Effect of curcumin on atherosclerosis in apoE/LDLR-double knockout mice.

It is widely appreciated that inflammation and oxidant stress contribute to atherogenesis. Curcumin, a polyphenolic natural compound has been reported to possess anti-inflammatory and anti-oxidant actions. We hypothesized that curcumin could inhibit the development of atherosclerosis in the apoE/LDLR-double knockout mice fed with Western diet (21% fat, 0.15% cholesterol w/w, without cholic acid). Curcumin (purity>or=98%), premixed with diet, was given for 4 months at a dose of 0.3 mg/ per day/ per mouse. In this model curcumin inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 19,2+/-0,6%, p<0,05) and "cross-section" method (565867+/-39764 microm2 vs. 299201+/-20373 microm2, p<0,05). Importantly, curcumin influenced neither the concentrations of cholesterol and triglycerides in blood nor animal body weight. To our knowledge, this is the first report that shows the anti-atherogenic effect of low dose of curcumin in fine model of atherosclerosis: gene-targeted apoE/LDLR-double knockout mice.     

Inhibition of nuclear factor-kappaB attenuates artherosclerosis in apoE/LDLR - double knockout mice.

Nuclear factor - kappaB (NF-kappaB) is a good therapeutic target for cardiovascular disease and numerous efforts are being made to develop safe NF-kappaB inhibitors. Nowadays many authors address NF-kappaB as a major therapeutic target in atherosclerosis, especially for preventive measures, in the light of two main hypothesis of atherosclerosis: oxidation and inflammation. We hypothesized that ammonium pyrrolidinedithioocarbamate (PDTC) - a well-known inhibitor of NF-kappaB could inhibit the development of atherosclerosis in this experimental model. We used apoE/LDLR - DKO mouse model, which is considered as a one of the best models to study the anti-atherosclerotic effect of drugs. In this model PDTC inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 15,63+/-0,6%) and "cross-section" method (565867+/-39764 microm2 vs. 291695+/-30384 microm2). Moreover, PDTC did not change the profile of cholesterol and triglycerides in blood. To our knowledge, this is the first report that shows the effect of PDTC on atherogenesis in gene-targeted apoE/LDLR - double knockout mice.     

Insulin receptor tyrosine kinase activity is unaltered in ob/ob and db/db mouse skeletal muscle membranes

Insulin binding and insulin receptor tyrosine kinase activity were examined in two rodent models with genetic insulin resistance using partially-purified skeletal muscle membrane preparations. Insulin binding activity was decreased about 50% in both 12-week (219 +/- 184 vs 1255 +/- 158 fmoles/mg, p less than 0.01) and 24-week old (2120 +/- 60 vs 1081 +/- 60 fmoles/mg, p less than 0.01) ob/ob mice. In contrast, insulin binding to membrane derived from 24-week old db/db mice was not significantly different from lean controls (1371 +/- 212 vs 1253 +/- 247 fmoles/mg). Insulin-associated tyrosine kinase activity of membranes from ob/ob skeletal muscle was decreased, compared to its normal lean littermate, when compared on a per mg of protein basis in both 12-week (37 +/- 3 vs 21 +/- 3 pmoles/min/mg, p less than 0.05) and 24-week old (71 +/- 5 vs 37 +/- 6 pmoles/min/mg, p less than 0.01) mice. However, no significant differences in kinase activities were observed when the data were normalized and compared on a per fmole of insulin-binding activity basis for the 12-week (12 +/- 1 vs 11 +/- 2) and 24-week (27 +/- 2 vs 20 +/- 3) age groups. Insulin receptor tyrosine kinase activity of db/db skeletal muscle membranes was not different than its normal lean littermate whether expressed on a protein (34 +/- 7 vs 30 +/- 3) or fmole of insulin-binding activity (21 +/- 4 vs 18 +/- 4) basis. These data suggest that insulin receptor tyrosine kinase is not associated with the insulin resistance observed in ob/ob and db/db mice and demonstrate differences in receptor regulation between both animal models.     

