Advanced glycation end products (AGEs) are proteins or lipids that are not enzymatically altered by the action of reducing sugars. AGEs are produced throughout life and are increased under conditions such as oxidative stress and hyperglycemia, and several studies suggest that AGEs play a role in diabetes-related complications.13,14 For example, AGEs promote intracellular and extracellular dysfunction through their ability to bind collagen and other extracellular matrix proteins irreversibly and by binding to its receptor, RAGE binding.15,16 AGE-RAGE activates genes involved in oxidative stress, inflammation, thrombosis and leukocyte recruitment16 . Therefore, the AGE-RAGE axis is involved in the pathogenesis and progression of vascular dysfunction in T2D. Non-pharmacological interventions such as diet and exercise training can prevent or delay the progression of T2D and related comorbidities17-20 by improving insulin sensitivity, glycated hemoglobin, glycemic parameters, lipid profile, blood pressure and body fat.21,22 In T2D/obese mice, exercise training reduced hepatic steatosis, adipogenic gene expression, and liver inflammation, independent of body fat, and increased the phosphorylation of acetyl-CoA-carboxylase and of oxidative genes, reversing T2D-induced complications in the liver.23 Furthermore, during exercise, skeletal muscles function as endocrine-like organs, producing myokines and exerting multiorgan effects on contractile and noncontractile tissues, including the liver and adipose tissue. , but the underlying mechanisms that contribute to the improvement of microcirculatory function remain unclear. A possible role of the AGE-RAGE pathway has recently been discussed, but the data are still controversial.25–28 Therefore, we aimed to investigate the effects of aerobic training on liver and adipose tissue microcirculation in diabetic C57BL/6 mice. We hypothesize that the underlying mechanism for the improvement of microcirculation by aerobic training involves the regulation of the AGE-RAGE pathway, the reduction of HSC and oxidative stress.
Materials and methods
Design and Animal Study
Male C57BL/6 mice (8 weeks old) were obtained from the central animal care unit of the Oswaldo Cruz Institution (RJ, Brazil) and maintained in standard cages at a controlled room temperature (22 ± 1 °C) and 12 h light-dark cycle ( dark begins at 6 pm). They were divided into two groups: control mice (CTL) fed a commercial cereal-based diet (Nuvilab-Quimtia) and diabetic mice (T2D) fed a high-carbohydrate diet (HCHF) and 25% fructose in the drinking water ad libitum (Figure 1A). The HCHF diet consisted of a modified cereal-based diet consisting of 55% fat, 35% carbohydrate.29 After 24 weeks, mice were further divided into four groups based on exercise: CTL (control diet without physical exercise, n= 10), CTL EX (control diet and exercise, n=10), T2D (HCHF diet plus 25% fructose no exercise, n=10) and T2D EX (HCHF diet plus 25% fructose exercise, n=10) . Mice underwent liver ultrasound, echocardiography, and microcirculation analysis 24 h after the last exercise session, while systolic blood pressure and oral glucose tolerance test (OGTT) were assessed 48 h after the last exercise session. After an overnight fast, liver and adipose tissue microcirculation was assessed in anesthetized mice (ketamine hydrochloride 100 mg/kg and xylazine 10 mg/kg, IP), blood was collected by cardiac puncture, and liver, heart, viscera and the subcutaneous WAT depots are harvested. Blood serum was obtained by centrifugation (700 × g for 15 min at 4 °C), and samples were stored at −80 °C for subsequent analysis. The Animal Protection Committee of the Oswaldo Cruz Foundation approved all experimental protocols (permit L-0012/18 A1), which were performed in accordance with the principles for the care and use of laboratory animals. Figure 1 Effect of physical exercise on hemodynamic and metabolic parameters in mice with type 2 diabetes (T2D). Study design (A) C57BL/6 mice were randomly divided into 4 groups: sedentary control group (CTL), which received a cereal-based diet throughout the experiment; the sedentary type 2 diabetic group, which had access to a high-carbohydrate, high-fat diet throughout the experiment (T2D); the exercise control group, which received normal food and underwent an exercise protocol (30-minute session, 3 times a week, 12 weeks) (CTL EX). and the type 2 diabetic exercise group, which had access to a high-carbohydrate, high-fat diet and underwent the same exercise protocol (T2D EX). At the end of the 36-week protocol, mice underwent in vivo analysis, including systolic blood pressure analysis, liver ultrasound, as well as liver and adipose tissue microcirculation assessments by in vivo microscopy and laser contrast imaging. The following parameters are shown: body weight during the experimental protocol (B), serum glucose levels during the oral glucose tolerance test (OGTT) (C) and AUC (D) of the CTL, CTL EX, T2D and T2D EX. *P < 0.05 T2D vs. CTL; **P <0.01 T2D vs. CTL; ***P < 0.001 T2D vs. CTL; #P < 0.05 T2D vs T2D EX; ##P < 0.01 T2D vs T2D EX. Figure 1A generated with BioRender.com.
