As obesity is a major risk factor for noninsulin-dependent diabetes mellitus, adipose tissue may generate a mediator that influences the activity of insulin on various target tissues. Recent evidence suggests that a cytokine, tumor necrosis factor-alpha (TNF alpha), may serve this role. This study investigates whether the expression of TNF alpha and its receptors is modulated during drug treatment to reduce insulin resistance. The effects of moderate weight loss by dietary restriction were also examined. We show here that a marked induction of TNF alpha mRNA occurs in adipose tissues from a mouse model of obesity-linked diabetes (KKAy) compared to that in nondiabetic mice (C57). Likewise, RNA transcripts encoding TNF R2 receptors (p75) were significantly increased in fat tissues of the obese diabetic animals. In muscle from these diabetic animals, RNA transcripts encoding both TNF R1 (p55) and R2 were significantly elevated, although R2 transcript abundance was less elevated than in fat. We also observed that the overexpression of mRNA for TNF alpha and both of its receptors could be at least partly normalized by treatment of the diabetic animals with the insulin-sensitizing agent pioglitazone. Treating of the obese diabetic animals by food restriction reduced the expression of mRNA for TNF R2 in muscle, but not fat. These results clearly indicate that gene expression for the TNF systems can be regulated by an insulin-sensitizing drug and reduction of body weight. Such findings support a role for this cytokine in the insulin-resistant diabetic state and show its modulation by therapies that reverse the disorder.
Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.
Protein tyrosine phosphatases (PTPs) constitute a diverse family of enzymes that, together with protein tyrosine kinases, control the level of intracellular tyrosine phosphorylation, thus regulating many cellular functions. PTP1B negatively regulates insulin signaling, in part, by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor, thereby attenuating receptor kinase activity. Inhibitors of PTP1B would therefore have the potential of prolonging the phosphorylated (activated) state of the insulin receptor and are anticipated to be a novel treatment of the insulin resistance characteristic of type 2 diabetes. We previously reported a series of small molecular weight peptidomimetics as competitive inhibitors of PTP1B, with the most active analogues having K(i) values in the low nanomolar range. Furthermore, we confirmed that the O-carboxymethyl salicylic acid moiety is a remarkably effective novel phosphotyrosine mimetic. Because of the low cell permeability of this compound class, it was important to investigate the possibility of replacing one or both of the remaining carboxyl groups while maintaining PTP1B inhibitory activity. The analogues described herein further support the importance of an acidic functionality at both positions of the tyrosine head moiety. An important discovery was the ortho tetrazole analogue 29 (K(i) = 2.0 microM), which was equipotent to the dicarboxylic acid analogue 2 (K(i) = 2.0 microM). Solution of the X-ray cocrystal structure of the ortho tetrazole analogue 29 bound to PTP1B revealed that the tetrazole moiety is well-accommodated in the active site and binds in a fashion similar to the ortho carboxylate analogue 2 reported previously. This novel monocarboxylic acid analogue revealed significantly higher Caco-2 cell permeability as compared to all previous compounds. Furthermore, compound 29 exhibited modest enhancement of insulin-stimulated 2-deoxyglucose uptake by L6 myocytes.
Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling in part by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor (IR), thereby attenuating receptor tyrosine kinase activity. Inhibition of PTP1B is therefore anticipated to improve insulin resistance and has recently become the focus of discovery efforts aimed at identifying new drugs to treat type II diabetes. We previously reported that the tripeptide Ac-Asp-Tyr(SO(3)H)-Nle-NH(2) is a surprisingly effective inhibitor of PTP1B (K(i) = 5 microM). With the goal of improving the stability and potency of this lead, as well as attenuating its peptidic character, an analogue program was undertaken. Specific elements of the initial phase of this program included replacement of the N- and C-termini with non-amino acid components, modification of the tyrosine subunit, and replacement of the tyrosine sulfate with other potential phosphate mimics. The most potent analogue arising from this effort was triacid 71, which inhibits PTP1B competitively with a K(i) = 0.22 microM without inhibiting SHP-2 or LAR at concentrations up to 100 microM. Overall, the inhibitors generated in this work showed little or no enhancement of insulin signaling in cellular assays. However, potential prodrug triester 70 did induce enhancements in 2-deoxyglucose uptake into two different cell lines with concomitant augmentation of the tyrosine phosphorylation levels of insulin-signaling molecules. Key elements of the overall SAR reported herein include confirmation of the effectiveness and remarkable PTP1B-specificity of the novel tyrosine phosphate bioisostere, O-carboxymethyl salicylic acid; demonstration that the tyrosine skeleton is optimal relative to closely related structures; replacement of the p-1 aspartic acid with phenylalanine with little effect on activity; and demonstration that inhibitory activity can be maintained in the absence of an N-terminal carboxylic acid. An X-ray cocrystal structure of an analogue bearing a neutral N-terminus (69) bound to PTP1B is reported that confirms a mode of binding similar to that of peptidic substrates.
The regulation of hexokinase II (HKII) was examined in fat and skeletal muscle of an animal model of non-insulin-dependent diabetes mellitus, the KKAY mouse. These tissues require insulin for facilitated transport of glucose and express the insulin-responsive transporter GLUT4. The combined data from two experiments (n = 12 for each experimental condition) demonstrated mean concentrations of plasma insulin in pmol/l and glucose in mmol/l of 122 and 7.2 (control nondiabetic C57 mouse) vs. 1,118 and 29.6 (diabetic mouse), respectively. The tissues of diabetic mice compared with control mice demonstrated a reduction of HKII mRNA abundance of 68% in epididymal fat (P = 0.0001) and 34% in the quadriceps muscles (P < 0.001), with concordant reduction in the abundance of GLUT4 mRNA of 60% in epididymal fat (P < 0.001). In comparison with the results in untreated diabetic mice, diabetic animals treated with the insulin-sensitizing drug pioglitazone demonstrated an increase in the abundance of HKII mRNA with a concordant increase of GLUT4 mRNA in epididymal fat (P = 0.03 and < 0.01, respectively), and an increase of HKII mRNA in the quadriceps muscles (P < 0.05). Separate experiments demonstrated a reduction of HKII protein abundance by 61% in epididymal fat (P < 0.001, n = 12 for each experimental condition) and by 71% in the quadriceps muscles (P < 0.001, n = 6 for each experimental condition). In comparison with untreated diabetic mice, there was an increase in the abundance of HKII protein in epididymal fat of animals treated with pioglitazone (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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