Sleeve gastrectomy improves cardiac function and glucose-lipid metabolism disorder in obese rats induced by a high-fat and high-sugar diet

Abstract

Obesity is affecting global health with multiple complications, including cardiac dysfunction. Currently, it is uncertain whether drug therapy should be applied in the early stages of obesity-induced cardiac dysfunction, with weight reduction as the first choice. Sleeve gastrectomy (SG) has been widely used to treat obesity and its complications, showing promising results. However, it remains unclear whether SG can alleviate obesity-induced cardiac dysfunction. A sudden decline in body weight and food intake was observed in both the obese and obese + SG groups, with a higher rate of increase observed in the Obese group. Elevated levels of plasma glucose, serum insulin, and glycated haemoglobin in obese rats were significantly reduced by SG. Markedly increased levels of alanine transaminase, aspartate transaminase, alkaline phosphatase albumin, total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels, elevated values of heart rate, left ventricular end-systolic pressure, left ventricular end-diastolic pressure, systolic pressure, and end diastolic pressure, and decreased value of stroke volume were observed in obese rats, which were sharply reversed by SG. Furthermore, enhanced pathological changes, including inflammatory cell infiltration and loss of cytoplasm striations, enhanced oil red O staining, increased TUNEL-positive cells, upregulated Bax and cleaved-caspase-3, and downregulated Bcl-2, were observed in obese rats, which were notably alleviated by SG. Lastly, the increased levels of relative proteins observed in obese rats were significantly reduced by SG. In conclusion, SG improved cardiac function and glucose-lipid metabolism disorders in obese rats induced by a high-fat and high-sugar diet.

OBESITY is a chronic disease closely related to lifestyle, characterized by excessive nutrition, insufficient exercise, and behavioral biases, leading to generalized excessive proliferation and accumulation of adipose tissue throughout the body [1]. It is a metabolic disease caused by multiple factors. The World Health Organization (WHO) defines obesity as having a body mass index (BMI) greater than 30 kg/m², while China’s standard for obesity is a BMI of 28 or higher. A cross-sectional study of 188 countries and regions over a 30-year period revealed that the global population of obese and overweight individuals has increased from 857 million in 1980 to 2.1 billion in 2016, accounting for approximately 30% of the global population, with this trend rapidly spreading worldwide [2, 3]. Each year, as many as 4.3 million people die from complications related to obesity. According to WHO data, China ranks second in the world in terms of the number of obese individuals, with 46 million obese adults and 300 million overweight adults [4]. Obesity is often accompanied by various complications, particularly an increased risk of heart dysfunction. Koehler et al. [5] reported that obese children exhibited significantly prolonged recovery time after submaximal treadmill exercise testing, with 25% of children showing EKG changes and early activation of cardiac reserve as a compensatory mechanism. Changes in hemodynamics can lead to ventricular hypertrophy, increased susceptibility to dilation, and subsequent increase in ventricular wall tension. If dilation occurs at a faster rate than hypertrophy, it can lead to the development of left ventricular systolic dysfunction. Conversely, if hypertrophy develops faster than dilation, it may lead to left ventricular diastolic dysfunction. Left ventricular posterior wall thickness, interventricular septum thickness, and left ventricular myocardial mass (LVM) are significantly increased in obese patients. LVM is positively correlated with body weight and blood pressure, while LVM index is significantly positively correlated with the percentage of overweight individuals [6, 7]. Although drugs used to treat chronic heart failure can potentially alleviate obesity-induced cardiac dysfunction, it remains uncertain whether drug therapy should be employed in the early stages of obesity-induced cardiac dysfunction. Currently, weight reduction strategies remain the primary choice for treating obesity patients.

Bariatric surgery, also known as metabolic surgery, is an effective surgical treatment that can achieve long-term weight loss in obese patients [8]. In 1966, Roux-en-Y gastric bypass (RYGB) was first reported for the treatment of morbid obesity. Until 2013, RYGB was the most commonly used surgical procedure worldwide in clinical practice and was once considered the gold standard [9]. Since 2014, sleeve gastrectomy (SG) has become the mainstream procedure worldwide due to its advantages of a low complication rate, short operation time, no change in gastrointestinal anatomy sequence, high patient acceptance, and feasibility of later corrective surgery [10]. In the past decade, studies have shown that the gastrointestinal changes associated with SG achieve weight loss not only through volume-limiting effects and absorption effects but also by treating and improving metabolic disorders, especially type 2 diabetes mellitus [11, 12]. However, it remains unclear whether SG can alleviate obesity-induced cardiac dysfunction by controlling body weight. In this study, we were aimed to explore and validate the feasibility of SG in treating and alleviating obesity-induced cardiac dysfunction in a rat model, providing potential treatment strategies for clinical practice.

