Experimental strategies in the study of Metabolism

goals Identify components in a reaction Understand regulation of reactions at the tissue of origin Identify physiological function of each reaction pathway Understand the control mechanisms in a pathway

Levels of organization by which metabolism can be studied Whole organism Isolated or perfused organs Isolated cells Cell-free systems Purified components Metabolic probes

whole animal studies Measures food and water intake (may be radiolabled precursors Measures metabolites from urine output/blood samples (biochemical assays) Metabolic cage

Isotope Tracers as Probes Substrates labeled with an isotopic form of some element can be fed to cells or whole organism and used to elucidate metabolic sequences Radioactive isotopes: 14C, 3H, 31P Stable ‘heavy’ isotopes: 18O, 15N

Disadvantages Impractical for screening purposes Requires a large number of animals

Isolated Tissues                                                                      Transducer Organ bath Printer

Isolated tissues Tissues are isolated from a newly sacrificed animal (guinea pig, rabbit, rats or mice) Proteins expressed by the cells in the tissues is similar to the proteins of the same tissues in the intact animal Effects observed in tissue or isolated organ assays may be predictive of effects in vivo

Cardiovascular pharmacology Isolated organ studies Isolated perfused rabbit heart Isolated auricles Isolated aortic rings

Limitations of the assay The value of the observed responses can be limited by the inability of an isolated tissue to react with other reciprocating organ-body systems (isolated tissues/organs excludes the effects of gastrointestinal system which influences the absorption of the test substance in vivo)

isolated cells as test system Suspension of cells or culture of cells derived from animal tissues or from continuously replicating cells lines.

Advantages of isolated cells Test substance can be examined in direct contact with the cell type of interest The concentration of of test substance can be rigorously controlled The secondary effects of extracellular influences such as metabolic factors , matrix and cell to cell contact can be excluded or controlled

Disadvantages Isolated cells may undergo mutation when kept for a long term growth All cell types do not respond similarly to a single test substance even if the cells originate from the cell organ

Cell free systems

Purified components Isolate enzyme from a specific pathway Allow reaction to proceed in the presence of substrate Duplicate a particular reaction in a pathway Reaction limited to those that do not require cellular structure

Metabolic probes Metabolic inhibitors- help to identify role of a reaction Mutation – identify the role of genes/ proteins in metabolism

Transgenic animal technology Animal whose genome has been modified -insertion of a new gene (“knock-in gene”) - target mutation of functional gene (‘knock-out genes”)c The objective is to produce animals which possess the transferred gene in their germ cells (sperm or ova). Such animals are able to act as "founder" stock to produce many offspring that carry a desirable gene or genes.

Applications Nude mice production for in vivo tumorigenicity

Pronuclear microinjection Pronuclear microinjection. Genetic material is injected directly into the fertilized egg which is then implanted back into a mouse and allowed to come to term.

Pronuclear microinjection Pronuclear microinjection. Genetic material is injected directly into the fertilized egg which is then implanted back into a mouse and allowed to come to term.