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Serum Betatrophin Level Changes in a Rat Model of Non-alcoholic Fatty Liver Disease

Serum betatrophin level changes in a rat model of non-alcoholic fatty liver disease

ABSTRACT

Background: Non-alcoholic fatty liver disease (NAFLD) is a common liver problem. Betatrophin, a liver derived hormone, was found to have a role in regulation of glucose and lipid metabolism. Few data were recorded about its serum level changes in both untreated and metformin treated NAFLD.

Aim

To investigate changes in serum betatrophin levels in both untreated and metformin treated NAFLD. Also, the relationship of betatrophin level to body mass index (BMI), liver histology, C-peptide, TNF, IL-6, liver enzymes and other metabolic (insulin resistance, lipid profile) parameters were also studied to identify the potential effect of betatrophin on the pathogenesis of NAFLD.

Methods

Rats were randomly divided into three groups of 8 rats each as follows: 1st group (control) in which rats were fed a standard diet for 14 weeks; 2nd group (fatty liver untreated,NAFLD) in which rats were fed high fat diet (HFD) for 14 weeks; and 3rd group (fatty liver metformin-treated, NAFLD+Metformin) in which rats were fed HFD for 14 weeks and were administered metformin daily by oral gavage from the start of the ninth week until the end of the 14th week. At the end of the experimental period, rats were fasted overnight before being anaesthetized with ether and then killed by decapitation. Blood was collected and serum was separated and stored to be studied.

Results

There was a significant (P<0.01) increase in BMI in NAFLD group in comparison to that in the control group, but, there was a significant (P<0.05) decrease in its value in the NAFLD+Metformin group in comparison to that in the NAFLD group. Also, there was a significant (P<0.001) increase in serum glucose, serum insulin and HOMA-IR in the NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001) decrease in their values in the NAFLD+Metformin group in comparison to that in the NAFLD group. Moreover, there was a significant (P<0.001) increase in serum betatrophin level in NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001)decrease in its level in NAFLD+Metformin group in comparison to that in NAFLD group. Furthermore, there was a significant (P<0.001) increase in serum C-peptide level in NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001) decrease in its level in the NAFLD+Metformin group in comparison to that in the NAFLD group. Also, there was a significant (P<0.001) increase in serum levels of both interleulin-6 (IL-6) and tumor necrosis factor- (TNF-in the NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001) decrease in their serum levels in the NAFLD+Metformin group in comparison to that in NAFLD group. Moreover, there was a significant (P<0.001) increase in triglycerides (TG), total cholesterol (TC), and low density lipoprotein-cholesterol (LDL-C) serum levels in the NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001) decrease in their serum levels in the NAFLD+Metformin group in comparison to that in the NAFLD groupFurthermore, there was a significant (P<0.001) decrease in serum level of high density lipoprotein-cholesterol (HDL-C) in NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001) increase in its serum level in the NAFLD+Metformin group in comparison to that in the NAFLD group. In addition, there was a significant (P<0.001) increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and -glutamyltransferase (GGT) serum levels in the NAFLD group in comparison to that in the control group, but, there was a significant (P<0.001) decrease in their serum levels in the NAFLD+Metformin group in comparison to that in the NAFLD group. Histopathological examination confirmed incidence of NAFLD with HFD in NAFLD group that was improved with metformin treatment as noticed in NAFLD+Metformin group.

Conclusion

In NAFLD, betatrophin levels were increased. This could be an important mechanism to explain the glucose intolerance during the course of NAFLD. It could be also considered as a mechanism by which NAFLD occurred. Also, serum betatrophin levels were significantly reduced after metformin treatment which indicated that it may be used as an indicator of NAFLD improvement. Thus, estimation of serum level of betatrophin can be considered as a biomarker that reflected the early occurrence of NAFLD and to follow up the effect of treatment.