Materials and Methods

Animals, modeling, and grouping

Eighteen male SD rats (7–9 weeks old) were obtained from Vital River (Beijing, China). The rats were divided into three groups: Control, Obese, and Obese + SG (n = 6/group). In the control group, rats were freely fed a normal diet. In the Obese and Obese + SG groups, rats were fed a high-fat and high-sugar diet for 14 weeks to induce obesity. SG was performed on rats in the Obese + SG group, while a Sham operation was conducted on rats in the Obese group. For SG, the rats were fully anesthetized until the pain reflex and corneal reflex disappeared. They were then placed in the supine position and secured on the operating table. The hair was shaved, the area was sterilized, a sterile drape was applied, and sterile gloves were worn. A 2 to 2.5 cm incision was made in the middle of the abdomen, starting 2 to 3 cm below the xiphoid process. Once the abdominal cavity was entered, the incision was covered with a saline-soaked gauze. The liver was gently separated using a sterile medical cotton swab to expose the stomach of the rat. Non-traumatic forceps were used to free the stomach from the abdominal cavity, and the hepatic and gastrosplenic ligaments were cut. The short gastric vessels and the gastroepiploic vessels adjacent to the pylorus were ligated and transected using a 5-0 silk suture. The greater curvature of the stomach was clamped using a non-traumatic bowel clamp, and then the fundus and 3/4 of the body of the stomach were resected starting 3 to 5 mm proximal to the pylorus and continuing to the angle of His. The residual stomach was cleaned using a saline-soaked cotton swab, and the gastric margin was continuously sutured using a 5-0 absorbable suture. Any bleeding points were intermittently sutured. After ensuring there was no active bleeding, the abdominal cavity was flushed with warm physiological saline 2 to 3 times and closed. For the Sham operation, the procedures of anesthesia, skin preparation, disinfection, draping, laparotomy, and dissection were the same as those for SG. However, no invasive procedures such as resection were performed. To eliminate the influence of anesthesia and surgical stress, the duration of surgery and anesthesia in the Sham operation group was prolonged to match that of SG. The body weights and food intake of each animal were recorded for 40 days post-surgery. Afterwards, the animals were euthanized, and blood and tissues were collected for analysis. All animal experiments were reviewed and approved by the Ethics Committee of Laizhou People’s Hospital. The high fat and high sugar diet consisted of a high-fat diet (46.9% of provided calories as fat, with 21% w/w cocoa butter containing 25% palmitic acid and 35% stearic acid) and free access to a 10% w/v fructose solution as the drinking beverage.

Detection of plasma glucose, glycated haemoglobin (HbA1c), and serum insulin

Blood was collected from each animal, and plasma and serum were obtained. A glucometer (Bayer, Germany) was used to measure plasma glucose level, while serum insulin level was detected using the method reported by Burgi [13]. The HbA1c level was measured using the method reported by Pattabiraman [14].

Detection of the cardiac function in rats

Cardiac function was detected by BL-420F biological function experiment system. The parameters included heart rate (HR), left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), systolic pressure (SP), end-diastolic pressure (EDP), and stroke volume (SV).

Blood biochemical tests

Rats were anesthetized with 4 mL/kg of 1% pentobarbital sodium injected intraperitoneally. Once the anesthesia took effect, the rats were placed in a supine position, and a sterile incision was made to open the abdomen. The abdominal aorta was then sampled and the serum was collected after centrifugation. The levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), albumin, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were measured using a blood biochemistry analyzer (Bayer, Germany).

HE staining

After sacrificing the rats, the left ventricles were removed and divided into two halves along the ligation line to preserve the area between the ligation line and the apex. The tissue was fixed with 4% paraformaldehyde, dehydrated with gradient alcohol, embedded in paraffin, and then made into 5 μm thick tissue slide specimens. Mayer’s hematoxylin was added and incubated for 10 minutes, followed by incubation with a 0.5% eosin solution for 3 minutes. Images were captured using a microscope (KEYENCE, Japan).