Effect of a novel leukotoriene synthesis inhibitor, BAY x1005, on the antigen- and LPS-induced airway hyperresponsiveness in guinea pigs

Due to the inhibition of 5-lipoxygenase-activating protein (FLAP), BAY x1005 is a new selective inhibitor of leukotriene synthesis. The effects of BAY x1005 on the antigen- and bacterial lipopolysaccharide (LPS)-induced airway hyperresponsiveness in guinea pigs were investigated. Six times provocation of aeroantigen caused biphasic increases in airway resistance which peaked at 1 hr (immediate phase reaction) and 4 hrs (late phase reaction). It also caused airway hyperreactivity to acetylcholine. BAY x1005 at doses of 10mg/kg and 30mg/kg significantly inhibited antigen-induced increase in respiratory resistance (Rrs) at 1 and 4 hrs after the last antigen challenge. Simultaneously, BAY x1005 inhibited the antigen-induced airway hyperresponsiveness at doses of 10 and 30mglkg and airway eosinophilia (bronchoalveolar lavage study) at a dose of 30 mg/kg. In addition, BAY x1005 at a dose of 30mg/kg inhibited bacterial LPS-induced airway hyperreactivity to acetylcholine. In this model, BAY x1005 did not affect the increase of the number of leukocytes in bronchoalveolar lavage fluid.These results suggest that BAY x1005 is a potent anti-asthmatic agent with an inhibitory action to airway hyperreactivity.     

The presence of leukocyte CC-chemokine receptor 2 in CCR2 knockout mice promotes atherogenesis

OBJECTIVE: To selectively determine the role of leukocyte CC-chemokine receptor 2 (CCR2) in atherogenesis. METHODS AND RESULTS: Bone marrow progenitor cells harvested from CCR2(+/+) mice were transplanted into irradiated CCR2(-/-) mice, representing the whole-body absence of CCR2 except in leukocytes. Transplantation of CCR2(-/-) bone marrow into CCR2(-/-) mice served as control. Eight weeks after bone marrow transplantation, the diet of regular chow was switched to a high-cholesterol diet for another 10 weeks in order to induce atherosclerosis. No significant differences in serum cholesterol and triglyceride levels were observed between the two groups. However, the mean cross-sectional aortic root lesion area of CCR2(+/+)-->CCR2(-/-) mice amounted up to 12.28+/-3.28x10(4) microm(2), compared with only 3.08+/-0.74 x 10(4) microm(2) observed in the CCR2(-/-)-->CCR2(-/-) group. Thus, the presence of CCR2 exclusively on leukocytes induces a fourfold increase in aortic lesion area. This extent of lesion development was comparable to C57Bl/6 mice receiving CCR2(+/+) bone marrow (10.08+/-3.30x10(4) microm(2)). CONCLUSION: These results point at a dominant role of leukocyte CCR2 in atherogenesis, implying that CCR2 from nonleukocyte sources, like endothelial cells or smooth muscle cells, is less critical in the initiation of atherosclerosis. Pharmacological inhibition of leukocyte CCR2 function might be a promising strategy to prevent atherosclerosis.     

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.     

IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils.

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.     

Epidermal growth factor in serum, urine, submandibular glands and kidneys of diabetic mice

Levels of epidermal growth factor (EGF) in serum were significantly decreased in streptozotocin (STZ)-diabetic mice (446 +/- 168 pg/ml after 1 week and 423 +/- 52 after 4 weeks vs 766 +/- 162 pg/ml in controls, P.002 and less than .001. respectively) and in genetically diabetic ob/ob mice (455 +/- 285 vs 962 +/- 453 pg/ml in nondiabetic ob/+ controls, P.043). The urinary excretion of EGF was significantly increased in STZ mice (104 +/- 53 vs 51 +/- 23 ng/h, P.013) but unchanged in ob/ob mice (33 +/- 9 vs 45 +/- 16 ng/h, P.134). However, when expressed per mg creatinine it was decreased in both cases: in STZ mice to 680 +/- 250 ng/mg at 1 week and 684 +/- 211 at 4 weeks vs 1250 +/- 303 ng/mg in controls (P less than .01); and in the ob/ob mice to 552 +/- 117 vs 1237 +/- 300 ng/mg in ob/+ controls (P less than .01). EGF content of the submandibular glands of STZ mice remained unchanged at 1 week (13.1 +/- 2.9 vs 11.0 +/- 1.8 micrograms/mg protein, P.170) but dropped by 4 weeks (4.7 +/- 1.2 micrograms/mg, P less than .001); in the ob/ob mice it was less than 20% that of controls (2.1 +/- 0.8 vs 12.2 +/- 3.6 micrograms/mg protein). In kidneys, the EGF content was not altered in either ob/ob (524 +/- 50 vs 571 +/- 33 pg/mg protein) or STZ mice (652 +/- 183 vs 665 +/- 80 pg/mg). The preproEGF mRNA level in STZ-treated mice was reduced after 4 weeks in submandibular glands but not in kidneys. The results show that diabetes affects EGF production, utilization and/or excretion in mice and that kidneys are spared from suppression of EGF synthesis that is pronounced in the submandibular glands.     

Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis

To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 +/- 25.0 vs 10.0 +/- 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 +/- 68 vs 325 +/- 18 x 10(3) microm(3) per glomerulus; p < 0.0001) and cellularity (47.1 +/- 8.9 vs 32.0 +/- 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 +/- 0.7 vs 0.02 +/- 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Hypercholesterolemia impairs reactive hyperemic vasodilation of 2A but not 3A arterioles in mouse cremaster muscle

Hypercholesterolemia and atherosclerosis have been associated with changes in the microvasculature, in particular with endothelial dysfunction. In the present study, the impact of atherogenic conditions on arteriolar vasomotor control was determined. Arteriolar [second-order (2A) and third-order (3A) arterioles; diameter range: 9-37 microm] responses during reactive hyperemia (RH) were determined in cremaster muscle of anesthetized mice. C57Bl/6 mice on normal rodent chow were used as controls and high-fat/high-cholesterol (HFC)-fed C57Bl/6 and ApoE3-Leiden mice as hypercholesterolemic mice. The HFC diet resulted in time-dependent increases in plasma cholesterol and triglyceride concentrations (P < 0.001), which were more pronounced in ApoE3-Leiden mice (P < 0.001). In control mice, inhibition of nitric oxide (NO) synthesis with Nomega-nitro-L-arginine (L-NNA) reduced baseline diameter from 17.9 +/- 1.2 to 15.9 +/- 1.3 microm (P < 0.05) and decreased the duration of RH [time to 50% (t50) of recovery: 23.3 +/- 3.6 vs. 12.5 +/- 1.3 s (P = 0.003)]. t50 was longer in 2A versus 3A arterioles (33 +/- 3 vs. 18 +/- 2 s, P < 0.001) and increased with wall shear rate at the beginning of RH in 2A arterioles only. Compared with control mice, RH duration was reduced in 2A arterioles of HFC mice (t50: 11 +/- 2 s, P < 0.001 vs. control) but not affected in 3A vessels. L-NNA did not affect baseline diameter in HFC mice and reduced t50 only in "slow" responders (t50 > or = 10 s). It is concluded that hypercholesterolemia results in an impairment of NO-mediated vasomotor control in 2A but not 3A arterioles during dynamic changes of perfusion like RH. 2A arterioles likely therefore represent the functional locus of endothelial dysfunction during atherogenic conditions.     

Type 2 diabetes mellitus in a non-obese mouse model induced by Meg1/Grb10 overexpression

We assessed the possibility of C57BL/6-Tg (Meg1/Grb10)isn(Meg1 Tg) mice as a non-obese type 2 diabetes (2DM) animal model. Meg1 Tg mice were born normal, but their weight did not increase as much as normal after weaning and showed about 85% of normal size at 20 weeks of age. Body mass index of Meg1 Tg mice was also smaller than that of control mice. The glucose tolerance test and insulin tolerance test showed that Meg1 Tg mice had reduced ability to normalize the blood glucose level. Blood urea nitrogen (BUN) in Meg1 Tg mice (19.6 +/- 1.2 mg/dl) was significantly lower than in controls (22.0 +/- 0.8 mg/dl), while plasma triglyceride, insulin, adiponectin, and resistin levels were significantly higher (202.0 +/- 23.4 mg/dl vs 146.3 +/- 23.4 mg/dl, 152.4 +/- 16.3 pg/ml vs 88.1 +/- 16.9 pg/ml, 74.4 +/- 10.9 microg/ml vs 48.3 +/- 7.0 microg/ml, and 4.0 +/- 0.2 ng/ml vs 3.6 +/- 0.2 ng/ml, respectively). Body, visceral fat weight and liver weights were significantly lower (19.6 +/- 0.4 g vs 24.3 +/- 0.3 g, 376.7 +/- 29.6 mg to 507.5 +/- 23.0 mg, and 906.0 +/- 41.8 mg to 1,001.0 +/- 15.1 mg, respectively). Thus, hyperinsulinemia observed in Meg1 Tg mice indicates that their insulin signaling pathway is somehow inhibited. With high fat diet, the diabetes onset rate of Meg1 Tg mice increased up to 60%. These results suggest that Meg1 Tg mice resemble human 2DM.     

