Insulin target tissues and cells.

Rodent adipose tissue and cells represent the targets exhibiting the most prominent insulin sensitivity (i.e., lowest EC/IC50) and responsiveness (i.e., highest fold stimulation/inhibition above basal) of the relevant insulin signaling cascades (e.g., insulin receptor activation) and metabolic end effector systems (e.g., lipolysis) in comparison to liver (e.g., gluconeogenesis) and muscle cells (e.g., glucose transport). This might be based in part on technical advantages of the adipose tissue/adipocyte preparation in comparison to that of muscle/myocytes. But more likely, it reflects the exquisite physiological role of the adipose tissue in the regulation and coordination of glucose and lipid metabolism, i.e., insulin stimulation of lipid synthesis (lipogenesis) and insulin inhibition of lipolysis. On the basis of their relatively easy technical preparation, functional adipose tissue fragments (epididymal fat pads) and primary adipocytes (isolated epididymal adipocytes) from rats as well as adipocyte cell lines derived from mice (3T3-L1, F442A) are the first choice for the development of robust and reliable cell-/tissue-based assay systems for insulin-like activity

Assays for insulin and insulin-like activity based on adipocytes.

Data from the metabolic assays (and signaling assays; see below) are calculated as stimulation factor above basal activity (absence of insulin/compound/drug candidate) for processes stimulated (e.g., lipogenesis, glucose transport, and GLUT4 translocation) or as difference between the basal and insulin/compound/drug candidate-induced values for processes downregulated (e.g., lipolysis). In each case, these data, which reflect the responsiveness of the metabolic effector system studied toward the respective stimulus (insulin/compound/drug candidate), are normalized to the basal (set at 0 %) and maximal insulin action (set at 100 %; elicited by maximally effective concentration of insulin). For characterization of the sensitivity of the metabolic effector system toward the respective stimulus, effective concentrations for the induction of 150 % (or higher) of the basal activity (set at 100 %) can be given. These so-called EC150-values facilitate the insulin-independent comparison of the relative potency of the insulin-like activity between compounds/drug candidates, in general, and in particular for those frequently observed stimuli, which do not elicit the same maximal response in % stimulation or inhibition and/or fail to approach the maximal insulin response

Assays for insulin and insulin-like metabolic activity based on hepatocytes, myocytes and diaphragms.

Despite the eminent importance of studies with primary and cultured adipocytes or adipose tissues on the basis of their physiological role in the regulation of lipid and carbohydrate metabolism in humans in combination with the relative low expenditure in preparing adipocytes of high quality and number, compounds and drug candidates for future antidiabetic and antiobesity therapy have to be analyzed for their effects in primary and cultured hepatocytes and myocytes or liver and muscle tissues, too. In principle, the majority of the assays described above for adipocytes can be adapted for the use with hepatocytes and myocytes. However, the following selection takes into account the relative contribution of each process monitored to its role in the whole-body regulation of intermediary metabolism in the normal and disease state. Moreover, technical aspects, such as requirement for a special equipment and applicability in throughput screening assays for drug discovery, were additional criteria