07-07-2012, 09:53 AM
Activation of Insulin Signal Transduction Pathway and Anti-diabetic Activity of Small Molecule Insulin Receptor Activators
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We recently described the identification of a non-peptidyl
fungal metabolite (L-783,281, compound 1), which
induced activation of human insulin receptor (IR) tyrosine
kinase and mediated insulin-like effects in cells, as
well as decreased blood glucose levels in murine models
of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski,
D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T.,
Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P.,
Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. &
Moller, D. E. (1999) Science 284, 974–977). Here we report
the characterization of an active analog (compound 2)
with enhanced IR kinase activation potency and selectivity
over related receptors (insulin-like growth factor
I receptor, epidermal growth factor receptor, and platelet-
derived growth factor receptor). The IR activators
stimulated tyrosine kinase activity of partially purified
native IR and recombinant IR tyrosine kinase domain.
Administration of the IR activators to mice was associated
with increased IR tyrosine kinase activity in liver.
In vivo oral treatment with compound 2 resulted in significant
glucose lowering in several rodent models of
diabetes. In db/db mice, oral administration of compound
2 elicited significant correction of hyperglycemia.
In a streptozotocin-induced diabetic mouse model.
EXPERIMENTAL PROCEDURES
Materials—All biological reagents used here were obtained from
commercial sources. Radiochemical were purchased from PerkinElmer
Life Sciences. Compound 1 (L-783,281) was prepared as described previously
(20). Compounds 2 and 3 were prepared synthetically as described
previously (21). Compound stock solutions were prepared in
100% dimethyl sulfoxide (Me2SO) and diluted in appropriate media just
prior to use.
Animals—Male db/db and db/1 mice were from Jackson Laboratories.
Male Harlan Sprague-Dawley rats were from Charles River Breeding
Laboratories, Inc. Animals were allowed access to standard rodent
chow and water ad libitum. Animals were treated orally (by gavage)
with vehicle or test compounds. Blood glucose level was measured using
One TouchTM Glucometer (Lifescan) or Accu-ChekTM blood glucose
monitoring system (Roche Molecular Biochemicals). Plasma glucose
concentration was measured using a glucose oxidase kit (Sigma).
Plasma insulin concentration was measured with a radioimmunoassay
kit (Linco). Animal care was in accordance with institutional guidelines.
RESULTS
Activation of Insulin Receptor Tyrosine Kinase and Signal
Transduction in CHO.T Cells—To identify a more potent and
selective analog of L-783,281 (compound 1) (Table I) derivatives
of this compound were synthesized and tested in a cell based
assay that monitors activation of IR tyrosine kinase activity in
CHO cells expressing human insulin receptor (CHO.IR) (20).
One of these derivatives (compound 2) increased the insulin
receptor tyrosine kinase activity in these cells with an EC50 of
300 nM, reflecting a greater than 10-fold improvement in the
potency compared with compound 1 (Table I).
DISCUSSION
The pathogenesis of Type 2 diabetes is complex, involving the
progressive development of insulin resistance and a relative
deficiency in insulin secretion, leading to overt hyperglycemia.
The molecular basis for insulin resistance in Type II diabetes is
not fully understood. However, abnormalities in insulin receptor
expression, structure (rarely), and function are present in
association with chronic insulin resistance and diabetes. Thus,
it has been described that patients with obesity and diabetes
have impaired insulin receptor binding in liver, muscle, and
adipose tissue (13–15, 29). The defect is exacerbated by additive
defect in insulin receptor kinase that is also present in
tissues of patients with overt diabetes (14, 29, 30). More recently,
defects in the insulin receptor-mediated signal transduction
pathway, including IRS-1 phosphorylation and PI 3-kinase
activation, have been found in tissues from Type 2
diabetic patients or rodent models (16, 31, 32). Thus, augmenting
insulin signaling by targeting insulin receptor activation
may represent a potential approach to alleviate insulin resistance
and improve glucose homeostasis in Type 2 diabetes.