Year : 2005 | Volume
: 11 | Issue : 1 | Page : 28--34
Angiogenic effects of leptin in patients with nonalcoholic steatohepatitis
Nagah Kamel Gaafar1, El-Sayed Ahmed Wasfi2, Yehia Ahmed El- Mashad1, Saad-El-Din Abd-El-Fattah Abou-El Noeman3, Mohamed Ahmed Hassanien1,
1 Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Biochemistry, King Khalid University Hospital, Riyadh, Saudi Arabia
Saad-El-Din Abd-El-Fattah Abou-El Noeman
Department of Medical Biochemistry, College of Medicine, King Saud University. P 0 Box 2925. Riyadh
Background: Nonalcoholic steatohepatitis (NASH) is characterized by morphological features indistinguishable from alcoholic hepatitis in individuals who do not consume excess alcohol. The role of leptin in the development of NASH is claimed to be through its inflammatory, fibrogenic and angiogenic effects.
Aim of the study: to evaluate the leptin status in patients with NASH with the emphasis on its angiogenic effects and its relation to basic fibroblast growth (BFGF). Patients and Methods: This study was conducted on twentyfive patients with NASH and twenty normal persons of matched age and sex as control. For both groups, lipid profile, fasting and postprandial blood glucose, Serum leptin and BFGF were determined. All the results were tabulated and statistically evaluated.
Results: Obesity, hyperlipidaemia and diabetes mellitus were prevalent among patients with NASH. There were significant increase of leptin and basic fibroblast growth factor in patients with NASH, as compared to control.
Conclusion: Leptin plays an important role in the pathogenesis of NASH through its metabolic, fibrogenic and angiogenic effects. It may also have a role in the development of complications
|How to cite this article:|
Gaafar NK, Wasfi ESA, El- Mashad YA, Abou-El Noeman SEA, Hassanien MA. Angiogenic effects of leptin in patients with nonalcoholic steatohepatitis.Saudi J Gastroenterol 2005;11:28-34
|How to cite this URL:|
Gaafar NK, Wasfi ESA, El- Mashad YA, Abou-El Noeman SEA, Hassanien MA. Angiogenic effects of leptin in patients with nonalcoholic steatohepatitis. Saudi J Gastroenterol [serial online] 2005 [cited 2022 Sep 27 ];11:28-34
Available from: https://www.saudijgastro.com/text.asp?2005/11/1/28/33334
Nonalcoholic steatohepatitis (NASH) is characterized by (i) fatty change (ii) hepatocyte injury, manifested by swollen or "ballooned cells" iii) lobular inflammation with inflammatory infiltrate, predominantly neutrophils with or without iv) fibrosis of the liver, typically periventicular/ pericellular in distribution  . NASH has the potential to progress to liver fibrosis, cirrhosis and even hepatocellular carcinoma  . The prevalence of NASH is higher in patients with obesity, Insulin resistance, type 2 diabetes mellitus and hyperlipidaemia ,,.
The high circulating leptin levels associated with obesity may contribute toNASH in two ways: 1) by promoting insulin resistance and elevated circulating insulin level and 2) by altering insulin signaling in hepatocytes so as to promote increased intracellular fatty acids - Leptin may also influence the progression from hepatic steatosis to NASH, as leptin could regulate inflammatory response and selectively enhances the secretion of certain inflammatory cytokines , .
Basic fibroblast growth factor (BFGF) is one of the most potent angiogenic factors. It was indicated that, progression of chronic liver diseases is associated with high level of BFGF  . Recently, it was found that leptin modulates angiogenic responses induced by BFGF, and leptin could produce synergistic effects with other angiogenic growth factors including BFGF  . To our knowledge, there are no reports in the medical literature with regards to circulating leptin, basic fibroblast growth factor and the correlation between them in patients with NASH, in order to contribute to clarification of pathogenic mechanisms in steatohepatitis.
