Inhibition of the chemokine (C-C motif) ligand 2/chemokine (C-C motif) receptor 2 pathway attenuates hyperglycaemia and inflammation in a mouse model of hepatic steatosis and lipoatrophy.

AIMS/HYPOTHESIS: Using a mouse model of lipoatrophic diabetes, we hypothesised that the chemokine (C-C motif) ligand 2 (CCL2)/chemokine (C-C motif) receptor 2 (CCR2) pathway contributes to hepatic macrophage accumulation and insulin resistance through induction of a chronic inflammatory state. METHODS: Metabolic variables of insulin resistance and inflammation were characterised in wild-type and lipoatrophic A-ZIP/F-1 transgenic (AZIP-Tg) mice. The AZIP-Tg mice were then treated with a CCR2 antagonist (RS504393, 2 mg kg(-1) day(-1)) or vehicle for 28 days via a subcutaneous mini-osmotic pump to examine the role of the CCL2/CCR2 pathway in lipoatrophic diabetes. RESULTS: The lipoatrophic AZIP-Tg mice were diabetic with high fasting glucose and serum insulin concentrations compared with littermate controls. The livers of AZIP-Tg mice were more than threefold enlarged and exhibited increased triacylglycerol content. CCL2 levels were highly elevated in both liver and serum of the AZIP-Tg mice compared with controls. In addition, the circulating CCL2 concentration was associated with increased macrophage accumulation and inflammation as documented by upregulation of Cd68 gene and Tnf-alpha [also known as Tnf] gene in livers from the AZIP-Tg mice. Treatment of the lipoatrophic AZIP-Tg mice with the CCR2 antagonist ameliorated the hyperglycaemia, hyperinsulinaemia and hepatomegaly in conjunction with a reduction in liver inflammation. CONCLUSIONS/INTERPRETATION: These findings demonstrate a significant role of the CCL2/CCR2 pathway in lipoatrophy-induced diabetes and provide clear evidence that metabolic improvements resulting from the inhibition of this inflammatory pathway are not adipose tissue-dependent.

Generation of soluble P- and E-selectins in vivo is dependent on expression of P-selectin glycoprotein ligand-1.

BACKGROUND: Factors contributing to the generation of soluble P- and E-selectins remain unclear. RESULTS: This work demonstrates that mice lacking P-selectin glycoprotein ligand-1 (Psgl-1(-/-)) are deficient in soluble P-selectin (sP-sel), which is due to a defective binding interaction between PSGL-1 and P-sel, because mice lacking alpha(1,3)-fucosyltransferase-VII are also deficient in sP-sel. Psgl-1(-/-) mice are also deficient in soluble E-selectin (sE-sel) indicating that leukocyte interactions with endothelial cells lead to the generation of sE-sel. The generation of sE-sel requires an interaction between PSGL-1 and P-sel, as deficiency of sE-sel is observed in both Psgl-1(-/-) and P-sel(-/-) mice. Bone marrow transplantation from Psgl-1(-/-) to Psgl-1(+/+) mice leads to deficiency of sP-sel and sE-sel in recipient mice, establishing the importance of bone marrow-derived PSGL-1 toward the generation of sP-sel and sE-sel. Bone marrow transplantation from P-sel(-/-) to P-sel1(+/+) mice does not lead to a significant reduction in sP-sel, confirming the importance of the endothelium toward the liberation of sP-sel. CONCLUSION: sP-sel and sE-sel reflect an interaction between leukocyte PSGL-1 and endothelial P-sel.

Effect of exercise duration on plasma endothelin-1 concentration.