Increased H2O2, vascular endothelial growth factor and receptors in the retina of the BBZ/Wor diabetic rat

Hyperglycemia in diabetes induces increased levels of hydrogen peroxide (H2O2), a reactive oxygen species generated by reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nontoxic levels of H2O2 increase endothelial cell permeability. Using a model of non-insulin-dependent diabetes, the BBZ/Wor rat, we investigated retinal levels of H2O2, vascular endothelial growth factor (VEGF) and its receptors, VEGF-R1 and VEGF-R2 by transmission electron microscopy at sites of the blood-retinal barrier (BRB). H2O2 localization was done by the cerium NADH oxidase method, and extravasation of endogenous serum albumin was used to document disruption of the BRB. Higher levels of H2O2 were detected in blood vessels of diabetic (78.7 +/- 4.84%) as compared with vessels from nondiabetic rats (39.0 +/- 4.47%). VEGF immunoreactivity was statistically higher in the inner BRB (24.67 +/- 0.33 colloidal gold particles/63 microm2 vs. 21.52 +/- 0.43 colloidal gold particles/63 microm2, p = .0001) and outer BRB (42.56 +/- 0.45 colloidal gold particles/63 microm2 vs. 15.51 +/- 0.51 colloidal gold particles/63 microm2, p = .0001) of diabetic rats as compared with age matched nondiabetic control rats. VEGF-R1 immunoreactivity was significantly higher in diabetic retinas in both the inner BRB (21.66 +/- 0.75 colloidal gold particles/63 microm2 vs. 12.69 +/- 0.61 colloidal gold particles/63 microm2, p = .0001) and outer BRB (22.76 +/- 2.36 colloidal gold particles/63 microm2 vs. 8.53 +/- 2.67 colloidal gold particles/63 microm2, p = .0013). VEGF-R2 was statistically higher in the inner BRB (8.97 +/- 0.57 colloidal gold particles/63 microm2 versus 7.03 +/- 0.65 colloidal gold particles/63 microm2, p = .0419) but not in the outer BRB (29.42 +/- 1.25 colloidal gold particles/63 microm2 vs. 28.07 +/- 1.42 colloidal gold particles/63 microm2, p = .4889). H2O2 levels correlated with increased VEGF (correlation coefficient = 0.82, p = .001) in this model of nonproliferative diabetic retinopathy. These results support that hyperglycemia is one factor that induces retinal endothelial cells in vivo to increase H2O2 via NADH oxidase and stimulates increases in VEGF resulting in disruption of the BRB.     

Monoamine transporter inhibitors and norepinephrine reduce dopamine-dependent iron toxicity in cells derived from the substantia nigra

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean +/- SD uptake of 100 microm 59FeCl3 in RCSN-3 cells was 25 +/- 4 pmol per min per mg, which increased to 28 +/- 8 pmol per min per mg when complexed with dopamine (Fe(III)-dopamine). This uptake was inhibited by 2 microm nomifensine (43%p < 0.05), 100 microm imipramine (62%p < 0.01), 30 microm reboxetine (71%p < 0.01) and 2 mm dopamine (84%p < 0.01). The uptake of 59Fe-dopamine complex was Na+, Cl- and temperature dependent. No toxic effects in RCSN-3 cells were observed when the cells were incubated with 100 microm FeCl3 alone or complexed with dopamine. However, 100 microm Fe(III)-dopamine in the presence of 100 microm dicoumarol, an inhibitor of DT-diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 microm nomifensine, 30 microm reboxetine and 2 mm norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe3+, (2) the uptake of Fe-norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe-norepinephrine complex even when DT-diaphorase is inhibited. These results support the proposed neuroprotective role of DT-diaphorase and norepinephrine.     