Oil Red O staining

The tissue slides were dewaxed and hydrated, followed by adding the oil red O staining solution. After a 30-minute incubation, the sections were washed with 70% isopropanol to remove any unbound dye. Images were captured using a microscope (KEYENCE, Japan).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)staining

The paraffin sections were dewaxed using xylene and gradient ethanol, followed by a 30-minute incubation with protease K without DNase. The sections were then washed with an immunostaining buffer for approximately 30 minutes, followed by a rinse with phosphate-buffered saline (PBS). After adding 50 μL of the TUNEL reaction solution, the sections were incubated in the dark for 60 minutes. They were then sealed with an anti-fluorescence quenching mounting medium and observed under a fluorescence microscope (KEYENCE, Japan), to assess myocardial cell apoptosis.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA was extracted from heart tissues using the TRIzol method (Invitrogen, USA). The content and purity of the RNA were assessed using an ultra-micro spectrophotometer. Subsequently, the RNA was reverse transcribed into cDNA using a reverse transcription kit (QIAGEN, Germany). The reaction system was prepared according to the manufacturer’s instructions, with 400 ng mRNA added to each reverse transcription system. Quantitative PCR was performed using a qPCR kit (QIAGEN, Germany). Glyceraldehyde phosphate dehydrogenase (GAPDH) served as the internal reference, and the gene levels were analyzed using the 2^–ΔΔCt method. The primer sequences are listed in Supplementary Table 1.

Western blotting assay

Heart tissues were treated with RIPA lysate and homogenized on ice using a glass homogenizer. Total proteins were extracted and the protein concentration was determined using the bicinchoninic acid method. Each well of a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel was loaded with 20 μg of protein for separation, and the proteins were then transferred onto a polyvinylidene fluoride membrane. After transfer, the membranes were blocked with 5% skim milk. Primary antibodies against Bax (1:1000), Caspase-3 (1:800), Bcl-2 (1:800), BDNF (1:800), ANP (1:1000), BNP (1:800), Myh7 (1:1000), CoL1 (1:800), CoL3 (1:800), and GAPDH (1:2000, CST, USA) were incubated with the membrane. Subsequently, the membranes were incubated with the secondary antibody (1:4000, CST, USA) and exposed to an electrochemiluminescence solution. The image J system was used to analyze the gray value of protein bands.

Statistical analysis

Data were expressed as mean ± standard deviation and analyzed using GraphPad software. The comparison was performed using the one-way analysis of variance method with Tukey’s test. A p-value of less than 0.05 was considered statistically significant.

Results

SG suppressed the body weight and food intake in obese rats

No significant differences in exercise were observed among all groups. Firstly, within 40 days after SG, the body weights and food intake were recorded and presented in Fig. 1A, B. Over time, a slight increase in body weight and minor changes in food intake were observed in the control group. A sudden decline in body weight and food intake was observed in both the obese and obese + SG groups, with a greater decline observed in the obese + SG group. Furthermore, as time went on, the rate of increase in body weight and food intake in obese rats was significantly suppressed by SG, suggesting a suppressive effect of SG on obesity in rats.

Fig. 1

SG suppressed the body weight and food intake in obese rats. (A) Changes in rat body weight during the 40-day post-surgery period. (B) Mean food intake of rats during the 40-day post-surgery period.

SG improved the glucose metabolism in obese rats

As depicted in Fig. 2A, the significantly elevated plasma glucose level, serum insulin level, and HbA1c level observed in obese rats were markedly reduced following SG. This indicates that SG effectively alleviates the impaired glucose metabolism in obese rats.

Fig. 2

SG improved the glucose metabolism, liver metabolic function, and dyslipidemia in obese rats. (A) Plasma glucose level, serum insulin level, and the HbA1c level were determined. (B) AST (IU/L), ALT (IU/L), ALP (IU/L), and albumin (g/L) were measured. (C) Total cholesterol (TC) level, Triglyceride (TG) in level, and Low-density lipoprotein cholesterol (LDL-C) level were detected. (* p < 0.05 vs. Control, ** p < 0.01 vs. Control, ^# p < 0.05 vs. Obese, ^## p < 0.01 vs. Obese).

SG improved the liver metabolic function in obese rats

Impaired hepatic function is commonly observed in the obese state [15]. The levels of AST, ALT, and ALP were significantly elevated, while the albumin level was greatly decreased in obese rats. However, these effects were significantly reversed following SG, indicating a restorative effect of SG on hepatic function in obese rats (Fig 2B).

