Maria das Graças Coelho de Souza

High fat diet induces central obesity, insulin resistance and microvascular dysfunction in hamsters

Microvascular dysfunction is an early finding in obesity possibly related to co-morbidities like diabetes and hypertension. Therefore we have investigated changes on microvascular function, body composition, glucose and insulin tolerance tests (GTT and ITT) on male hamsters fed either with high fat (HFD, n=20) or standard (Control, n=21) diet during 16 weeks. Total body fat and protein content were determined by carcass analysis, aorta eNOS and iNOS expression by immunoblotting assay and mean blood pressure (MAP) and heart rate (HR) by an arterial catheter. Microvascular reactivity in response to acetylcholine and sodium nitroprusside, functional capillary density (FCD), capillary recruitment induced by a hyperinsulinemic status and macromolecular permeability after 30 min ischemia was assessed on either cheek pouch or cremaster muscle preparations. Compared to Control, HFD animals have shown increased visceral fat (6.0 ± 0.8 vs. 13.8 ± 0.6g/100g BW), impaired endothelial dependent vasodilatation, decreased FCD (11.3 ± 1.3 vs. 6.8 ± 1.2/field) and capillary recruitment during hyperinsulinemia and increased macromolecular permeability after ischemia/reperfusion (86.4 ± 5.2 vs.105.2 ± 5.1 leaks/cm(2)), iNOS expression and insulin resistance. MAP, HR, endothelial independent vasodilatation and eNOS expression were not different between groups. Our results have shown that HFD elicits an increase on visceral fat deposition, microvascular dysfunction and insulin resistance in hamsters.

Substitution of drinking water by fructose solution induces hyperinsulinemia and hyperglycemia in hamsters

PURPOSE To test the possibility of obtaining a practical and stable model of hyperinsulinemia and hyperglycemia in hamsters, substituting the drinking water by 10% or 20% fructose solutions for a period of 2, 4, or 6 months. METHODS Male hamsters were divided into 3 main groups, further divided in 3 subgroups: Two months: Group Ia control (n = 51) received filtered water, Group Ib (n = 49) received 10% fructose solution instead of water, Group Ic (n=8) received 20% fructose solution instead of water. Four months: Group IIa control (n=8), Group IIb 10% fructose (n = 7), Group IIc 20% fructose (FIIc, n = 7). Six months: Group IIIa control (n = 6), Group IIIb 10% Fructose (n = 6), Group IIIc 20% Fructose (n = 5). All groups were fed with the same laboratory diet. The animals were weighed every 2 weeks during the study period. On the final day of each experiment (61st, 121st, and 181st day after the beginning of the study, respectively), the animals were weighed and anesthetized for blood collection to determine plasma glucose and insulin after at least a 12-h fast. Ten animals of group Ia and 10 of group Ib were evaluated to determine changes in macromolecular permeability induced by ischemia/reperfusion as measured in the cheek pouch microcirculation. RESULTS Compared to controls, the animals that drank the 10% or 20% fructose solution had significantly greater weight gain (P < .001), fasting plasma glucose (P < .001) Reperfusion, after 30 min ischemia, resulted in an immediate but reversible increase in postcapillary leakage (L) of 89.0 +/- 2.0 L/cm(2) (group Ia - controls), and 116.5 +/- 4.8 L/cm(2) (group Ib 10% fructose), P < .001. These results suggest that chronic administration of either 10% or 20% fructose solutions could be used to experimentally induce a stable hamster model of hyperinsulinemia and hyperglycemia. CONCLUSION The model might facilitate the study of basic mechanisms of hyperglycemia and hyperinsulinemia affecting the microvasculature as demonstrated by the findings regarding ischemia/reperfusion after only 2 months of treatment.