Patients and Methods
A total of 25 patients with NASH and 20 healthy persons as controls were enrolled in the study. NASH patients and control persons were selected from the Tropical Medicine Department and outpatient clinic at Tanta University Hospital. All patients with NASH underwent detailed clinical and laboratory evaluation, including liver function tests and hepatitis markers. All subjects underwent upper abdominal ultrasonography. Liver biopsy was done in two selected cases. Exclusion of bilharziasis was done by indirect haemagglutination. None of the studied subjects had renal dysfunction, drug toxicity, active infection, or recent gastrointestinal bleeding. They did not receive antibiotics or vasoactive drugs at the time of study. Menopausal females, alcoholic, or cardiac patients were excluded. All of the patients and controls were subjected to proper history taking and clinical examination. BMI was calculated as weight (in kilograms) divided by the square of height (in meters) in all patients and controls. Overweight was defined as BMI between 25-29.9 kg/m 2 , and obesity was defined as BMI above 29.9 kg/m 2 Furthermore, obesity is classified as class I obesity with BMI between 30-34.9 kg/m 2 , class 2 obesity with BMI between 35-39.9 kg/m 2 and extreme obesity with BMI equal to or more than 40 kg/m 2 . All subjects received the same dietary protein intake at least one day before the study. On the day of study, all of them were fasting for at least 12 hours and rested in bed. Then blood samples were taken for various laboratory investigations after their consent.
Laboratory investigations were done in the form of, lipid profile, fasting and postprandial blood glucose, serum leptin and (BFGF).
Serum leptin level was measured by a commercially available ELISA kit (The DSL-10-23100 human leptin ELISA Kit; Diagnostic System laboratories, Webster. Texas). This assay is a direct Sandwich ELISA based, sequentially, on capture of human leptin molecules from samples to the wells of a microtiter plate coated by pretitered amount of polyclonal rabbit antihuman leptin antibodies, and wash away of unbound materials from samples. Then, binding of a biotinylated monoclonal antibody to the captured human leptin, and conjugation of alkaline phosphatase to biotinylated antibodies. After that, wash away of free antibody-enzyme conjugates. Finally, quantification of immobilized antibody-enzyme conjugates by monitoring alkaline phosphatase activities in the presence of the substrate p-nitrophenyl phosphate. The enzyme activity is measured spectrophotometrically by the increased absorbency at 405 nm due to production of the yellow colored product p-nitrophenol. Since the increase in absorbency is directly proportional to the amount of captured human leptin in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of human leptin , .Serum basic FGF level was measured by a commercially available EIA kit (The Accucyte Human bFGF; Cytimmune, Sciences INC, Greenmead Drive). This assay is a competitive enzyme immunoassay (IA), which measures the natural and recombinant forms of the cytokine BFGF. With this assay system, goat anti-rabbit antibodies are used to capture a specific BFGF complex in each sample consisting of BFGF antibody, biotinylated BFGF, and sample/standard. The biotinylated BFGF conjugate (competitive ligand) and sample or standard compete for BFGF specific antibody binding sites. Therefore, as the concentration of BFGF in the sample increases, the amount of biotinylated BFGF captured by the antibody decreases. The assay is visualized using a streptavidin alkaline phosphatase conjugate and an ensuing chromagenic substrate reaction. The amount of BFGF detected in each samples is compared to a BFGF standard curve which demonstrates an inverse relationship between optical density (OD) and cytokine concentration e. he igher the OD the lower the cytokine concentration in the sample ,
All results are expressed as mean ± SD. The mean BMI and other serum parameters among control and patient group were compared by the independent samples-tand the Leveene's test. All analysis were two tailed and were conducted using a computer-based statistics software (SPSS for windows 8.0, 1977, SPSS, Chicago, IL) A p-value of less than 0.05 was accepted as statistically significant.