SG improved the dyslipidemia in obese rats

Dyslipidemia is a critical characteristic in obesity [16]. The levels of TC, TG, and LDL-C in obese rats were significantly reduced by SG, indicating the protective effect of SG on the disorder of lipid metabolism in obese rats (Fig 2C).

SG alleviated the cardiac function in obese rats

To assess the impact of SG on cardiac function in obese rats, the BL-420F biological function experiment system was used to measure various parameters in each animal 40 days after SG. Obese rats exhibited significantly elevated values of HR, LVESP, LVEDP, SP, and EDP, as well as a decreased SV value. However, after SG, there was a notable reduction in HR, LVESP, LVEDP, and EDP values, along with an increase in SV (Fig. 3A–F). These findings suggest that SG has a reparative effect on cardiac function in obese rats.

Fig. 3

SG alleviated the cardiac function in obese rats. (A) Heart rate (HR); (B) Left ventricular end systolic pressure (LVESP); (C) Left ventricular end-diastolic pressure (LVEDP); (D) Systolic pressure (SP); (E) End diastolic pressure (EDP); (F) Stroke volume (SV). (* p < 0.05 vs. Control, ** p < 0.01 vs. Control, ^# p < 0.05 vs. Obese, ^## p < 0.01 vs. Obese).

SG improved the pathological state in left ventricles of obese rats

As shown in Fig. 4A, the control group exhibited neatly and densely arranged myocardial cells with visible cytoplasmic striations and normal nuclear morphology. The intercellular structures were clear, and the connective tissue between muscle fibers was normally arranged. In contrast, the Obese group displayed distorted myocardial cell morphology, characterized by cell enlargement, disorganized arrangement, loss of cytoplasmic striations, increased extracellular matrix, local infiltration of inflammatory cells, nuclear condensation, and even fragmentation and dissolution of nuclei. Following SG, the extent of myocardial cell damage was reduced compared to the Obese group. The nuclear morphology was approximately normal, although there was a slight disordered arrangement of local myocardial cells and mild infiltration of inflammatory cells. As shown in Fig. 4B, the percentage of oil red O staining in the Obese group was markedly increased, which was notably reduced in the Obese + SG group, suggesting suppressive effect of SG on fat deposits in myocardial cells.

Fig. 4

SG improved the pathological state in left ventricles of obese rats. (A) HE staining was utilized to determine the pathological state of left ventricles in obese rats. (B) Oil Red O staining was used to detect fat deposits in the left ventricles of obese rats.

SG inhibited the apoptosis in left ventricles of obese rats

To assess the apoptotic status in the left ventricles, the TUNEL staining assay was performed (Fig. 5A). The percentage of TUNEL-positive cells in the left ventricles was significantly elevated in obese rats but was markedly reduced after SG (Fig. 5B). Additionally, the increased levels of Bax and Caspase-3, along with the decreased level of Bcl-2 observed in obese rats, were notably reversed by SG (Fig. 5C). These findings indicated that SG effectively suppressed apoptosis in the left ventricles of obese rats.

Fig. 5

SG inhibited the apoptosis in left ventricles of obese rats. (A) TUNEL staining was utilized to assess the apoptosis state in left ventricles in obese rats. (B) The percentage of TUNEL-positive cells in left ventricles of obese rats. (C) Protein levels of Bax, Bcl-2, and Cleaved-caspase-3 were determined by Western blotting assay. (** p < 0.01 vs. Control, ^# p < 0.05 vs. Obese, ^## p < 0.01 vs. Obese).

SG suppressed the expression of cardiac hypertrophy and fibrosis related factors in cardiac tissues

To further validate the protective effects of SG against cardiac hypertrophy and fibrosis in obese rats, mRNA and protein levels of related factors were determined. In obese rats, significantly increased levels of ANP, BNP, Myh7, CoL1, and CoL3 were observed, which were markedly attenuated by SG (Fig. 6A, B).

Fig. 6

SG suppressed the expression of cardiac hypertrophy and fibrosis-related factors in cardiac tissues. (A) mRNA levels of ANP, BNP, Myh7, CoL1, and CoL3 were detected using the RT-qPCR assay. (B) Protein levels of ANP, BNP, Myh7, CoL1, and CoL3 were determined by Western blotting assay. (** p < 0.01 vs. Control, ^# p < 0.05 vs. Obese, ^## p < 0.01 vs. Obese).