Novel findings in the cephalic phase of digestion: A role for microcirculation?☆

The cephalic phase of digestion (CPD) has been extensively investigated in terms of digestion and metabolism. Nevertheless, microcirculatory changes required to prepare peripheral tissues in order to dispose nutrients have never been assessed. In this study, microvascular function has been evaluated to determine its behavior and potential association to hormonal secretions during CPD. Thirty-nine healthy male subjects, 23.4 ± 0.5 years (mean ± SD) and BMI of 23.3 ± 2.3 kg/m(2), were randomized into receiving cognitive-sensorial stimuli to elicit CPD (CPD group, n=20) or not (control group, n=19), after a 12-h overnight fast. Main outcomes were differences in resting and peak functional capillary density (FCD, cap/mm(2)); resting red blood cell velocity (RBCV), peak RBCV (RBCV(max)) and time taken to reach it (TRBCV(max)); peak flow and vasomotion, before and after CPD and their associations with insulin and/or pancreatic polypeptide (PP). In the CPD group, basal FCD (24.9 ± 7.6 to 28.3 ± 8.1, p=0.005), peak FCD (27.8 ± 6.3 to 32.6 ± 7.1, p=0.002), RBCV (0.306 ± 0.031 to 0.330 ± 0.027 mm/s, p=0.005), RBCV(max) (0.336 ± 0.029 to 0.398 ± 0.292 mm/s, p=0.005) and peak flow (23.5 ± 14.3 to 26.9 ± 15.8 PU, p<0.01) increased while TRBCV(max) decreased (4.9 ± 1.5 to 3.5 ± 1.2s, p=0.01). No significant changes could be detected in the control group. Groups have not presented differences for insulin, but PP significantly increased in the CPD group and was positively associated to basal FCD increase (rho=0.527, p=0.03). In conclusion, neurally-mediated anticipatory responses of digestion elicited functional capillary recruitment associated to PP in healthy men, suggesting a precocious role for microcirculation in the physiology of digestion and nutrient homeostasis.

Effects of Resistance Training on Obese Adolescents.

PURPOSE The effects of resistance training (RT) alone upon endothelial function, metabolic and hemodynamic profiles, physical fitness, body composition, and inflammatory biomarkers in nondiabetic obese adolescents were investigated. METHODS Adolescents were assigned into nonobese control (CG, n = 20; 14.7 ± 1.4 yr) and obese (OB, n = 24; 14.1 ± 1.0 yr) groups. Muscle and skin endothelial reactivity, body composition, at-office and 24-h ambulatory blood pressure, metabolic profile, adipocytokines, aerobic and strength fitness were assessed before and after 12 wk of RT (CG, only at admission). RESULTS After RT, body mass did not change in OB, but significant reductions in body fat (1.6%; P = 0.01), waist circumference (2.9%; P < 0.001), waist-to-hip ratio (3.3%; P < 0.001), homeostasis model assessment for insulin resistance (15.4%; P = 0.02), endothelin-1 (14.2%; P = 0.04), and fibrinogen (6.9%; P = 0.03) were found. Both at-office and ambulatory blood pressure decreased, whereas skin endothelium-dependent vasodilation (32%; P = 0.02), VO2 (14.3%; P = 0.04), and HR (5.3%; P = 0.04) during submaximal exercise and isokinetic strength (extension, 21.3%; flexion, 29.9%; P < 0.0001) increased. Forearm vascular conductance increased at rest (28.1%; P = 0.03) and during postocclusive reactive hyperemia (25.2%; P = 0.02). After RT differences between CG and OB at admission were no longer detected for most outcomes. CONCLUSIONS RT alone improved endothelial function, hemodynamic and metabolic profiles, body composition, and physical fitness in nondiabetic obese adolescents regardless of changes in body mass.

Chronic aerobic exercise associated to dietary modification improve endothelial function and eNOS expression in high fat fed hamsters.

