Cardiac metabolism in mice: tracer method developments and in vivo application revealing profound metabolic inflexibility in diabetes

Studies of cardiac fuel metabolism in mice have been almost exclusively conducted ex vivo. The major aim of this study was to assess in vivo plasma FFA and glucose utilization by the hearts of healthy control (db/+) and diabetic (db/db) mice, based on cardiac uptake of (R)-2-[9,10-(3)H]bromopalmitate ([3H]R-BrP) and 2-deoxy-D-[U-14C]glucose tracers. To obtain quantitative information about the evaluation of cardiac FFA utilization with [3H]R-BrP, simultaneous comparisons of [3H]R-BrP and [14C]palmitate ([14C]P) uptake were first made in isolated perfused working hearts from db/+ mice. It was found that [3H]R-BrP uptake was closely correlated with [14C]P oxidation (r2 = 0.94, P < 0.001). Then, methods for in vivo application of [3H]R-BrP and [14C]2-DG previously developed for application in the rat were specially adapted for use in the mouse. The method yields indexes of cardiac FFA utilization (R(f)*) and clearance (K(f)*), as well as glucose utilization (R(g)'). Finally, in the main part of the study, the ability of the heart to switch between FFA and glucose fuels (metabolic flexibility) was investigated by studying anesthetized, 8-h-fasted control and db/db mice in either the basal state or during glucose infusion. In control mice, glucose infusion raised plasma levels of glucose and insulin, raised R(g)' (+58%), and lowered plasma FFA level (-48%), K(f)* (-45%), and R(f)* (-70%). This apparent reciprocal regulation of glucose and FFA utilization by control hearts illustrates metabolic flexibility for substrate use. By contrast, in the db/db mice, glucose infusion raised glucose levels with no apparent influence on cardiac FFA or glucose utilization. In conclusion, tracer methodology for assessing in vivo tissue-specific plasma FFA and glucose utilization has been adapted for use in mice and reveals a profound loss of metabolic flexibility in the diabetic db/db heart, suggesting a fixed level of FFA oxidation in fasted and glucose-infused states.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Binding, internalization, and intracellular processing of protein ligands. Derivation of rate constants by computer modeling

Information about ligand binding, dissociation, internalization, and intracellular processing and about receptor turnover, processing, and insertion into the membrane is contained in the time-dependent changes in concentrations of membrane-associated and internalized ligand. Single experiments similar in design to those typically performed for Scatchard analyses of binding data conducted at physiological temperature and in the absence of inhibitors of ligand-receptor complex internalization and degradation can provide kinetic data sufficient to permit derivation of all the respective rate constants by numerical methods. We developed an analytical solution of the kinetic model which assumes that all of these processes follow first order kinetics. The model represents interactions of surface receptors (R)s, the surface ligand-receptor complex (LR)s and internalized receptor-ligand complex (LR)I: d[R]S/dt = Vr - kt[R]S - ka[L] [R]S + kd [LR]S; d[LR]S/dt = ka[L] [R]S - kd[LR]S - ke[LR]S; d[LR]I/dt = ke[LR]S - kh[LR]I; Vr is the constant rate of insertion of receptors into the membrane, kt is the internalization rate constant for free receptors, ka and kd are association and dissociation rate constants for ligand-surface receptor interaction, ke is the internalization rate constant for ligand-receptor complexes, and kh is the intracellular ligand decomposition rate constant. The interaction of radioiodinated human recombinant interferon-alpha 2a with the human alveolar lung carcinoma cell line, A549, was adequately accounted for by the model. The rate constants, numerically derived from time-dependent concentrations of surface-bound and internalized ligand of other systems taken from the literature, were in agreement with values of these rate constants individually measured by steady-state experiments. In cases where the fate of internalized radioactivity was more complex than assumed by the model, the parameters ka, kt, (kd + ke) and Vr could be derived from the time dependence of [LR]S.     

