Metabolism Core

Through Washington University’s Diabetes Center, we have access to two Seahorse instruments, the 24 well and 96 well formats, for measurement of mitochondrial respiration and glycolysis. The Seahorse is ideally suited for adherent cells though in some cases can be adapted for use in tubules ex vivo. The Core can provide guidance for experimental design and proper controls to help the user best answer their specific question. There is also expertise at the Core for using the Seahorse on primary proximal tubules.

These assays are designed to assess substrate oxidation using tissue ex vivo and radioactive substrates. As an example, we have used injured kidney tissue to measure fatty acid oxidation using ³H-labeled palmitate. If the palmitate is fully oxidized, then the ³H will be incorporated into ³H₂O, which can be quantified using a scintillation counter. A similar technique can measure glucose/pyruvate oxidation as well.

Substrate-dependent respiration in kidney tissue can be measured using the Oroboros Oxygraph 2K through Washington University’s Nutrition Obesity Research Center (NORC). The Core has optimized methods for measuring respiration in permeabilized kidney tissue.

Analyses involve use of standardized reagents and an optimized sequential multiple substrate-uncoupler-inhibitor titration (SUIT) protocol for analysis of oxidative phosphorylation. Routine respiration is measured after addition of Complex I substrates (glutamate or malate), Complex II substrate (succinate) and ADP (state 3), followed by LEAK/State 4 respiration after addition of oligomycin and uncoupling measurements after added FCCP. Depending upon the project, palmitoylcarnitine or pyruvate may be used as well.

Global profiling of metabolites and lipids will be accomplished by using liquid chromatography/mass spectrometry (LC/MS). The platform can be applied to biofluids such as blood and urine or tissues such as those from the kidney.

This technique is useful to get a snapshot of compounds involved in glycolysis, the TCA cycle, the pentose-phosphate pathway, one-carbon metabolism, amino acid metabolism, fatty acid synthesis and oxidation, complex lipids, ceramides, sphingomyelins, and many more.

The metabolomics experiments will be performed in the Patti laboratory, which houses 20 mass spectrometers for metabolomics. We will use well-established protocols that have been developed by the Patti team over the past 15 years.  Analysis can also be performed by the Patti lab, which has pioneered interpretation of metabolomics data.  

Whereas metabolomics provides information about the relative concentration of metabolites at a comprehensive scale, isotope tracer experiments provide information about the dynamics of a specific pathway of interest. The appropriate isotope tracer (e.g. glucose versus glutamine, uniformly labeled versus positionally labeled) and exact duration will depend upon each project’s goals.

In general, the isotope tracing capabilities will support three unique goals: (i) determining the metabolic flux of a pathway, (ii) mapping the fate of a particular nutrient, and (iii) quantitating how much of a given nutrient is metabolized by each pathway. The Patti laboratory regularly performs in vitro and in vivo isotope tracing using a variety of metabolic substrates.