Glucokinase (GK) plays a critical role for maintaining glucose homeostasis with regulating glucose uptake in liver and insulin secretion in pancreas. GK activators have been reported to decrease blood glucose levels in patients with type 2 diabetes mellitus. However, clinical development of GK activators has failed due to the loss of glucose-lowering effects and increased plasma triglyceride levels after chronic treatment. Here, we generated a novel GK activator, TMG-123, examined its in vitro and in vivo pharmacological characteristics, and evaluated its risks of aforementioned clinical issues. TMG-123 selectively activated GK enzyme activity without increasing Vmax. TMG-123 improved glucose tolerance without increasing plasma insulin levels in both insulin-deficient (Goto-Kakizaki rats) and insulin-resistant (db/db mice) models. The beneficial effect on glucose tolerance was greater than results observed with clinically available antidiabetic drugs such as metformin and glibenclamide in Zucker Diabetic Fatty rats. TMG-123 also improved glucose tolerance in combination with metformin. After 4 weeks of administration, TMG-123 reduced the Hemoglobin A1c levels without affecting liver and plasma triglyceride levels in Goto-Kakizaki rats and Diet-Induced Obesity mice. Moreover, TMG-123 sustained its effect on Hemoglobin A1c levels even after 24 weeks of administration without affecting triglycerides. Taken together, these data indicate that TMG-123 exerts glucose-lowering effects in both insulin-deficient and -resistant diabetes, and sustains reduced Hemoglobin A1c levels without affecting hepatic and plasma triglycerides even after chronic treatment. Therefore, TMG-123 is expected to be an antidiabetic drug that overcomes the concerns previously reported with other GK activators.
objective: This study was designed to examine the effect of peroxisome proliferator-activated receptor-α (PPAR-α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR-α ligands (Wy-14,643 and fenofibrate) were added to 3T3-L1 adipocytes, RAW264 macrophages, or co-culture of 3T3-L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) mRNA expression and secretion were examined. PPAR-α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR-α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR-α-deficient mice. Results: In the co-culture of 3T3-L1 adipocytes and RAW264 macrophages, PPAR-α ligands reduced MCP-1 and TNF-α mRNA expression and secretion in vitro relative to vehicle-treated group. The anti-inflammatory effect of Wy-14,643 was observed in adipocytes treated with macrophage-conditioned media or mouse recombinant TNF-α and in macrophages treated with adipocyte-conditioned media or palmitate. Systemic administration of PPAR-α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy-14,643 also exerted an anti-inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR-α-deficient mice. Discussion: This study provides evidence for the anti-inflammatory effect of PPAR-α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity-related metabolic syndrome.
The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser 603 residue (site 3) is considered to be a pivotal site targeted by Ca 2؉ /calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser 603 residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKIIinvolved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser 603 without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser 603 -synapsin I (pS603-Syn I-Ab) and phospho-Thr 286/287 -CaMKII. The bradykinin-evoked phosphorylation of Ser 603 was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser 603 kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca 2؉ /calmodulin-independent Ser 603 kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated 32 P into synapsin I in a Cdc42/ GTP␥S-dependent manner, and its phosphorylation site was confirmed as Ser 603 using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser 603 residue in the presence of Cdc42/GTP␥S. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat ␣PAK (PAK1) in PC12 cells evokes the phosphorylation of Ser 603 even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative ␣PAK quenches the phosphorylation. These results raise the possibility that Ser 603 on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.
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