Role of insulin resistance in the pathogenesis and development of type 2 diabetes in WBN/Kob-Lepr(fa) rats.

WBN/Kob-Lepr(fa) (fa/fa) rats have been identified as a new animal model of type 2 diabetes (T2DM), as they are characterized by impaired pancreatic insulin secretion and severe insulin resistance. Our previous study demonstrated impaired insulin secretion and its involvement in hyperglycemia in fa/fa rats. The present study was aimed at elucidating the role of insulin resistance in the development and progression of diabetes in these animals. Troglitazone (TGZ) was used as an insulin sensitizer. Insulin resistance and insulin secretory capacity were measured by a homeostasis model assessment of insulin resistance and the area under the blood concentration-time curve for plasma insulin levels after intravenous glucose tolerance testing, respectively. The fa/fa rats exhibited marked insulin resistance between 5 and 11 weeks of age, compared with age-matched Wistar rats. The insulin secretory capacity of fa/fa rats was higher than that of Wistar rats at 5 weeks of age, but decreased by 50% between 9 and 11 weeks of age. The fa/fa rats were fed a standard diet, with or without 0.2% w/w TGZ, for 4 weeks. Treatment with TGZ significantly improved insulin resistance, hyperglycemia and hypertriglyceridemia in both prophylactic and therapeutic study groups. These results suggest that insulin resistance is markedly involved in the development and progression of T2DM in fa/fa rats.

Absorption, elimination, and metabolism of CS-1036, a novel α-amylase inhibitor in rats and monkeys, and the relationship between gastrointestinal distribution and suppression of glucose absorption.

The absorption, metabolism, and excretion of (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-d-glucopyranosyl)-α-d-glucopyranoside (CS-1036), a novel and potent pancreatic and salivary α-amylase inhibitor, were evaluated in F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of CS-1036 were low (2.67-3.44 ml/min/kg and 0.218-0.237 l/kg for rats and 2.25-2.84 ml/min/kg and 0.217-0.271 l/kg for monkeys). After intravenous administration of [(14)C]CS-1036 to rats and monkeys, radioactivity was mainly excreted into urine (77.2% for rats and 81.1% for monkeys). After oral administration, most of the radioactivity was recovered from feces (80.28% for rats and 88.13% for monkeys) with a low oral bioavailability (1.73-2.44% for rats and 0.983-1.20% for monkeys). In rats, intestinal secretion is suggested to be involved in the fecal excretion as a minor component because fecal excretion after intravenous administration was observed (15.66%) and biliary excretion was almost negligible. Although intestinal flora was involved in CS-1036 metabolism, CS-1036 was the main component in feces (70.3% for rats and 48.7% for monkeys) and in the intestinal contents (33-68% for rats up to 2 hours after the dose) after oral administration. In Zucker diabetic fatty rats, CS-1036 showed a suppressive effect on plasma glucose elevation after starch loading with a 50% effective dose at 0.015 mg/kg. In summary, CS-1036 showed optimal pharmacokinetic profiles: low oral absorption and favorable stability in gastrointestinal lumen, resulting in suppression of postprandial hyperglycemia by α-amylase inhibition.

Plasma profiles of glucose, insulin and lipids in the male WBN/Kob-Lepr(fa) rat, a new model of type 2 diabetes with obesity.

Plasma profiles of glucose, insulin and lipids were examined in the male WBN/Kob-Lepr(fa) (fa/fa) rat, a new model of type 2 diabetes (T2D), in comparison with age-matched original male WBN/Kob (lean) rats. The fa/fa rats developed hypertriglycemia, obesity and hyperglycemia from 5, 7, and 9 weeks of age, respectively. Plasma insulin levels in fa/fa rats were significantly higher than those in lean rats at 5 weeks of age, but after 11 weeks of age gradually declined to the levels in lean rats. HOMA-IR, a measure of insulin resistance status, showed that fa/fa rats had insulin resistance. The fa/fa rat has the potential to become an important animal model of T2D with obesity.

Contributions of hepatic gluconeogenesis suppression and compensative glycogenolysis on the glucose-lowering effect of CS-917, a fructose 1,6-bisphosphatase inhibitor, in non-obese type 2 diabetes Goto-Kakizaki rats.