Obesity is epidemic in the western world and central adipose tissue deposition points to increased cardiovascular morbidity and mortality, independently of any association between obesity and other cardiovascular risk factors. Physical exercise has been used as non-pharmacological treatment to significantly reverse/attenuate obesity comorbidities. In this study we have investigated effects of exercise and/or dietary modification on microcirculatory function, body composition, serum glucose, iNOS and eNOS expression on 120 male hamsters treated for 12 weeks with high fat chow (HF, n = 30) starting on the 21st day of birth. From week 12 to 20, animals were randomly separated in HF (no treatment change), return to standard chow (HFSC, n = 30), high fat chow associated to an aerobic exercise training program (AET) (HFEX, n = 30) and return to standard chow+AET (HFSCEX, n = 30). Microvascular reactivity in response to acetylcholine and sodium nitroprusside and macromolecular permeability increase induced by 30 minutes ischemia followed by reperfusion were assessed on the cheek pouch preparation. Total body fat and aorta eNOS and iNOS expression by immunoblotting assay were evaluated on the experimental day. Compared to HFSC and HFSCEX groups, HF and HFEX ones presented increased visceral fat [(mean±SEM) (HF)4.9±1.5 g and (HFEX)4.7±0.9 g vs. (HFSC)*3.0±0.7 g and (HFSCEX)*1.9±0.4 g/100 g BW]; impaired endothelial-dependent vasodilatation [Ach 10−8 M (HF)87.9±2.7%; (HFSC)*116.7±5.9%; (HFEX)*109.1±4.6%; (HFSCEX)*105±2.8%; Ach10−6 M (HF)95.3±3.1%; (HFSC)*126±6.2%; (HFEX)*122.5±2.8%; (HFSCEX)*118.1±4.3% and Ach10−4 M (HF)109.5±4.8%; (HFSC)*149.6±6.6%; (HFEX)*143.5±5.4% and (HFSCEX)*139.4±5.2%], macromolecular permeability increase after ischemia/reperfusion [(HF)40.5±4.2; (HFSC)*19.0±1.6; (HFEX)*18.6±2.1 and (HFSCEX)* 21.5±3.7 leaks/cm2), decreased eNOS expression, increased leptin and glycaemic levels. Endothelial-independent microvascular reactivity was similar between groups, suggesting that only endothelial damage had occurred. Our results indicate that an aerobic routine and/or dietary modification may cause significant improvements to high fat fed animals, diminishing visceral depots, increasing eNOS expression and reducing microcirculatory dysfunction.

Aerobic exercise improves microvascular dysfunction in fructose fed hamsters

Fructose is a major diet component directly related to severe damages to the microcirculation and to diseases such as obesity, diabetes and hypertension to which physical activity is pointed out as an important non-pharmacological treatment since its positive effects precede anthropometric improvements. In this study we have investigated the effects of a light/moderate aerobic exercise training (AET) on microcirculatory dysfunction elicited by carbohydrate overload during a period of 5 months. Male hamsters (Mesocricetus auratus) whose drinking water was substituted (F) or not (C) by 10% fructose solution, during 20 weeks, associated or not to AET in the last 4 weeks (EC and EF subgroups) had their microcirculatory function evaluated on the cheek pouch preparation, glucose and insulin tolerance (GTT and ITT) tested. Arterial blood was collected for pO2, pCO2, HCO3(-), pH, total CO2, saturated O2 and lactate determinations. Liver fragments were observed using an electron microscope. Microcirculatory responses to acetylcholine [Ach, an endothelium-dependent vasodilator; 10(-8)M - *123.3±7.5% (C), 119.5±1.3% (EC), *98.1±3.2% (F) and 133.6±17.2% (EF); 10(-6)M - *133.0±4.1% (C), 135.6±4.3% (EC), *103.4±4.3% (F) and 134.1±5.9% (EF); 10(-4)M - *167.2±5.0% (C), 162.8±5.4% (EC), *123.8±6.3% (F) and 140.8±5.0% (EF)] and to sodium nitroprusside [SNP, an endothelium-independent vasodilator; 10(-8)M - 118.8±6.8% (C), 114.0±5.0% (EC), 100.2±2.9% (F), 104.9±4.4% (EF); 10(-6)M - 140.6±11.7% (C), 141.7±5.5% (EC), 125.0±4.7% (F), 138.3±2.8% (EF); 10(-4)M - 150.4±10.9% (C), 147.9±6.5% (EC), 139.2±7.3% (F), 155.9±4.7% (EF)] and macromolecular permeability increase induced by 30 min ischemia/reperfusion (I/R) procedure [14.4±3.5 (C), 30.0±1.9 (EC), *112.0±8.8 (F) and *22.4±0.9 leaks/cm(2) (EF)] have shown that endothelium-dependent vasodilatation was significantly reduced and I/R induced macromolecular permeability augmented in sedentary fructose (F) subgroup and both improved after AET. Electron microscopy analysis of the liver showed significant differences between exercised and sedentary subgroups with greater amount of glycogen in F subgroups compared to other ones. No significant changes on mean arterial pressure, heart rate or blood gase between subgroups could be detected. Our results point out that AET could normalize microcirculatory dysfunction elicited by long term substitution of drinking water by 10% fructose solution.