The results of main parameters studied are shown in [Table 1] and [Table 2]. The main risk factors for NASH were overweight and obesity, hyperlipidaemia and diabetes mellitus. There was significant increase in BMI in patients with NASH (32.1±3.3 Kg/m2) as compared to control (23.6±1.56 Kg/m2). According to BMI 80% were obese and 20 % were overweight. The obese patients were further categorized as 52% class I obesity and 28% class II obesity. There was significant increase of serum cholesterol ( 5.3±0.32 mmol/L ), triglyceride (1.62±0.42 mmol/L ) and LDL (3.4±0.28 mmol/L) as compared to controls ( 4.5±0.25 mmol/L ), ( 0.97±0.14 mg/dl) and ( 1.09±0.1 mg/dl) for cholesterol, triglycerides and LDL respectively. Sixty percent of the patients showed serum cholesterol over 5.2 mmol/L and 56% of the patients showed serum triglycerides equal or over 1.7mmol/L. Type 2 diabetes mellitus represented 40% of the patients in the present study. Serum leptin level showed significant increase in patients with
NASH (20.15±2.78 ng/ml) as compared to control group (7.9±0.57 ng/ml). Basic fibroblast growth factor was significant by increased level in patients with NASH (3.7±1.17 ng/ml) compared to controls (1.44±0.5 ng/ml).
In the present study, we demonstrated that risk factors such as obesity, diabetes mellitus and dyslipidemia were associated with NASH. These risk factors play a role in the development of NASH. This observation was reported by many workers and established as the most important factors in developing the disease ,,
These metabolic disorders may reflect the presence of insulin resistance that in turn may explain some of the pathophysiological mechanisms of NASH. In these metabolic disorders the balance between storage of triglyceride in the liver and in adipose tissue is distorted in favor of net transfer of triglyceride from adipose tissue to the liver leading to hepatic steatosis (fatty liver).
Leptin is closely involved in the regulation of food intake, body composition, and energy expenditure (and therefore in the pathogenesis of obesity  ~. Recent experiments showed that leptin promotes insulin resistance, elevates circulating insulin levels, and augments inflammatory and profibrogenic response in the murine liver exposed to hepatotoxic chemicals ,, . These data, in conjunction with the observation that leptin is expressed and synthesized by the activated hepatic stellate cells  , support the notion that leptin might play a role in the pathogenesis of NASH.
In the present study, significantly high serum leptin in patient with NASH were found which to our surprise, did not correlate with other studied parameter. Our present finding correlated with the previous reports suggest in higher levels of leptin in NASH patient , . On the other hand, our results contrast with previous report by Chalasani et al  , who failed to detect statistically significant difference in serum leptin levels between patients with NASH and their control. Failure to detect an association between serum leptin and NASH in their study is likely to be due to the similarity of percent body fat and subcutaneous fat in patients with NASH and their controls. In the present study the percentage of obese subjects were 80% among NASH patients, indicating that those patients might have had higher body fat or higher subcutaneous fat than controls to account for the higher levels of serum leptin seen in patients with NASH in the present study. It has been shown that the amount of total body and subcutaneous fat are critical in determining the serum levels of leptin in obese patients  . Leptin modulates and activates the angiogenic effects of many cytokines and growth factors including BFGF. Furthermore, leptin synergistically stimulates angiogenesis with BFGF and vascular endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors  . In patients with NASH, this may be related to occurrence of complications including liver fibrosis, cirrhosis and even hepatocellular carcinoma  . the present study is considered a novel study reporting a significant increase of BFGF in patients with NASH as compared to controls.
As the growth of any solid tumor depends on angiogenesis, so the angiogenic effects of leptin and its relation to other angiogenic growth factor especially BFGF, must be considered in studying the development and the progress of hypervascular tumors as hepatocellular carcinoma HCC)and metastatic liver tumors , . The high levels of serum leptin and BFGF, in patients with NASH, make a hypothesis of the possibility of development of HCC in those patients. So they can be used as useful diagnostic and prognostic markers for HCC and for evaluating progression of chronic benign liver diseases.
It can be concluded that, obesity, noninsulin-dependent diabetes mellitus and dyslipidemia are important risk factors for the development of hepatic steatosis and NASH. Leptin plays an important role in the pathogenesis of NASH through its metabolic, fibrogenic and angiogenic effects. Leptin, may also have a role in the development of complications as liver cirrhosis and HCC in patients with steatohepatitis, as leptin mediates and regulates the action of many cytokines and growth factors including BFGF. Increase serum leptin levels itself can be considered as a risk factor for other vascular injuries. Finally, BFGF may play a role in the development of liver fibrosis, cirrhosis and even hepatocellular carcinoma in patients with NASH.
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