AIM: Endothelin-1 (ET-1) is a potent vasoconstricting peptide released mostly from vascular endothelial cells. Isolated exercise sessions of relatively long duration (=or>30 min) have produced increases in plasma ET-1 concentration while shorter exercise sessions usually have not. The purpose of the present study was to verify an effect of exercise duration at a steady work rate on plasma ET-1 concentration. METHODS: Eleven endurance-trained males (age 27+/-6 years; maximal oxygen consumption--VO2max--56+/-7 mLxkg-1xmin-1, body fat 11+/-5%; mean+/-SD) exercised on a treadmill at 70% VO2max on 2 occasions separated by at least 2 weeks. During a short-duration session, subjects expended approximately 3,360 kJ (60+/-2 min). During a long-duration session, subjects expended approximately 6,300 kJ (112+/-4 min). Six of the subjects performed the 3,360 kJ session before the 6,300 kJ session while the other 5 subjects performed the 6,300 kJ session first. RESULTS: The short-duration session did not cause plasma ET-1 concentration to change immediately after exercise (0.23+/-0.01 pmolxL-1 before exercise, 0.22+/-0.02 pmolxL-1 after exercise, mean+/-SE). However, 10 of 11 subjects had increased ET-1 after the long-duration session (0.28+/-0.02 pmolxL-1 before exercise, 0.32+/-0.02 pmolxL-1 after exercise, P=0.0004). A treatment-by-time effect was present (P=0.003). CONCLUSION: These results demonstrate an effect of exercise duration on plasma ET-1 concentration. Exercise duration is, therefore, an essential consideration when investigating exercise's effect on ET-1.

Pioglitazone protects against thrombosis in a mouse model of obesity and insulin resistance.

BACKGROUND: As arterial thrombosis accounts for the vast majority of cardiovascular complications in obese, insulin resistant patients, we hypothesized that improving insulin sensitivity may be effective in reducing the thrombotic response following vascular injury. OBJECTIVES: We investigated the effect of the thiazolidinedione drug, pioglitazone, on the thrombotic response to injury in obese, insulin resistant mice. METHODS: Insulin-resistant, obesity-prone mice (KK strain) were treated with pioglitazone, placebo, or the sulfonylurea compound, glipizide, for 2.5 weeks and then subjected to photochemical injury of the carotid artery. RESULTS: KK mice have a significant increase in adiposity (7 weeks: 25.6%; 15 weeks: 34.4%; P < 0.0001) and thrombotic tendency (7 weeks: 21.2 +/- 1.9 min; 15 weeks: 13.7 +/- 1.7 min; P < 0.01) with age. Pioglitazone provided significant protection from thrombosis at both time points, prolonging the time to occlusive thrombosis by 40% and 68%, at 7 and 15 weeks of age, respectively (P < 0.05). Similarly, following a diet-challenge to promote diabetes, pioglitazone provided protection from occlusive thrombus formation (Placebo: 11.3 +/- 1.0 min; Pioglitazone: 22.3 +/- 3.9 min; P < 0.05). However, despite a salient effect of glipizide on the hyperglycemia of the mice, there was no effect on the time to occlusive thrombus formation (13.2 +/- 0.9 min, n = 4) compared with placebo-treated mice. The pioglitazone protection was paralleled by significantly lower soluble P-selectin and platelet P-selectin expression providing evidence of an antiplatelet effect. CONCLUSIONS: We conclude that pioglitazone treatment provides protection against arterial thrombosis in an obese, insulin resistant, prothrombotic mouse model.

Deficiency of tissue factor pathway inhibitor promotes atherosclerosis and thrombosis in mice.

BACKGROUND: Tissue factor initiates blood coagulation after atherosclerotic plaque disruption. Tissue factor pathway inhibitor (TFPI) inhibits tissue factor activity and may reduce thrombus formation in this setting. We evaluated the effect of heterozygous TFPI deficiency on the development of atherosclerosis and thrombosis in atherosclerosis-prone mice. METHODS AND RESULTS: Mice with a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency (TFPI(+/-)/apoE(-/-)) were generated by crossbreeding, and they were analyzed for atherosclerosis throughout the vascular tree. Compared with mice with a normal TFPI genotype (TFPI(+/+)/apoE(-/-)), mice with a TFPI deficiency exhibited a greater atherosclerotic burden involving the carotid and common iliac arteries. Staining for active tissue factor within the plaque revealed more activity in TFPI(+/-)/apoE(-/-) mice compared with TFPI(+/+)/apoE(-/-) mice. Consistent with increased plaque tissue factor activity, the time to occlusive thrombosis after photochemical carotid plaque injury was significantly decreased in TFPI(+/-)/apoE(-/-) mice. CONCLUSIONS: These observations indicate that TFPI protects from atherosclerosis and is an important regulator of the thrombosis that occurs in the setting of atherosclerosis.