Relationships between emerging cardiovascular risk factors, z‐BMI, waist circumference and body adiposity index (BAI) on adolescents

The body adiposity index (BAI) has been recently proposed as an alternative index to body mass index (BMI) and waist circumference (WC) to evaluate adiposity in adults, with special focus on its ability to discriminate gender specificities on adiposity. Endothelial dysfunction, circulating endothelial cells (CECs), endothelin‐1 and adipocytokines are all related to atherosclerosis and nowadays considered as markers of emerging cardiovascular (CV) risk. This study aimed to determine in normal weight and obese adolescents which measures of body composition (BAI and z‐BMI) or distribution (WC) correlate better with emerging CV risk markers.

Arteriolar diameter and spontaneous vasomotion: Importance of potassium channels and nitric oxide

Arterioles display cyclic variations in diameter, termed vasomotion initiated by smooth muscle cells (SMCs), but the endothelium should also be evaluated due to its modulatory role on vessel tone. Since nitric oxide (NO) and prostacyclin (PGI2) regulate SMC tone and activate K(+) currents, we have investigated their role on vasomotion, by observing effects of topical application of N(ω)-nitro-l-arginine (L-NA, NO synthesis inhibitor), glibenclamide (KATP channel inhibitor), sodium nitroprusside (SNP, NO donor), iloprost (PGI2 analogue) and methylene blue (MB, cGMP production inhibitor) on the cheek pouch preparation of anesthetized male hamsters. L-NA (10(-10)-10(-6)M) induced vasoconstriction, reduction and abolition of vasomotion. MB (10(-7) to 10(-5)M) reduced mean arteriolar diameter with no changes on vasomotion. In the presence of 10(-6)M of MB, addition of 10(-6)L-NA totally abolished vasomotion without further constriction. Glibenclamide (10(-6)M) in the presence of L-NA at equimolar concentration restored both vasomotion frequency and amplitude. This effect was not observed in the presence of TEA 5mM. SNP (10(-10)-10(-6)M) induced a dose-dependent increase of arteriolar diameter and decreased vasomotion. Iloprost (10(-12)-10(-6)M) induced a concentration dependent increase of arteriolar diameter, reduced vasomotion frequency, but in lower concentrations (10(-12)-10(-10)M) increased its amplitude and in higher concentrations (10(-9)-10(-6)M) decreased it. SNP and iloprost inhibited vasomotion at 10(-7)M; however, at this concentration SNP and iloprost induced an increment of 35% and 50% of the initial arteriolar diameter, respectively. In the presence of L-NA (10(-6)M), vasomotion was restored by SNP at 10(-10)M and iloprost 10(-12)M, which corresponded to 80% of the initial diameter value. Around the initial (control) arteriolar diameter value, vasomotion presented its highest frequencies and amplitudes. Cessation of vasomotion occurred with L-NA (10(-6)M) in the presence of SNP (10(-6)M) and iloprost (10(-7)M) when arteriolar diameter reached 150% and 120% of its initial value, respectively. In conclusion, the present study strongly suggests that vasomotion (1) is not solely related to vascular tone, (2) needs an interplay between vascular tone and membrane currents and (3) could be modulated by NO (but not cGMP) and KATP channels. In addition, our results point to the existence of dissociation between vasomotion frequency and amplitude.

