Effects of Gastric Sleeve Surgery on the Serum Levels of GH, IGF-1 and IGF-binding Protein 2 in Morbidly Obese Patients

Background Bariatric surgery is an effective treatment for severe obesity. It also ameliorates diabetes independently of weight loss through mechanisms that are not fully understood. In this study, we investigated the levels of GH, IGF-1 and IGF-binding protein (IGFBP-2) after gastric sleeve surgery in healthy obese individuals. Method This study was conducted in 33 obese (BMI >38.3) healthy male subjects aged 25 to 50 years undergoing sleeve gastrectomy. GH, IGF-1 and IGFBP-2 levels were evaluated by ELISA at baseline and 6-12 months after surgery. Other parameters, such as glucose, BMI, insulin, HOMA-IR and lipid profile, were also investigated. Results Systemic GH (12.32 vs. 50.97 pg/mL, p < 0.001) and IGFBP-2 levels (51.86 vs. 68.81 pg/mL, p < 0.001) were elevated after bariatric surgery. There was no change in IGF-1 level from before to after surgery. BMI (52.18 vs. 40.11, p = 0.001), insulin (19.35 vs. 8.80 mIU/L, p < 0.001) and HOMA-IR index (6.48 to 2.52, p < 0.001) were reduced after surgery. Lipid profile analysis revealed that total cholesterol (4.26 vs. 5.12 mmol/L, p < 0.001) and high-density lipoprotein (HDL) (0.90 to 1.55 mmol/L, p < 0.001) were increased, while triglycerides were decreased, after surgery (1.62 vs. 1.05 mmol/L p < 0.001). GH, IGF-1, and IGFBP-2 were not correlated with insulin or lipid parameters. Conclusions Our study suggests that improved circulating GH and IGFBP-2 levels may mediate the beneficial effects of gastric sleeve surgery in improving insulin sensitivity and reducing insulin demand. a small number of homogenous males without medical complications. Future studies should be conducted in a larger sample including women and control lean subjects undergoing non-bariatric laparoscopic surgery for comparison. Other relevant metabolic molecular pathways and molecules in different fat depots should also be explored.

regulation, reproduction and ageing. GH is produced by the anterior pituitary gland in response to growth hormone-releasing hormone (GHRH), which is released by the hypothalamus as a normal reflection of multiple features, such as hypoglycaemia, low free fatty acids in the blood, high amino acids, good exercise and sleep [2]. All these features are diminished in adult subjects with high BMI [3]. Following an increase in BMI, GH secretion is reduced, and lipid metabolism is disturbed, leading to increased T2D and cardiovascular disease risk [4]. IGF-1 reflects GH levels and mediates its growth effects, while the metabolic effects of GH, including stimulation of lipolysis and inhibition of insulin signalling in fat and muscle, are induced directly through the GH receptor [5]. IGF-1 in the circulation binds to IGF-binding proteins (IGFBPs). These IGFBPs act as transporter proteins, modulate IGF-1 actions and regulate its clearance [6]. In obesity, non-esterified fatty acids and insulin inhibit IGFBP production, which increases free IGF-1 in circulation [7].
IGFBP-2 is one of the most abundant IGFBPs and is responsible for several cellular processes, such as cell proliferation, migration, and adhesion, which play a significant role in cancer establishment and progression [8][9]. It is secreted by differentiating preadipocytes. Plasma IGFBP-2 level can be used as a biomarker of insulin sensitivity, as it helps in glucose metabolism by improving insulin sensitivity [10]. Increased IGFBP2 has had a strong negative association with the risk of T2D and BMI [11][12].
Lower circulating levels of IGFBP-2 have been linked with an increased risk of developing metabolic syndrome and increased levels of triglyceride-rich particles [13].
Bariatric surgery is an effective treatment for severe obesity that leads to the improvement and remission of many obesity-related comorbidities, sustained weight loss over time, improvement in quality of life and prolonged survival [14][15]. Bariatric surgery reduces body weight and improves glycaemic control through reduced nutrient intake and malabsorption. However, other mechanisms, such as changes in the secretion and activity of hormones and neurotransmitters involved in appetite, energy expenditure and glucose metabolism, also add to the beneficial effects of bariatric surgery [16][17].
The underlying mechanisms of how bariatric surgery influences the physiological metabolic process pre-and post-surgery are not fully understood. Therefore, the aim of this study was to assess the activity of the GH/IGF-1 axis and IGFBP-2 levels in obese patients before and 6-12 months after gastric sleeve surgery and their correlations with other anthropometric parameters and lipid profile.