Mechanisms for antidiabetic effect of gingerol in cultured cells and obese diabetic model mice

There have been studies on health beneficial effects of ginger and its components. However, there still remain certain aspects that are not well defined in their anti-hyperglycemic effects. Our aims were to find evidence of possible mechanisms for antidiabetic action of [6]-gingerol, a pungent component of ginger, employing a rat skeletal muscle-derived cell line, a rat-derived pancreatic β-cell line, and type 2 diabetic model animals. The antidiabetic effect of [6]-gingerol was investigated through studies on glucose uptake in L6 myocytes and on pancreatic β-cell protective ability from reactive oxygen species (ROS) in RIN-5F cells. Its in vivo effect was also examined using obese diabetic db/db mice. [6]-Gingerol increased glucose uptake under insulin absent condition and induced 5′ adenosine monophosphate-activated protein kinase phosphorylation in L6 myotubes. Promotion by [6]-gingerol of glucose transporter 4 (GLUT4) translocation to plasma membrane was visually demonstrated by immunocytochemistry in L6 myoblasts transfected with glut4 cDNA-coding vector. [6]-Gingerol suppressed advanced glycation end product-induced rise of ROS levels in RIN-5F pancreatic β-cells. [6]-Gingerol feeding suppressed the increases in fasting blood glucose levels and improved glucose intolerance in db/db mice. [6]-Gingerol regulated hepatic gene expression of enzymes related to glucose metabolism toward decreases in gluconeogenesis and glycogenolysis as well as an increase in glycogenesis, thereby contributing to reductions in hepatic glucose production and hence blood glucose concentrations. These in vitro and in vivo results strongly suggest that [6]-gingerol has antidiabetic potential through multiple mechanisms.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9730-3) contains supplementary material, which is available to authorized users.     

大黄酸和大黄素的热分析及其动力学研究

本文采用热重法(TG)和差热分析法(DTA)测定了大黄酸和大黄素的DTA,TG-DTG曲线。两者的DTA曲线中皆有两个较为明显的吸热峰,第一个在熔化过程中出现,第二个发生在热分解过程中并伴随有明显的失重现象。TG曲线均有一个失重平台,失重率在90%以上。用TG-DTG法对两者在非等温条件下进行热分解动力学研究,把从TG-DTG曲线中取得的数据和31个不同的方程采用Achar微分法和Madhusudanan-Krishnan-Ni-nan(MKN)积分法对其进行非等温分解动力学研究,得到动力学参数活化能(E和指前因子A)和分解动力学机理及方程。得出结论:大黄酸和大黄素的动力学方程为dα/dt=Aexp(-E/RT)3/2(1-α)4/3[1/(1-α)1/3-1]-1和dα/dt=Aexp(-E/RT)3/2(1-α)2/3[1/(1-α)1/3]-1,其分解等合3D抗理。二者的活化能E(kJ/mol)分别为117.6和86.79,lnA/s-1分别是36.72和27.44。     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Dose-responsive insulin regulation of glucose transport in human skeletal muscle

Glucose transport is regarded as the principal rate control step governing insulin-stimulated glucose utilization by skeletal muscle. To assess this step in human skeletal muscle, quantitative PET imaging of skeletal muscle was performed using 3-O-methyl-[11C]glucose (3-[11C]OMG) in healthy volunteers during a two-step insulin infusion [n = 8; 30 and 120 mU.min(-1).m(-2), low (LO) and high (HI)] and during basal conditions (n = 8). Positron emission tomography images were coregistered with MRI to assess 3-[11C]OMG activity in regions of interest placed on oxidative (soleus) compared with glycolytic (tibialis anterior) muscle. Insulin dose-responsive increases of 3-[11C]OMG activity in muscle were observed (P < 0.01). Tissue activity was greater in soleus than in tibialis anterior (P < 0.05). Spectral analysis identified that two mathematical components interacted to shape tissue activity curves. These two components were interpreted physiologically as likely representing the kinetics of 3-[11C]OMG delivery from plasma to tissue and the kinetics of bidirectional glucose transport. During low compared with basal, there was a sixfold increase in k3, the rate constant attributed to inward glucose transport, and another threefold increase during HI (0.012 +/- 0.003, 0.070 +/- 0.014, 0.272 +/- 0.059 min(-1), P < 0.001). Values for k3 were similar in soleus and tibialis anterior, suggesting similar kinetics for transport, but compartmental modeling indicated a higher value in soleus for k1, denoting higher rates of 3-[11C]OMG delivery to soleus than to tibialis anterior. In summary, in healthy volunteers there is robust dose-responsive insulin stimulation of glucose transport in skeletal muscle.     