Contributions of gluconeogenesis suppression in liver, kidney, and intestine as major gluconeogenic organs to the glucose-lowering effect of CS-917, a fructose 1,6-bisphosphatase inhibitor, was evaluated in overnight-fasted Goto-Kakizaki (GK) rats. CS-917 decreased plasma glucose by suppressing glucose release and lactate uptake from liver but not from kidney and intestine. These results suggest that hepatic gluconeogenesis suppression predominantly contributes to the glucose-lowering effect of CS-917 in GK rats. Moreover, the mechanism by which CS-917 decreased plasma glucose more in overnight-fasted GK rats than in non-fasted ones was investigated. Lactate uptake from liver was suppressed by 15 mg/kg of CS-917 in both states, but glucose release from liver and plasma glucose were decreased only in the overnight-fasted state. CS-917 at 30 mg/kg decreased hepatic glycogen content in both states and depleted it in the overnight-fasted state. In the non-fasted GK rats, co-administration of CS-917 with CP-91149, a glycogen phosphorylase inhibitor, suppressed hepatic glycogen reduction by CS-917 and decreased plasma glucose more than single administration of CS-917. These results suggest that gluconeogenesis suppression by CS-917 was counteracted by hepatic glycogenolysis especially in the non-fasted state and that combination therapy with CS-917 and CP-91149 is efficacious to decrease plasma glucose in GK rats.

Dynamic changes of adiponectin and S100A8 levels by the selective peroxisome proliferator-activated receptor-gamma agonist rivoglitazone.

OBJECTIVE: Accumulating evidence indicates that the regimen to increase adiponectin will provide a novel therapeutic strategy for metabolic syndrome. Here, we tested the effect of a potent and selective peroxisome proliferator-activated receptor-γ agonist, rivoglitazone (Rivo), a newly synthesized thiazolidinedione derivative, on adiponectin, insulin resistance, and atherosclerosis. METHODS AND RESULTS: ob/ob mice, apolipoprotein E knockout (apoE KO) mice, and apoE and adiponectin double knockout mice were administered pioglitazone, Rivo, or no compound. Remarkable elevation of plasma adiponectin was observed, especially in Rivo-treated ob/ob mice. Rivo ameliorated insulin resistance in ob/ob mice and reduced atherosclerotic areas in apoE KO mice compared with the pioglitazone group but failed to decrease atherosclerotic areas in double knockout mice. Among adipose mRNAs, adipose S100A8, which activates Toll-like receptor 4-dependent signal transduction cascades and locates upstream of inflammation, was markedly increased in ob/ob mice, and its increase was completely reversed by Rivo treatment. In RAW264.7 macrophage cells and 3T3-L1 adipocytes, Rivo significantly reduced S100A8 mRNA levels. CONCLUSIONS: The peroxisome proliferator-activated receptor-γ agonist Rivo remarkably enhanced adiponectin in plasma and decreased adipose S100A8 mRNA levels in obese mice. Rivo treatment apparently ameliorated insulin resistance in ob/ob mice and reduced atherosclerosis in apoE KO mice, partly through adiponectin.

Discovery of potent and orally active tricyclic-based FBPase inhibitors.

With the aim of exploring the effect of tricyclic-based FBPase inhibitors in cells and in vivo, a series of prodrugs of tricyclic phosphonates was designed and synthesized. Introducing prodrug moieties into tricyclic-based phosphonates led to the discovery of prodrug 15c, which strongly inhibited glucose production in monkey hepatocytes. Furthermore, prodrug 15c lowered blood glucose levels in fasted cynomolgus monkeys.

A prodrug approach towards the development of tricyclic-based FBPase inhibitors.

For the purpose of reducing the strong CYP3A4 inhibitory potency of diamide prodrug 4, cyclic prodrugs of tricyclic-based FBPase inhibitors were synthesized. Extensive SAR studies led to the discovery of pyridine-containing cyclic prodrug 20, which strongly inhibited glucose production in monkey hepatocytes and also showed weak CYP3A4 inhibitory potency.

Metformin primarily decreases plasma glucose not by gluconeogenesis suppression but by activating glucose utilization in a non-obese type 2 diabetes Goto-Kakizaki rats.