Acute hepatic steatosis in mice by blocking beta-oxidation does not reduce insulin sensitivity of very-low-density lipoprotein production

Accumulation of triglycerides (TG) in the liver is generally associated with hepatic insulin resistance. We questioned whether acute hepatic steatosis induced by pharmacological blockade of beta-oxidation affects hepatic insulin sensitivity, i.e., insulin-mediated suppression of VLDL production and insulin-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) and PKB. Tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyl transferase-1 (CPT1), was used for this purpose. Male C57BL/6J mice received 30 mg/kg TDGA or its solvent intraperitoneally and were subsequently fasted for 12 h. CPT1 inhibition resulted in severe microvesicular hepatic steatosis (19.9 +/- 8.3 vs. 112.4 +/- 25.2 nmol TG/mg liver, control vs. treated, P < 0.05) with elevated plasma nonesterified fatty acid (0.68 +/- 0.25 vs. 1.21 +/- 0.41 mM, P < 0.05) and plasma TG (0.39 +/- 0.16 vs. 0.60 +/- 0.10 mM, P < 0.05) concentrations. VLDL-TG production rate was not affected on CPT1 inhibition (74.9 +/- 15.2 vs. 79.1 +/- 12.8 mumol TG.kg(-1).min(-1), control vs. treated) although treated mice secreted larger VLDL particles (59.3 +/- 3.6 vs. 66.6 +/- 4.5 nm diameter, P < 0.05). Infusion of insulin under euglycemic conditions suppressed VLDL production rate in control and treated mice by 43 and 54%, respectively, with formation of smaller VLDL particles (51.2 +/- 2.5 and 53.2 +/- 2.8 nm diameter). Insulin-induced insulin receptor substrate (IRS)1- and IRS2-associated PI3-kinase activity and PKB-phosphorylation were not affected on TDGA treatment. In conclusion, acute hepatic steatosis caused by pharmacological inhibition of beta-oxidation is not associated with reduced hepatic insulin sensitivity, indicating that hepatocellular fat content per se is not causally related to insulin resistance.     

Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1

It is well known that nonselective, nonsteroidal anti-inflammatory drugs inhibit renal renin production. Our previous studies indicated that angiotensin-converting enzyme inhibitor (ACEI)-mediated renin increases were absent in rats treated with a cyclooxygenase (COX)-2-selective inhibitor and in COX-2 -/- mice. The current study examined further whether COX-1 is also involved in mediating ACEI-induced renin production. Because renin increases are mediated by cAMP, we also examined whether increased renin is mediated by the prostaglandin E(2) receptor EP(2) subtype, which is coupled to G(s) and increases cAMP. Therefore, we investigated if genetic deletion of COX-1 or EP(2) prevents increased ACEI-induced renin expression. Age- and gender-matched wild-type (+/+) and homozygous null mice (-/-) were administered captopril for 7 days, and plasma and renal renin levels and renal renin mRNA expression were measured. There were no significant differences in the basal level of renal renin activity from plasma or renal tissue in COX-1 +/+ and -/- mice. Captopril administration increased renin equally [plasma renin activity (PRA): +/+ 9.3 +/- 2.2 vs. 50.1 +/- 10.9; -/- 13.7 +/- 1.5 vs. 43.9 +/- 6.6 ng ANG I x ml(-1) x h(-1); renal renin concentration: +/+ 11.8 +/- 1.7 vs. 35.3 +/- 3.9; -/- 13.0 +/- 3.0 vs. 27.8 +/- 2.7 ng ANG I x mg protein(-1) x h(-1); n = 6; P < 0.05 with or without captopril]. ACEI also increased renin mRNA expression (+/+ 2.4 +/- 0.2; -/- 2.1 +/- 0.2 fold control; n = 6-10; P < 0.05). Captopril led to similar increases in EP(2) -/- compared with +/+. The COX-2 inhibitor SC-58236 blocked ACEI-induced elevation in renal renin concentration in EP(2) null mice (+/+ 24.7 +/- 1.7 vs. 9.8 +/- 0.4; -/- 21.1 +/- 3.2 vs. 9.3 +/- 0.4 ng ANG I x mg protein(-1) x h(-1); n = 5) as well as in COX-1 -/- mice (SC-58236-treated PRA: +/+ 7.3 +/- 0.6; -/- 8.0 +/- 0.9 ng ANG I x ml(-1) x h(-1); renal renin: +/+ 9.1 +/- 0.9; -/- 9.6 +/- 0.5 ng ANG I x mg protein(-1) x h(-1); n = 6-7; P < 0.05 compared with no treatment). Immunohistochemical analysis of renin expression confirmed the above results. This study provides definitive evidence that metabolites of COX-2 rather than COX-1 mediate ACEI-induced renin increases. The persistent response in EP(2) nulls suggests involvement of prostaglandin E(2) receptor subtype 4 and/or prostacyclin receptor (IP).     