Maximum exercise capacity
Animals were first acclimatized to treadmill walking (Hectron Fitness Equipment, Brazil) through a 15-min session at a speed of 12 m/min for three consecutive days at the 23rd week of the study. At week 24, the maximal exercise capacity test was performed on a treadmill at a progressive speed to exhaustion (10 m/min increased by 3 m/min every 3 min). Exhaustion was determined when the animal could no longer maintain the pace and remained on the shock grid at the end of the carpet for at least 5 s. Exercise intensity was determined based on the maximum speed achieved during the test.30,31 At week 30, mice underwent a second test to adjust the intensity of exercise training (data not shown).
Physical exercise
Physical exercise began on the 24th week of the diet and lasted 12 weeks. Animals were exercised in the morning (8 a.m. to 12 a.m.) on a treadmill (Hectron Fitness Equipment, Brazil) at 0% incline, three times a week, with 30 minutes per session, at 80% of the maximum speed achieved at maximal exercise capacity test (~75 to 80% of maximal oxygen uptake). This strain has previously been shown to be capable of restoring cardiac microvascular insufficiency in obese animals with metabolic syndrome.31
Blood Pressure
Noninvasive blood pressure measurements were performed in the mouse tail by photoplethysmography with automatic data acquisition (Insight, Brazil). Before the first measurement, animals were acclimated to the containment vessel to minimize stress and blood pressure fluctuations. The measurement protocol started by pre-warming the animals to a temperature of 36 °C for 5 min. Mice were then placed in the device to measure systolic blood pressure, and the result was expressed as the average of three measurements.32
Ultrasound of the Heart and Liver
Mice were anesthetized using 2% isoflurane, abdominally shaved, and placed supine on a heated table. Ultrasound was performed using a sound-conducting gel (Carbogel, Brazil) applied to the mid-ventricle and the VEVO 770 system (VisualSonics, Canada) connected to a 30 MHz transducer. Ejection fraction, fractional area change, stroke volume, end-diastolic and systolic volumes were assessed during echocardiography. Liver ultrasound assessed the echogenicity of the liver parenchyma. All measurements were determined by a single observer who was blinded to the study design.33,34
Endoscopic microscopy
The left lateral lobe of the liver and epididymal fat were excised by laparotomy followed by microcirculation analysis. A screen displayed the images for analysis using a 10x objective lens for in vivo microscopy (Olympus BX150WI, EUA). To examine the interaction between leukocytes and the endothelium, the number of labeled leukocytes (0.3 mg/kg rhodamine 6G, iv) rolling or adhering to sinusoidal and postatrial venules was measured. Leukocytes were counted for 30 seconds in an area of 170 µm2. Leukocytes with a velocity less than that of blood flow were classified as rolling and those that remained stationary were classified as adherent cells.35,36
Laser Speckle Contrast Imaging (LSCI)
LSCI (Pericam System PSI, Sweden) assessed microvascular blood flow in the liver and epididymal fat.37 LSCI provides an index of microcirculatory perfusion that corresponds to the mean concentration and velocity of blood cells while assessing microvascular blood flow in real time. 10 The mice were kept in constant …