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.     

Effect of maternal glucose concentration on uteroplacental glucose consumption and transfer in pregnant sheep

The present study was designed to measure the relationships between maternal arterial glucose concentration [( GI]A) and fetal arterial glucose concentration [( GI]a), uteroplacental glucose consumption (UPGC), and the rate of uteroplacental glucose transfer to the fetus (UPGT) in pregnant sheep in late gestation. [GI]A was controlled by a glucose clamp technique and the glucose flux rates of the uteroplacenta were quantified by the Fick principle. [GI]A varied from 1.81 to 154.7 mg/dl; [GI]a was directly related to [GI]A: [GI]a = 0.374 [GI]A + 1.81, r = 0.873, P less than 0.001. Fetal arterial blood oxygen content decreased with [GI]A (P less than 0.05) and fetal arterial blood lactate concentration increased with [GI]A (P less than 0.001). There was no significant effect of [GI]A on the rates of uteroplacental lactate production, uteroplacental oxygen consumption, fetal oxygen consumption, or uterine or umbilical blood flow. Both UPGC and UPGT were directly related to [GI]A: UPGC = -2.221 x 10(-3) chi 2 + 0.646 x -6.016, r = 0.80; UPGT = -1.208 x 10(-3) chi 2 + 0.405 x -2.416, r = 0.90. UPGC and UPGT were approximately parallel over the range of [GI]A studied (UPGC = 1.19 UPGT + 3.79, r = 0.764). These results demonstrate the importance of UPGC to maternal-fetal glucose homeostasis and indicate that factors regulating uteroplacental glucose consumption and transfer to the fetus become limiting at comparable levels of [GI]A and [GI]a. The estimated kinetic constants for UPGC represent the metabolism of glucose by the uteroplacental tissues, but the estimated kinetic constants for UPGT represent the metabolism of glucose by the fetus as well as the transfer of glucose by the uteroplacenta to the fetus.     

Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing.

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi--Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf-1/3, and the values extrapolated to infinite flow rate (vf-1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.     

Glucose kinetics and substrate oxidation during exercise in the follicular and luteal phases.

The purpose of this investigation was to determine whether plasma glucose kinetics and substrate oxidation during exercise are dependent on the phase of the menstrual cycle. Once during the follicular (F) and luteal (L) phases, moderately trained subjects [peak O(2) uptake (V(O(2))) = 48.2 +/- 1.1 ml. min(-1). kg(-1); n = 6] cycled for 25 min at approximately 70% of the V(O(2)) at their respective lactate threshold (70%LT), followed immediately by 25 min at 90%LT. Rates of plasma glucose appearance (R(a)) and disappearance (R(d)) were determined with a primed constant infusion of [6,6-(2)H]glucose, and total carbohydrate (CHO) and fat oxidation were determined with indirect calorimetry. At rest and during exercise at 70%LT, there were no differences in glucose R(a) or R(d) between phases. CHO and fat oxidation were not different between phases at 70%LT. At 90%LT, glucose R(a) (28.8 +/- 4.8 vs. 33.7 +/- 4.5 micromol. min(-1). kg(-1); P < 0.05) and R(d) (28.4 +/- 4.8 vs. 34.0 +/- 4.1 micromol. min(-1). kg(-1); P < 0.05) were lower during the L phase. In addition, at 90%LT, CHO oxidation was lower during the L compared with the F phase (82.0 +/- 12.3 vs. 93.8 +/- 9.7 micromol. min(-1) .kg(-1); P < 0.05). Conversely, total fat oxidation was greater during the L phase at 90%LT (7.46 +/- 1.01 vs. 6.05 +/- 0.89 micromol. min(-1). kg(-1); P < 0.05). Plasma lactate concentration was also lower during the L phase at 90%LT concentrations (2.48 +/- 0.41 vs. 3.08 +/- 0.39 mmol/l; P < 0.05). The lower CHO utilization during the L phase was associated with an elevated resting estradiol (P < 0.05). These results indicate that plasma glucose kinetics and CHO oxidation during moderate-intensity exercise are lower during the L compared with the F phase in women. These differences may have been due to differences in circulating estradiol.     