Clinical Examinations and Blood collection
All patients were clinically examined by a physician, a psychologist and a nutritionist before surgery and attended surgery and nutrition clinics at 3, 6 and 12 months after surgery. The patients were not on medication for kidney, thyroid, or liver disorders and were taking oral vitamins. BMI was recorded at each visit, and patients were classified according to their BMI results. Blood was taken from patients in the fasting state one day before surgery and 6-12 months later. Serum was separated following centrifugation at 1500×g for 10 minutes and stored at -80°C in aliquots within 30 minutes of collection. Other parameters, such as routine CBC, lipid profile, glucose, insulin, and liver function test results, were retrieved from the hospital files.

Enzyme-Linked Immunosorbent Assay (ELISA)
GH, IGF-1 and IGFBP-2 levels were analysed by indirect Simple Step Human ELISA kits (GH Ab190811, IGF-1 Ab100545 and IGFBP-2 Ab100540) following the manufacturer's instructions (abcam, Cambridge, UK). Briefly, patient samples (33 pre-and 33 post-bariatric surgery) and standards were reacted with specific antibodies coated in the microplates for each protein under investigation and incubated at room temperature (18-25°C) for 1 hour on a plate shaker. Next, the cocktail of antibodies (capture and detector antibodies) was added and incubated as before. One hundred microliters of TMB substrate was added to the microplate and incubated as previously described. The reactions were stopped by adding 100 μl stop solution to each well, and the absorbance was read by a microplate reader (EL 800, BioTek Instruments, USA) at 450 nm.

Statistical analysis
Data were analysed using SPSS (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.). Categorical data are expressed as absolute numbers and percentages.
Numerical data are expressed as mean, median, standard error of the mean (SEM) and range.
Student's t-test was used to compare the data pre-and post-surgery. A p value < 0.05 was considered statistically significant.