Discussion

SG has become the most widely utilized technique for weight loss surgery due to its relative safety, simplicity, and effectiveness in reducing weight and improving metabolism. The procedure involves removing a large portion of the stomach along the greater curvature, reducing its volume to approximately 20% of its original size, resulting in a corresponding decrease in food intake for patients after SG [17]. However, SG is not solely a restrictive surgery. The resection of the stomach alters the synthesis and release of gastrointestinal hormones, which can impact glucose and lipid metabolism, as well as eating behavior. Moreover, although SG does not directly modify the gastrointestinal structure, changes in hormone levels, improved glucose and lipid metabolism, and altered food preferences following the surgery also lead to changes in the composition of intestinal bacteria. These factors interact with each other and act as causative factors, ultimately leading to postoperative weight loss and improvement in metabolic-related conditions [18, 19]. In this study, we observed a significant decrease in body weight during the first two weeks after SG, followed by a gradual increase. It may be attributed to allowing rats to have unrestricted access to food while changing their diet from high-fat to normal feed postoperatively. These findings indirectly emphasize the importance of postoperative dietary management for patients undergoing SG in clinical practice. Over time, the beneficial effects of postoperative changes in gastrointestinal hormones may gradually diminish or be counteracted by other pathways [20]. Additionally, consistent with previously published data [21], severe impairments in liver metabolic function and lipid metabolism were observed in obese rats, which were significantly alleviated by SG. Indeed, the protective effects of SG against obesity have been reported in several studies [22-24], further confirming our findings in the present study.

Studies have shown that obesity or overweight affects myocardial systolic and diastolic function through changes in hemodynamics and activation of inflammatory responses, leading to adverse cardiovascular outcomes. The hearts of obese or overweight patients are exposed to sustained stress overload, especially as heart volume and pressure load, as well as blood viscosity, increase with obesity, which increases the risk of ventricular wall thickening, thereby leading to ventricular hypertrophy and wall thickening [25, 26]. Even if obese patients have normal blood pressure, increased cardiac load may still lead to heart damage. It has been reported that changes in the quality, structure, and function of the heart in obese patients are closely related to their BMI [27]. Studies by Grossman [28] and Zarich [29] have found that obesity can act as an independent risk factor in the development and progression of cardiovascular disease. The increase in the prevalence rate of cardiovascular disease is closely related to the degree of obesity, which affects the structure and function of the heart in obese patients. It is mainly manifested as an increase in left ventricular diameter, thickening of the left ventricular wall and septum, an increase in the left ventricular mass index, and impaired left ventricular function. In this study, impaired cardiac function, severe pathological changes in the left ventricles, and an enhanced apoptotic state in the left ventricles were observed in obese rats, which were consistent with the data presented by Lee [30]. After SG, the cardiac function was repaired, accompanied by an alleviation of the pathological state and apoptosis in the left ventricles, revealing the cardiac protective function of SG. Moreover, as mentioned by Bartosova [31], severe diastolic dysfunction, as revealed by increased LVESP and EDP, was observed in obese rats. However, it was sharply repressed by SG, suggesting an alleviating effect of SG on diastolic dysfunction in obese rats.

Cardiac hypertrophy and fibrosis are critical risk factors for myocardial infarction [32, 33]. The development of cardiac remodeling and dysfunction is often accompanied by increased levels of cardiac hypertrophy factors (such as ANP, BNP, and Myh7) [34] and fibrosis factors (such as CoL1 and CoL3) [35]. In this study, we observed that the cardiac dysfunction in obese rats was associated with exacerbated cardiac hypertrophy and fibrosis, which was consistent with previous findings by Daher [36] and Martinez [37]. Following SG, the cardiac dysfunction and fibrosis were significantly alleviated, providing further evidence for the cardiac protective effect of SG in obese rats. In future studies, we will explore the potential mechanisms underlying the protective properties of SG on cardiac dysfunction in obese rats.

Conclusion

In conclusion, SG effectively improved cardiac function and alleviated glucose-lipid metabolism disorders in obese rats induced by a high-fat and high-sugar diet.

Acknowledgments

Not applicable.

Author Contributions

XJL and ZWZ conceived and supervised the project. XJL, WW, YS and LMQ performed the biological experiments. LY and ZWZ analyzed data and wrote the manuscript.

Funding

No funding.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the ethics committee of Laizhou People’s Hospital.

Competing of interest

The authors declare that there is no conflict of interest.

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