Fed-state clamp stimulates cellular mechanisms of muscle protein anabolism and modulates glucose disposal in normal men

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-(13)C]leucine and [3-(3)H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine R(d) [rate of disappearance (synthesis)] was stimulated more (45 +/- 4 vs. 24 +/- 4 micromol/min, P < 0.01) and endogenous R(a) [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 +/- 6 vs. 49 +/- 2 micromol/min, P < 0.001). Glucose R(d) was similar; thus Hyper-3 metabolic clearance rate (331 +/- 23 vs. 557 +/- 41 ml/min, P < 0.0005) and R(d)/insulin (M, 0.65 +/- 0.10 vs. 1.25 +/- 0.10 mg.min(-1).pmol(-1).l, P < 0.001) were less, despite higher insulin (798 +/- 74 vs. 450 +/- 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70(S6K1); P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E).4E-BP1 complex (P = 0.01), these are consistent with increased mTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal.     

Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.     

2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscle capillary blood flow kinetics estimated from pulmonary O2 uptake and near-infrared spectroscopy.

The near-infrared spectroscopy (NIRS) signal (deoxyhemoglobin concentration; [HHb]) reflects the dynamic balance between muscle capillary blood flow (Q(cap)) and muscle O(2) uptake (Vo(2)(m)) in the microcirculation. The purposes of the present study were to estimate the time course of Q(cap) from the kinetics of the primary component of pulmonary O(2) uptake (Vo(2)(p)) and [HHb] throughout exercise, and compare the Q(cap) kinetics with the Vo(2)(p) kinetics. Nine subjects performed moderate- (M; below lactate threshold) and heavy-intensity (H, above lactate threshold) constant-work-rate tests. Vo(2)(p) (l/min) was measured breath by breath, and [HHb] (muM) was measured by NIRS during the tests. The time course of Q(cap) was estimated from the rearrangement of the Fick equation [Q(cap) = Vo(2)(m)/(a-v)O(2), where (a-v)O(2) is arteriovenous O(2) difference] using Vo(2)(p) (primary component) and [HHb] as proxies of Vo(2)(m) and (a-v)O(2), respectively. The kinetics of [HHb] [time constant (tau) + time delay [HHb]; M = 17.8 +/- 2.3 s and H = 13.7 +/- 1.4 s] were significantly (P < 0.001) faster than the kinetics of Vo(2) [tau of primary component (tau(P)); M = 25.5 +/- 8.8 s and H = 25.6 +/- 7.2 s] and Q(cap) [mean response time (MRT); M = 25.4 +/- 9.1 s and H = 25.7 +/- 7.7 s]. However, there was no significant difference between MRT of Q(cap) and tau(P)-Vo(2) for both intensities (P = 0.99), and these parameters were significantly correlated (M and H; r = 0.99; P < 0.001). In conclusion, we have proposed a new method to noninvasively approximate Q(cap) kinetics in humans during exercise. The resulting overall Q(cap) kinetics appeared to be tightly coupled to the temporal profile of Vo(2)(m).     

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study.

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).     

Synthesis and biological activity of novel pyrimidinone containing thiazolidinedione derivatives

A series of pyrimidinone derivatives of thiazolidinediones were synthesized. Their biological activity were evaluated in insulin resistant, hyperglycemic and obese db/db mice. In vitro PPARgamma transactivation assay was performed in HEK 293T cells. PMT13 showed the best biological activity in this series. PMT13 (5-[4-[2-[2-ethyl-4-methyl-6-oxo-1,6-dihydro-1-pyrimidinyl]ethoxy]phenylmethyl]thiazolidine-2,4-dione) showed better plasma glucose, triglyceride and insulin-lowering activity in db/db mice than rosiglitazone and pioglitazone. PMT13 showed better PPARgamma transactivation than the standard compounds. Pharmacokinetic study in Wistar rats showed good systemic exposure of PMT13. Twenty-eight day oral toxicity study in Wistar rats did not show any treatment-related adverse effects.     