Effects of acute exercise on plasma erythropoietin levels in trained runners.

PURPOSE: The purpose of this study was to investigate further the influence of exercise on erythropoietin. METHODS: We observed the effects of high intensity running on plasma erythropoietin concentration in competitive distance runners. A repeated measures design was used to compare the responses of intermittent high intensity (HIGH) exercise to continuous moderate intensity (MOD) exercise and rest (REST). The HIGH treatment consisted of 60 min of exercise alternating 5 min of running at approximately 90% of VO2max with 5 min of brisk walking. The MOD treatment consisted of a continuous 60-min run on the treadmill at 60% of VO2max. Blood samples were collected immediately before the exercise (PRE), immediately following the exercise (POST), and 4 (heart rate (4HR), 12 (12HR), 24 (24HR), and 48 (48HR)) h following the exercise. The variables examined included plasma erythropoietin concentration ([EPO]), hemoglobin (Hb) concentration ([Hb]), hematocrit (Hct), red blood cell count (RBC), and mean corpuscular volume (MCV). RESULTS: ANOVA revealed the expected treatment-by-time interaction for Hct and [Hb] suggesting a hemodilution at 24 and 48 h postexercise for the MOD and HIGH treatments. However, no significant treatment-by-time interactions were observed for [EPO], RBC, or MCV. CONCLUSION: These results indicate that intermittent high intensity exercise does not have a significant effect on [EPO] in trained distance runners.

Red cell volume and [erythropoietin] responses during exposure to simulated microgravity.

In order to test the hypothesis that normal gravity is an important influence on human serum [Erythropoietin] ([Epo]), the hematologic response to 16 d of 6 degrees head-down tilt (HDT, n = 6 men) was compared with 16 d of normal gravity exposure (CON, n = 7 men). Prior to bed rest, CON and HDT subjects, respectively, were similar in the following characteristics (mean +/- SD): age = 40 +/- 3, 39 +/- 6 yr; height = 181 +/- 5, 182 +/- 6 cm; weight = 88.5 +/- 11.3, 81.7 +/- 12.0 kg; maximal oxygen consumption in supine 6 degrees head-down tilt position (VO2max) = 2.63 +/- 0.38, 2.67 +/- 0.52 L.min-1; hematocrit = (Hct) 41.6 +/- 2.4, 43.0 +/- 3.4%; hemoglobin ([Hb]) = 15.1 +/- 1.0, 14.5 +/- 1.0 g.100 ml-1; plasma volume (PV) = 3829 +/- 857, 3768 +/- 512 ml; and [Epo] = 11.6 +/- 2.9, 10.0 +/- 6.2 mU.ml-1. Calculated red cell volume (RCV) was greater in HDT than CON (2845 +/- 410 vs. 2139 +/- 253 ml, p < 0.05) at baseline. Decreases in PV (-15%, 580 ml, p < 0.05) and an insignificant decrease in RCV (-12%, 354 ml, p = 0.07) were observed in the HDT group, with a concurrent 6% increase in [Hb] (p < 0.05). PV, RCV and [Hb] remained unchanged in the CON group. [Epo] remained unchanged during HDT (12.2 +/- 3.2; 10.8 +/- 3.8; 11.2 +/- 3.1; 11.2 +/- 2.6 mU.ml-1 for HDT days 1, 2, 8 and 16, respectively). There was no difference between CON and HDT groups in [Epo] before or during HDT. It was concluded that the insignificant change (-12%) in RCV observed during HDT was insufficient to stimulate an increase in [Epo], probably because the content of oxygen in arterial blood remained unaffected. The observation that [Epo] remained unchanged despite this loss of RCV during HDT also suggests a possible decrease in the responsiveness of the erythropoietic system to [Epo].