Beneficial effects of the micronized purified flavonoid fraction (MPFF, Daflon® 500 mg) on microvascular damage elicited by sclerotherapy

Objectives To evaluate if the micronized purified flavonoid fraction (MPFF) treatment could reduce the side effects of sclerotherapy (a procedure frequently used to treat venous disease manifestations) by minimizing the inflammatory response within the surrounding tissues. Method Twenty-two male New Zealand rabbits were treated by gavage with micronized purified flavonoid fraction (MPFF; 300 mg/kg/day) or vehicle (10% lactose solution) during 21 consecutive days, starting 7 days before sclerotherapy. The sclerotherapy consisted of an injection containing 5% ethanolamine oleate solution in the rabbit’s dorsal ear vein. Before and after sclerotherapy, venular and arteriolar diameters, microvascular permeability, functional capillary density (FCD), number of rolling and sticking leukocytes were evaluated on ear microcirculation. Images of the sclerotherapy site were taken before and after the procedure. Results Compared to placebo, MPFF treatment prevented the increase in venular diameter, preserved FCD (P < 0.001) and reduced the number of leaky sites (P < 0.001) and sticking leukocytes (P < 0.001). Imaging confirmed these effects on thrombosis and perivascular edema of the sclerosed vein, 14 days after procedure. Conclusion MPFF treatment limited the postsclerotherapy inflammation in surrounding microvascular network, suggesting that MPFF may prevent undesirable secondary effects of the procedure in this animal model. This study warrants further investigation for its use in clinical conditions.

Low testosterone levels are associated with endothelial dysfunction in oophorectomized early postmenopausal women

BACKGROUND The actual consequences of low testosterone levels in women remain uncertain. OBJECTIVE To assess endogenous testosterone influence on body composition, vascular and metabolic function in recent postmenopausal women. DESIGN We studied 81 postmenopausal women under transdermal estradiol (E2) replacement therapy, 36 with bilateral oophorectomy (group O), and 45 controls (group C) through venous occlusion plethysmography, bioimpedance, DEXA, biochemical, hormonal, and inflammatory profile. RESULTS Total testosterone level (TT) in group O was 11.0 (4.0-17.75) vs 23.0 (10.0-42.5) ng/dl in group C (P=0.001). Forearm blood flow, in ml/min/100  ml tissue, was lower in group O compared to group C at baseline (1.57 (1.05-2.47) vs 2.19 (1.59-2.66) P=0.036), following reactive hyperemia response (endothelium-dependent flow mediated dilatation, 3.44 (2.38-4.35) vs 4.3 (3.09-5.52), P=0.031) and following nitroglycerin (endothelium-independent dilation, 1.39 (0.99-1.7) vs 1.76 (1.15-2.0), P=0.025), with a positive correlation between TT and all parameters except for the reactive hyperemia response (r=0.233-0.312, P=0.036-0.004). The sVCAM1 levels were negatively correlated with TT (r=-0.320, P=0.005). E2 and other hormone levels, biochemical parameters and body composition did not differ between groups. Multiple linear regressions showed that the levels of TT, compared with other confounding variables, may explain the variation observed on endothelial parameters, with low explanatory power. CONCLUSION The absence of ovarian testosterone production in recent postmenopausal oophorectomized women was associated with deleterious effects on endothelial function.