The potentially enhanced mitogenic activity of insulin analogs represents a safety risk that requires detailed analysis of new analogs considered for therapeutic applications. We assessed the signaling properties and mitogenic potency of two novel rapid-acting insulin analogs, Lys Insulin therapy of diabetic patients aims to achieve tight blood glucose control to reduce the progression of long-term complications (1). However, the pharmacokinetic characteristics of currently available insulin preparations are unable to mimic the pattern of endogenous insulin secretion and make it impossible to achieve sustained normoglycemia (2). Great efforts have been made to develop novel insulin molecules with altered pharmacodynamic characteristics that might lead to improved glycemic control using recombinant DNA technology (rev. in 3-5). One limiting factor is the slow absorption of conventional unmodified insulin from subcutaneous tissues because of the slow dissociation rate of hexameric insulin complexes into monomers at the injection site (6,7). Modification of the B26ϪB30 region of the insulin molecule, particularly substitution of amino acids with charged residues at the association sites, allows the production of a range of insulin analogs with reduced selfassociation that exhibit no profound perturbations of insulin receptor recognition (4,8). This has been demonstrated for insulin analogs such as Lispro (Lys B28 ,Pro B29 ) insulin and insulin aspart (Asp B28 insulin), two rapidacting insulins that are in clinical use and improve postprandial glycemic control (3,5).A major concern related to the long-term use of insulin analogs stems from the observation that modification of the insulin molecule in the B10 and B26ϪB30 regions alters the affinity for the IGF-I receptor more than for the insulin receptor and may lead to enhanced mitogenic activity of these analogs (9). This potential safety risk was first recognized for the analog Asp B10 insulin, which was found to exhibit a tumor-promoting activity in SD rats (10) and to induce a profound mitogenic effect in many cell systems (11-13). The enhanced mitogenic signaling profile of an insulin analog may result from 1) an increased affinity toward the IGF-I receptor, resulting in augmented IGF-I receptor signaling (9); 2) the so-called time-dependent specificity that describes a distinct correlation between the mitogenic potential and the occupancy time at the insulin receptor for a given insulin analog (14); and 3) a combination of both IGF-I and insulin receptorϪ mediated processes. Most recent data suggest that the mitogenic properties correlate better with the IGF-I recep-

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Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.

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We have recently shown that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of actin microfilaments in rat cardiomyocytes, and that inhibition of 12-lipoxygenase abrogates insulin-stimulated GLUT4 translocation in these cells. In the present study, we used mice that were null for the leukocyte 12/15-lipoxygenase to explore the implications of this enzyme for insulin action under IN VIVO conditions. Insulin induced a profound reduction in blood glucose in both control and knockout mice. However, significantly higher serum insulin levels were observed in these animals. GLUT4 expression in heart and skeletal muscle was unaffected in KO mice. Insulin-regulated serine phosphorylation of Akt and GSK3alpha and GSK3beta was unaltered in heart and skeletal muscle of knockout mice, suggesting unaltered insulin signaling. Fractionation of hind limb muscles showed that insulin had induced a prominent translocation of GLUT4 to skeletal muscle plasma membranes in control mice. However, this response was largely reduced in knockout animals. Our data show that the lack of leukocyte 12/15-lipoxygenase does not lead to the development of an insulin-resistant phenotype. However, perturbation of GLUT4 translocation in skeletal muscle of knockout mice may indicate latent insulin resistance, and supports our hypothesis that eicosanoids are involved in insulin-mediated regulation of muscle glucose transport.