Metformin is an anti-diabetic agent that has been reported to decrease plasma glucose by multiple mechanisms, such as decreasing hepatic glucose production and activating peripheral glucose utilization. In order to elucidate the primary glucose-lowering mechanism of metformin, the present study focused on a comparison of the acute effect between metformin and CS-917 as a direct gluconeogenesis inhibitor. We examined the effect of metformin and CS-917 on glucose turnover in intravenous glucose-loaded Goto-Kakizaki (GK) rats, and on gluconeogenesis and glucose utilization in rat hepatocytes. Moreover, the glucose-lowering effect of metformin and CS-917 was compared in a fed and a fasted state in GK rats. In intravenous glucose-loaded GK rats, metformin and CS-917 lowered plasma glucose by increasing the glucose disappearance rate and by decreasing the glucose appearance rate, respectively. In rat hepatocytes, CS-917 but not metformin suppressed gluconeogenesis (IC(50)=0.136microM). Instead, metformin dose-dependently increased glucose uptake and the following lactate production at 30 to 100microM. Metformin decreased plasma glucose more in a fed state than in a fasted state in GK rats. CS-917, however, decreased plasma glucose more in a fasted state. These results confirm that metformin primarily decreases plasma glucose not by gluconeogenesis inhibition but by activating glucose utilization in GK rats. Moreover, metformin and CS-917 have different glucose-lowering effects depending on the nutrient state, which may be related to differences in their mechanisms of action. Such differences in action may have implications for metformin and CS-917 in the treatment of type 2 diabetes patients.

CS-917, a fructose 1,6-bisphosphatase inhibitor, improves postprandial hyperglycemia after meal loading in non-obese type 2 diabetic Goto-Kakizaki rats.

Postprandial hyperglycemia is one of the features of type 2 diabetes. Increased hepatic gluconeogenesis is a predominant cause of postprandial hyperglycemia in type 2 diabetes. In this study, we evaluated the effect of gluconeogenesis inhibition on postprandial hyperglycemia using CS-917, a novel inhibitor of fructose 1,6-bisphphosphatase (FBPase) which is one of the rate-limiting enzymes of gluconeogenesis. The suppressive effect of CS-917 on postprandial hyperglycemia was evaluated in a meal loading test in Goto-Kakizaki (GK) rats, non-obese type 2 diabetic animal model characterized by impaired insulin secretion. In addition, we describe acute effect of CS-917 on fasting hyperglycemia in overnight-fasted GK rats and chronic effect of CS-917 in multiple dosing GK rats.CS-917 suppressed plasma glucose elevation after meal loading in a dose-dependent manner at doses ranging from 10 to 40 mg/kg. In an overnight-fasted state, CS-917 decreased the plasma glucose levels dose-dependently at doses ranging from 2.5 to 40 mg/kg. Consistent with the inhibition of FBPase, glucose-lowering was associated with an accumulation of hepatic d-fructose 1,6-bisphosphate and a reduction in hepatic d-fructose 6-phosphate. Chronic treatment of CS-917 decreased plasma glucose significantly, and no significant increase in plasma lactate and no profound elevation in plasma triglycerides were observed by both acute and chronic treatment of CS-917 in GK rats.These findings suggest that enhanced gluconeogenesis contributes to hyperglycemia in postprandial conditions as well as in fasting conditions, and that CS-917 as an FBPase inhibitor corrects postprandial hyperglycemia as well as fasting hyperglycemia.

Pressure effects on the heat-induced aggregation of equine serum albumin by FT-IR spectroscopic study: Secondary structure, kinetic and thermodynamic properties.

Pressure can restrain the heat-induced aggregation and dissociate the heat-induced aggregates. We investigated the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by Fourier transform infrared spectroscopy. The results suggest that the alpha-helical structure collapses at the beginning of heat-induced aggregation, then the rearrangement of structure from partially unfolded structure to the intermolecular beta-sheet takes place through the activated state. We determined the activation volume for the heat-induced aggregation (DeltaV( not equal)=+92+/-8 ml mol(-1)) and the partial molar volume difference between native state and heat-induced aggregates (DeltaV(N-->HA)=+32 ml mol(-1)). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular beta-sheet is unfavorable under high pressure. We also determined the free energy profile of ESA. This energy profile explains the restriction of the formation of heat-induced aggregates by pressure. These results explain the structural differences between heat-induced aggregates with intermolecular beta-sheet and pressure-induced aggregates without intermolecular beta-sheet.