Establishment of a basic method for manipulating preantral follicles: effects of retrieval method on in vitro growth of preantral follicles and intrafollicular oocytes

The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.     

Phosphodiesterase 4 inhibitor GPD-1116 markedly attenuates the development of cigarette smoke-induced emphysema in senescence-accelerated mice P1 strain

Phosphodiesterase 4 (PDE4) is an intracellular enzyme specifically degrading cAMP, a second messenger exerting inhibitory effects on many inflammatory cells. To investigate whether GPD-1116 (a PDE4 inhibitor) prevents murine lungs from developing cigarette smoke-induced emphysema, the senescence-accelerated mouse (SAM) P1 strain was exposed to either fresh air or cigarette smoke for 8 wk with or without oral administration of GPD-1116. We confirmed the development of smoke-induced emphysema in SAMP1 [air vs. smoke (means +/- SE); the mean linear intercepts (MLI), 52.9 +/- 0.8 vs. 68.4 +/- 4.2 microm, P < 0.05, and destructive index (DI), 4.5% +/- 1.3% vs. 16.0% +/- 0.4%, P < 0.01]. Emphysema was markedly attenuated by GPD-1116 (MLI = 57.0 +/- 1.4 microm, P < 0.05; DI = 8.2% +/- 0.6%, P < 0.01) compared with smoke-exposed SAMP1 without GPD-1116. Smoke-induced apoptosis of lung cells were also reduced by administration of GPD-1116. Matrix metalloproteinase (MMP)-12 activity in bronchoalveolar lavage fluid (BALF) was increased by smoke exposure (air vs. smoke, 4.1 +/- 1.1 vs. 40.5 +/- 16.2 area/microg protein; P < 0.05), but GPD-1116 significantly decreased MMP-12 activity in smoke-exposed mice (5.3 +/- 2.1 area/microg protein). However, VEGF content in lung tissues and BALF decreased after smoke exposure, and the decrease was not markedly restored by oral administration of GPD-1116. Our study suggests that GPD-1116 attenuates smoke-induced emphysema by inhibiting the increase of smoke-induced MMP-12 activity and protecting lung cells from apoptosis, but is not likely to alleviate cigarette smoke-induced decrease of VEGF in SAMP1 lungs.     

The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.     

The effect of hypoxia on uptake and degradation of low density lipoproteins by cultured human arterial smooth muscle cells.

Atheroma have been produced in experimental animal by systemic hypoxia. This study assessed the effects of hypoxia on binding, uptake and degradation of human low density lipoprotein (LDL) by human arterial smooth muscle cells, the cell involved in atherogenesis. The LDL content of the smooth muscle cell grown in the usual conditions (95% air [20% O2], 5% CO2) increased with the incubation time of LDL in the medium (7.5 mug protein/ml of medium); the trypsin releasable LDL "binding" reached a plateau by 24 h (2.2 +/- 1.3 [x +/- S.D.]) ng/mug LDL protein added per 10(6) cells whereas the LDL in the cell after trypsinization ("net uptake") continued to increase up to 48 h (6.5 +/- 4.6 ng/mug LDL protein added per 10(6) cells at 48 h). LDL protein degradation increases rapidly between 7 and 48 h (10.4 ng/mug LDL protein added per 10(6) cells at 24 h) after an initial delay of approximately 7 h. Smooth muscle cells grown under hypoxic conditions (5%02) had similar LDL "binding " but showed increased "net uptake" (10.7 +/- 4.8 ng/mug LDL protein added per 10(6) cells) and a 36 +/- 13% decrease in degradation (p less than 0.05; n =8). The impaired degradation of lipoprotein by smooth muscle cells may, in part, explain the role of hypoxia in atherogenesis.