiratory chain on the mitochondrial inner membrane. Assays to examine mitochondrial respiration provide a direct readout of cellular metabolic activity.

Assessing Mitochondrial Integrity

Mitochondrial integrity is essential for the regulation of cellular energy the regulation of apoptosis.

Measuring Amino Acid Metabolism

Amino acids are the building blocks of cellular proteins and also serve as key nutrients to fuel cellular processes. For example, Glutamine is an important metabolic fuel that helps rapidly proliferating cells meet their demands for ATP. Direct links to CST products available to study amino acid metabolism, including key enzymes like GLS, IDH1, and IDH2 are accessible via the CST Glutamine Metabolism Interactive Pathway.

Measuring mTOR Activity

The mechanistic target of rapamycin (mTOR) is a serine threonine kinase that serves a master regulator of cell growth and metabolism. mTOR integrates signaling from extracellular growth factors, cytokines, and acts as a nutrient sensor to affect multiple cellular processes, including cell proliferation, survival, cell growth and protein translation. mTOR exists in two distinct signaling complexes, termed mTORC1 and mTORC2, that receive signaling inputs primarily via the PI3K/AKT, ERK1/2, and AMPK pathways. Key downstream targets of mTORC1 include p70/S6K and 4E-BP1/2, while mTORC2 transduces signals via SGK1, PKCa, and AKT. Components of mTOR signaling can be queried using reagents from the CST mTOR substrate sampler kit which includes:

 

mTOR

Serine/Threonine Kinase

Phospho-p70 S6 kinase (Thr389)

Downstream effector of mTORC1, Thr389 correlates with kinase activity

Phospho-p70 S6 kinase (Ser371)

Downstream effector of mTORC1, Ser371 correlates with kinase activity

Phospho-4E-BP1 (Thr37/46)

Downstream effector of mTORC1, inhibits cap-dependent translation,Thr37/46 are readouts of mTOR activity

Phospho-mTOR (Ser2448)

mTOR Ser2448 is phosphorylated via PI3K/AKT activation

 

Assessing AMPK Activity- A Master Regulator of Metabolism

AMP-activated protein kinase (AMPK) is master regulator of cellular energy homeostasis that modulates both glucose and lipid metabolism. AMPK phosphorylates downstream targets to regulate glucose metabolism (e.g. PFKFB3, GYS1), lipid metabolism (e.g. HMGR, ACC1, PLD1), transcription (e.g. (HDAC4/5/7, p300, Srebp1), and cell growth/autophagy (e.g. Raptor, ULK1, Becilin-1). Components of AMPK signaling can be queried using reagents from the CST AMPK substrate sampler kit which includes:

 

AMPKa

Serine/Threonine Kinase

Phospho-AMPKa (Thr172)

Serine/Threonine Kinase, phosphorylation at Thr172 is essential for activation

ULK1

Serine/Threonine Kinase and downstream effector of AMPK linked to autophagy

Phospho-ULK1(Ser555)

Phosphorylation of ULK1 by AMPK at Ser555 is critical for starvation-induced autophagy, cell survival under conditions of low nutrients and energy, and mitochondiral homeostasis

Raptor

AMPK substrate and component of mTORC1

Phospho-Raptor (Ser 792)

Ser 792 is phosphorylated by AMPK to inhibit mTORC1

Beclin-1

Downstream effector of AMPK linked to autophagy

Phospho-Beclin-1 (Ser93)

Ser 93 (in humans) is phosphorylated by AMPK induce autophagy. Corresponds to murine Ser 91

 

Measuring the Warburg Effect

The Warburg Effect is a metabolic adaptation of many cancers that promotes cancer cell growth and survival. Specifically, cancer cells tend to favor metabolism via glycolysis over the more efficient and commonly used oxidative phosphorylation pathway even in the presence of oxygen. Several signaling pathways including activation of the PI3K/AKT and RAS contribute to the Warburg effect. In addition, a dimeric form of the pyruvate kinase isoenzyme M2 (PKM2) is thought to play a central role in the Warburg effect. One hallmark of the Warburg effect is increased production of lactate. Key component contributing to the Warburg effect and products to assay their expression and activity can be directly access via the CST Warburg Effect Signaling Pathway.

Assessing Insulin Receptor (IR) Signaling

Insulin, the major hormone controlling cellular energy functions such as glucose and lipid metabolism, acts by binding to and activating the insulin receptor tyrosine kinase.   Receptor activation induces the recruitment of the IRS family of adaptor proteins and downstream activation predominantly through the PI3K/AKT and ERK1/2 pathways to affect numerous cellular processes. Components of insulin receptor signaling can be assayed using reagents in the CST Insulin Receptor Substrate Sampler Kit, which includes:

IRS-1

Insulin Receptor signaling adaptor protein

Phospho-IRS-1(Ser307)

Ser 307 is phosphorylated by JNK and IKK

Phospho-IRS-1(Ser612)

Ser 612 phosphorylation is mediated by the PKC an mTOR pathways

Phospho-IRS-1(Ser318)

Downstream effector of mTORC1, inhibits cap-dependent translation,Thr37/46 are readouts of mTOR activity

IRS-2

Insulin Receptor signaling adaptor protein

 

Additional Chemical-Based Assays to Measure Metabolism

Several additional methods exist to measure the levels and activities of key metabolic processes in cells. These include:

  • The detection of oxidative cofactors including NAD+/NADH and NADP+/NADPH, typically done via colorimetric analysis of cell extracts on a microplate reader
  • Quantification of S-Adenosylmethionine and S-Adenosylhomocysteine, central components of the methylation cycle, by HPLC or colorimetric assay of cell lysates.

Learn more about Metabolism.

Chris S
Chris S
Chris Sumner is the Editor-in-Chief of Lab Expectations. When he's not reading/writing about curing disease, he's hiking in the woods, playing guitar, or searching for the world's best lobster roll.

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