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

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

Isotope Tracers as Probes

Disadvantages Impractical for screening purposes Requires a large number of animals

                                             

                      

Organ bathTransducer

Printer

Isolated Tissues

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