The secondary structure of pressure- and temperature-induced aggregates of equine serum albumin studied by FT-IR spectroscopy.

The protein aggregation is divided into amyloid fibrils and amorphous aggregates. Amyloid fibrils are composed of the 3-dimensional ordered structure and are bound to thioflavin T and Congo red dyes. The amorphous aggregates with the disordered structure do not bind to these dyes. We have investigated the pressure- and heat-induced aggregates of equine serum albumin (ESA) from the secondary structural viewpoint using FT-IR spectroscopy. We show the secondary structural differences between heat- and pressure-induced aggregates of ESA. The heat-induced irreversible aggregates of ESA are composed of the intermolecular beta-sheet structure without binding thioflavie T and Congo red to be amorphous form. On the other hand, the pressure-induced reversible aggregates are composed of the random structure to be also amorphous form. From the comparison of pressure effects on ESA in native and reducing conditions of disulfide bridges, we demonstrate that the restriction of structural flexibility by disulfide bridges is an important factor for the reversibility of the pressure-induced aggregation.

Cold denaturation of ubiquitin at high pressure.

Cold-induced conformational transition of ubiquitin was studied at pH 4.5 under a constant pressure of 2 kbar using variable pressure one-dimensional 1H and two-dimensional 15N/1H NMR spectroscopy as well as IR spectroscopy. Although a tendency for preferential stabilization of a peculiar locally disordered and partially hydrated conformer I, identical with that previously found with variable-pressure NMR at 0 degrees C, is recognized, the transition of the folded conformer N to the unfolded conformer U occurs largely cooperatively with decreasing temperature, reaching near completion at - 21 degrees C. NMR spectral features as well as the analysis of NMR relaxation parameters indicate that the polypeptide chain is almost fully unfolded, fairly well-hydrated and floppy at - 21 degrees C, whereas the IR spectrum shows a substantial decrease of the beta-sheet. The Gibbs energy change from the folded state (a mixture of N and I) to the unfolded state at 2 kbar obtained from the 1H NMR data is fitted well with a single DeltaCp value of 2.43 +/- 0.13 (kJ/K mol) for the entire temperature range between - 21 and 90 degrees C, covering both the cold denaturation and heat denaturation, showing that the two denatured states actually belong to a single thermodynamic phase of the protein. The DeltaCp value determined at 2 kbar is substantially smaller than the DeltaCp determined at 1 bar (3.8-5.8 (kJ/Kmol), which is consistent with the fact that the denaturation takes place from a mixture of N and I at 2 kbar rather than from pure N at 1 bar.

Identification of multiple serine racemase (SRR) mRNA isoforms and genetic analyses of SRR and DAO in schizophrenia and D-serine levels.

BACKGROUND: We previously reported a reduction in serum levels of D-serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA) receptor, in schizophrenia, supporting the hypofunction hypothesis of NMDA neurotransmission in schizophrenia. In this study, we examined the genetic roles of serine racemase (SRR), an enzyme catalyzing the formation of D-serine from L-serine, and D-amino-acid oxidase (DAO) in the susceptibility to schizophrenia and the regulation of serum D-serine levels. METHODS: We determined the complete cDNA and genomic structures of SRR and performed mutation screening. Single nucleotide polymorphisms (SNPs) in SRR and DAO were tested for their association with schizophrenia in both case-control and family-based designs and for correlation with serum levels of D-serine. RESULTS: Genomic analyses revealed that human brain SRR transcripts consist of four isoforms with one major species, which were derived from alternative use of various 5' end exons. Genetic association analyses showed no significant association between SRR/DAO and schizophrenia. We replicated the decreased serum D-serine levels in schizophrenia in the sample set, but D-serine levels did not correlate with SRR/DAO genotypes. CONCLUSIONS: The SRR/DAO are not likely to be major genetic determinants in the development of schizophrenia or control of serum D-serine levels.

Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.