Introduction
Type 2 diabetes (T2D) is a common term for heterogeneous metabolic disorders, and its main characteristic is hyperglycemia.1 It is characterized by insulin resistance, and is closely associated with obesity. T2D is strongly associated with metabolic alterations in various tissues, including the liver, leading to the development of non-alcoholic fatty liver disease (NAFLD). in up to 70% of patients with T2D, and is responsible for accelerating the progression of the disease.4,5 In addition, as the disease progresses, the risk of developing macrovascular and microvascular complications increases.6 We have shown that the Type 1 diabetic (T1D) rats have hepatic microcirculatory dysfunction, which can be prevented by oral hypoglycemic agents.7 We also demonstrated that NAFLD is closely related to hepatic microcirculatory abnormalities, such as increased leukocyte recruitment and decreased of hepatic microvascular blood flow.8–10 In addition, patients with T2D have alterations in the microcirculation of adipose tissue, which is associated with obesity, insulin resistance, hyperglycemia and dyslipidemia.11,12.
Advanced glycation end products (AGEs) are proteins or lipids altered non-enzymatically 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 dysfunction intracellular and extracellular through its ability to irreversibly bind collagen and other extracellular matrix proteins and by binding to its receptor, RAGE.15,16 The AGE-RAGE junction 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 its associated comorbidities17–20 by improving insulin sensitivity, glycosylated hemoglobin, glycemic parameters, lipid profile , blood pressure and body fat21,22. obese mice, exercise training reduced hepatic steatosis, lipogenic gene expression, and hepatic inflammation, independent of body adiposity, and increased phosphorylation of acetyl-CoA carboxylase and oxidative genes , reversing complications caused by T2D in the liver23. During exercise, skeletal muscles act as endocrine-like organs, producing myokines and exerting a multiorgan effect on contractile and noncontractile tissues, including liver and adipose tissue.24 Several molecular and metabolic mechanisms appear to be involved. in the beneficial effects of exercise on T2D. , but the underlying mechanisms that contribute to the improvement of microcirculatory function are still unclear. A possible role of the AGE-RAGE pathway was recently 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 improving microcirculation by aerobic training involves modulation of the AGE-RAGE pathway, downregulation of HSC, and oxidative stress.
Materials and methods
Design of studies and animals
Male C57BL/6 mice (8 weeks old) were obtained from the central animal care facility of the Oswaldo Cruz Foundation (RJ, Brazil) and maintained in standard cages at controlled room temperature (22 ± 1 ° C) and 12 hours of dark light. cycle (dark from 6 p.m.). They were divided into two groups: control mice (CTL) receiving a commercial grain-based diet (Nuvilab-Quimtia) and diabetic mice (T2D) receiving a high-carbohydrate diet (HCHF) and 25% fructose a l drinking water ad libitum (Figure 1A). The HCHF diet consisted of a modified grain-based diet consisting of 55% fat and 35% carbohydrate.29 After 24 weeks, the mice were 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 without exercise, n=10) and T2D EX (HCHF diet plus 25% fructose exercise, n=10 ). Mice underwent liver ultrasound, echocardiogram, and microcirculatory analysis 24 hours after the last exercise session, while systolic blood pressure and oral glucose tolerance test (OGTT) were assessed 48 hours 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 the liver, heart , visceral and subcutaneous WAT. collected deposits. Blood serum was obtained by centrifugation (700 × g for 15 min at 4°C) and aliquots were stored at −80°C for further analysis. The Animal Welfare Committee of the Oswaldo Cruz Foundation approved all experimental protocols (license L-0012/18 A1), which were performed following the principles of 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 grain-based diet throughout the experiment; the sedentary type 2 diabetes 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 min session, 3 times per week, 12 weeks) (CTL EX); and the exercise group of type 2 diabetics, who 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 assessments of liver microcirculation and adipose tissue by in vivo microscopy and laser spot 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 groups 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 created with BioRender.com.
Maximum exercise capacity
Animals were first acclimated to treadmill walking (Hectron Fitness Equipment, Brazil) using a 15-min session at 12 m/min for three consecutive days during study week 23. At week 24, the maximal exercise capacity test was performed on a treadmill at 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 in the shock grid at the end of the mat 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
The physical exercise started at the 24th week of dietary feeding and lasted for 12 weeks. The animals were exercised in the morning (8:00 a.m. to 12:00 p.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 maximum. exercise capacity test (~75 to 80% of maximal oxygen uptake). This intensity has previously been shown to be able to restore cardiac microvascular rarefaction in obese animals with metabolic syndrome.31
blood pressure
Noninvasive blood pressure measurements were performed in the mouse tail using photoplethysmography with automatic data acquisition (Insight, Brazil). Before the first measurement, the animals were adapted to the containment container to minimize stress and blood pressure fluctuations. The measurement protocol began with 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
Heart and liver ultrasound
Mice were anesthetized with 2% isoflurane, underwent abdominal hair removal, and were placed in the supine position on a heated table. Ultrasound was performed using a sound-conducting gel (Carbogel, Brazil) applied to the mid-abdomen and the VEVO 770 system (VisualSonics, Canada) connected to a 30 MHz transducer. Ejection fraction, fractional area change, systolic volume, end-diastolic and systolic volumes were evaluated during the echocardiogram. 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
Intravital microscopy
The left lateral lobe of the liver and the epididymal fat pad were exteriorized by laparotomy followed by microcirculation analysis. A monitor displayed the images for analysis using a 10x objective for intravital microscopy (Olympus BX150WI, USA). To examine the interaction between leukocytes and endothelium, the number of labeled leukocytes (0.3 mg/kg rhodamine 6G, iv) rolling or adhering to sinusoidal and postsinusoidal venules was counted. Leukocytes were counted for 30 seconds in an area of 170 μm2. Leukocytes with a velocity lower than blood flow were classified as rolling, and those that remained stationary were classified as adherent cells35,36.
Laser speckle contrast imaging (LSCI)
LSCI (Pericam System PSI, Sweden) assessed the microvascular blood flow of the liver and epididymal fat.37 LSCI provides an index of microcirculation perfusion corresponding to the average concentration and velocity of blood cells, while assesses microvascular blood flow in real time.10 Mice were kept stable. …