Anthropometric and biochemical assessments
Anthropometric data of the 33 obese male patients are shown in Table 1 Table 2). There was no significant difference in glucose level before vs. after surgery.
Analysis of GH, IGF-1 and IGFBP-2 levels in serum before and after surgery   and IGFBP-2 levels were increased after bariatric surgery, while IGF-1 level was not altered. Insulin sensitivity was increased, as reflected by a decrease in insulin level and HOMA-IR index.
The somatotropic axis has an important role in maintaining healthy conditions, and it is suppressed in obesity due to reduced GH and IGF-1 levels in the body. Hyperinsulinaemia and high circulating free fatty acids reduce IGFBP-1 production by the liver, which is responsible for reduced GH production from the pituitary and consequently low IGF-1 levels [19].
IGF-1 has an important role in GH activity and is related to its serum levels. Increased IGF-1 levels in obesity have a negative effect and cause GH suppression [20][21]. In the current study, GH was elevated after gastric sleeve surgery, while IGF-1 was not changed. Insulin can bind to the IGF-1 receptor, and IGF-1 can bind to the insulin receptor, and both stimulate growth and hypoglycaemic effects. Moreover, hybrid heterodimeric receptors can be formed consisting of an insulin and an IGF-1 receptor α-β dimer, which signal mainly IGF-1 [22]. After bariatric surgery, GH was increased, but IGF-1 was not. It seems that IGF-1 sensitivity was improved since HOMA-IR was also improved; therefore, IGF-1 signalling was increased despite unchanged plasma levels. High, low and normal levels of IGF-1 have been reported in obese populations [19][20]. Our results are in agreement with previous studies reporting no change in IGF levels postoperatively [23,24]. However, an earlier decrease (3 weeks to 1 month) and an increase 1 year after bariatric surgery have also been reported [25,26]. In obese children, reduced GH is not associated with decreased levels of IGF-1 or reduced somatic growth [21].
In our study, cholesterol, HDL and LDL were increased after bariatric surgery, but they were still within the desired physiological levels. An increase in the lipid profile can be explained by the lipolytic effects of GH and the release of free fatty acids from visceral adipose tissue and, to a lesser extent, from subcutaneous fat by increasing hormone-sensitive lipase (HSL). Furthermore, GH maintains triglyceride storage in the liver by either inhibiting triglyceride lipolysis via HSL or oxidation by PPARγ [27]. GH also stimulates triglyceride uptake into skeletal muscle to be used for energy or stored as intramyocellular lipids [28]. Furthermore, as a result of bariatric surgery, calorie intake is reduced, which might lead to increased GH levels since its secretion is stimulated by hypoglycaemia. Increased GH after bariatric surgery has beneficial effects on maintaining proper glucose levels and preventing liver steatosis. In the liver, GH stimulates autophagy and preserves plasma glucose levels in chronically starved mice [29]. Moreover, GH signalling in the liver is essential to regulate intrahepatic lipid metabolism, while IGF-1 helps in reducing the catabolic effects of GH [30]. In the current study, the levels of TC, LDL and HDL were physiologically increased and thought to be beneficial outcomes of increased growth hormone.
IGFBP-2 is the main IGF-binding protein associated with regulating body weight and homeostasis and protects against obesity and insulin resistance [10, [31][32][33]. Obesity-related hyperinsulinaemia increases IGF-1 and inhibits IGFBP-2 secretion [34]. In our study, IGFBP-2 was increased, while insulin level, HOMA-IR index and BMI were reduced 6-12 months after gastric sleeve surgery. IGFBP-2 concentration has been associated with improvements in insulin sensitivity, BMI and lipid profile in obesity-related studies. A recent 20-year longitudinal study of ageing has shown that IGFBP-2 level increases with age, positively correlates with insulin sensitivity, and negatively correlates with BMI at baseline and follow-up [34]. In obese children, circulating levels of IGFBP-2 correlate negatively with body mass and positively with insulin sensitivity [35]. A recent animal study has shown that metformin upregulates IGFBP-2 production through activation of the AMPK-Sirt1-PPARα signalling pathway [36]. Metformin-treated diabetic patients have higher IGFBP-2 levels and lower serum IGF1 levels than untreated patients [36]. This highlights IGFBP-2 as a novel target for metformin action and AMPK-Sirt1-PPARα as a novel pathway to control metabolic syndrome. IGFBP-2 was shown to be increased after biliopancreatic diversion in obese patients and was associated with improved glucose and lipid levels that were sustained even after one year of follow-up [37]. IGBP-2 has also been shown to be regulated by leptin and may mediate some of leptin's antidiabetic effects [38]. Early increased levels of IGFBP-2 were noticed after gastric bypass but normalized shortly after [38]. It was recently reported that higher basal levels of IGBP-2 were associated with lower risk of metabolic syndrome and type 2 diabetes and its levels increased after bariatric surgery [39]. In the current study, sleeve gastrectomy resulted in sustained higher levels of IGFBP-2 in a healthy male population, indicating an important role of this protein in improving insulin sensitivity, and it resulted in increased levels of GH.

Conclusions
In conclusion, our study suggests that improved circulating GH and IGFBP-2 levels may be an endocrine response that mediates the beneficial effects of gastric sleeve surgery by improving insulin sensitivity and reducing insulin demand. The increased levels of total cholesterol, LDL, and HDL after bariatric surgery in this study were within the desired physiological levels that would help in sparing glucose.

Limitation and Recommendation
Our study subjects were a small number of homogenous males without medical complications. Future studies should be conducted in a larger sample including women and control lean subjects undergoing non-bariatric laparoscopic surgery for comparison. Other relevant metabolic molecular pathways and molecules in different fat depots should also be explored.