5,5-Difluoro- and 5-Fluoro-5-methyl-hexose-based C-Glucosides as potent and orally bioavailable SGLT1 and SGLT2 dual inhibitors

(2S,3R,4R,5S,6R)-2-Aryl-5,5-difluoro-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diols and (2S,3R,4R,5S,6R)-2-aryl-5-fluoro-5-methyl-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diols were discovered as dual inhibitors of sodium glucose co-transporter proteins (e.g. SGLT1 and SGLT2) through rational drug design, efficient synthesis, and in vitro and in vivo evaluation. Compound 6g demonstrated potent dual inhibitory activities (IC₅₀ = 96 nM for SGLT1 and IC₅₀ = 1.3 nM for SGLT2). It showed robust inhibition of blood glucose excursion in an oral glucose tolerance test (OGTT) in Sprague Dawley (SD) rats when dosed at both 1 mg/kg and 10 mg/kg orally. It also demonstrated postprandial glucose control in db/db mice when dosed orally at 10 mg/kg.     

Involvement of heparan sulfate in the renoprotective effects of imidapril,an angiotensin-converting enzyme inhibitor,in diabetic db/db mice

Abstract

We investigated the renoprotective effects of imidapril hydrochloride ((-)-(4?S)-3-[(2?S)-2-[[(1?S)-1-ethoxycarbonyl-3-phenylpropyl] amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril), an angiotensin-converting enzyme inhibitor, in a diabetic animal model. We used BKS.Cg-+Lepr^db/+Lepr^db (db/db) mice, a genetic animal model of obese type 2 diabetes. Diabetic db/db mice suffered from glomerular hyperfiltration, albuminuria and hypoalbuminemia. Oral administration of 5?mg/kg/day of imidapril for 3 weeks suppressed renal hyperfiltration, reduced albuminuria and normalized hypoalbuminemia. Imidapril did not influence body weights, blood pressure or blood glucose concentrations in db/db mice. Urinary excretion of heparan sulfate (HS) in non-treated 11-week-old db/db mice was significantly lower than that in age-matched non-diabetic db/+m mice. HS is a component of HS proteoglycans, which are present in glomerular basement membranes and glycocalyx of cell surfaces. Reduced urinary HS excretion indicated glomerular HS loss in db/db mice. Imidapril increased urinary excretion of HS to concentrations observed in db/+m mice, indicating that imidapril prevented the loss of renal HS. These results suggest that imidapril ameliorates renal hyperfiltration and loss of renal contents of HS. Improvement of filtration function and maintenance of HS, which is an important structural component of glomeruli, may contribute to renoprotective effects of imidapril.     

Augmented lipopolysaccharide-induced TNF-alpha production by peritoneal macrophages in type 2 diabetic mice is dependent on elevated glucose and requires p38 MAPK

Dysregulated inflammation is a complication of type 2 diabetes (T2D). In this study, we show that augmented LPS-induced TNF-alpha production by resident peritoneal macrophages (PerMphi) in type 2 diabetic (db/db) mice is dependent on elevated glucose and requires p38 MAPK. Intraperitoneal LPS administered to db/db and nondiabetic (db/+) mice induced 3- and 4-fold more TNF-alpha in the peritoneum and serum, respectively, of db/db mice as compared with db/+ mice. Examination of the TLR-4/MD2 complex and CD14 expression showed no difference between db/db and db/+ PerMphi. Ex vivo stimulation of PerMphi with LPS produced a similar 3-fold increase in TNF-alpha production in db/db PerMphi when compared with db/+ PerMphi. PerMphi isolated from db/+ mice incubated in high glucose (4 g/L) medium for 12 h produced nearly 2-fold more TNF-alpha in response to LPS than PerMphi incubated in normal glucose medium (1 g/L). LPS-dependent stimulation of PI3K activity, ERK1/2 activation, and p38 kinase activity was greater in PerMphi from db/db mice as compared with db/+ mice. Only inhibition of p38 kinase blocked LPS-induced TNF-alpha production in PerMphi from db/db mice. Taken together, these data indicate that augmented TNF-alpha production induced by LPS in macrophages during diabetes is due to hyperglycemia and increased LPS-dependent activation of p38 kinase.