Key Words: Betatrophin, Non-Alcoholic Fatty Liver Disease, Metformin, C Peptide, Insulin Resistance

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Introduction

Betatrophin, or lipasin, is a circulating protein angiopoietin-like 8 (ANGPTL8) that is produced by liver and adipose tissue (Quagliarini, Wang et al. 2012, Yi, Park et al. 2013, Cahová, Habart et al. 2017) and increases pancreatic  cell proliferation. In animal models of type II diabetes, there was increased pancreatic  cell mass (Bock, Pakkenberg et al. 2003), and betatrophin mRNA was upregulated 3 to 4 folds in their liver. Non-alcoholic fatty liver disease (NAFLD) is a worldwide public health problem because obesity prevalence is increasing, populations are aging, and sedentary lifestyles dominate (Loomba and Sanyal 2013). NAFLD was defined as the accumulation of liver fat, mainly triglycerides, in more than 5% of hepatocytes with no evidence of alcohol intake or other secondary etiology of liver disease (Leng, Jiang et al. 2011, Takahashi and Fukusato 2014). In NAFLD, liver hypertrophy is caused by the accumulation of fat. It can be divided into simple steatosis and non-alcoholic steatohepatitis (NASH). Simple steatosis was observed as lipid accumulation in hepatocytes with little or no inflammation and fibrosis. NASH included inflammation and fibrosis (Leng, Jiang et al. 2011). Although NAFLD was closely related with obesity, yet, 20-40% of NAFLD patients were not obese (Shin and Jung 2016). Hepatic cholesterol accumulation, increased serum levels of inflammatory cytokines and incidence of insulin resistance were observed in rat models of NALFD (Manco, Marcellini et al. 2007, Shin and Jung 2016). In NAFLD, decreased adiponectin secretion resulted in decreased 5′ adenosine monophosphate-activated protein kinase (AMPK) activation. Thus, fatty acid clearance in the liver was low and the fatty acid remnants accumulated in hepatocytes or were released into the bloodstream (Larter and Farrell 2006). Increased delivery of free fatty acids to the liver was caused by insulin resistance which increased peripheral adipose tissues lipolysis (Choi and Diehl 2008). With the increased prevalence of metabolic syndrome in the general population, NAFLD had become the most common cause of liver diseases (Angulo 2002). Metformin, a biguanide oral hypoglycemic agent, was commonly used in treatment of type II diabetes mellitus. It exerted its activity by decreasing both glucose absorption from gastrointestinal tract and glucose synthesis by the liver, but, it increased glucose uptake by cells without increasing insulin level (Cusi and DeFronzo 1998). Yi et al found that betatrophin dramatically promoted the proliferation of pancreatic islet beta cells, increased functional beta cells mass over time and improved glucose tolerance in mice (Yi, Park et al. 2013). Also, some reports showed that betatrophin inhibited lipoprotein lipase activity which caused a decrease in the clearance of triglycerides (TG), and increased serum TG in mice (Fu, Berhane et al. 2014). Therefore, betatrophin is likely to regulate glucose homeostasis and lipid metabolism in mice. Pereira, Salsamendi et al. (2015) declared that NAFLD pathogenesis can be explained by the three-hit theory which stated that the 1st hit started with triglycerides accumulation in the liver causing hepatic injury, then, steatohepatitis and/or fibrosis occurred as a 2nd hit which was mediated by an increase in the proinflammatory cytokines, then, the 3rd hit included the decrease in proliferation of hepatocyte progenitor cells. Therefore, we speculated that betatrophin played a role in the occurrence and development of NAFLD. However, few studies focused on the role of betatrophin in the occurrence of NAFLD and its progression. Also, although prevalence of NAFLD is high, yet, its detection and screening depends primarily on either imaging devices, as ultrasonography, or estimation of liver enzymes, which were inaccurate (Fracanzani, Valenti et al. 2008). Thus, there was a requirement for reliable biomarkers for early detection and follow up of NAFLD. Moreover, considering that obesity, type 2 diabetes mellitus (T2DM), and NAFLD share insulin resistance as a common pathophysiologic mechanism (Utzschneider and Kahn 2006) and that the dominant expression of betatrophin is in the liver (Zhang 2012), we hypothesized that circulating betatrophin levels might be elevated in NAFLD. Therefore, the present study investigated changes in serum levels of betatrophin, liver enzymes, proinflammatory cytokines, C-peptide, metabolic parameters along with the histopathological features of liver in untreated, and metformin-treated NAFLD in comparison to control rats. Also, this study was designed to assess the possible role of betatrophin in the pathogenesis of NAFLD in a rat model.

Materials and Methods

The experimental work was performed from 18th of January 2017 to 9th of July 2017 in the Physiology Department, Faculty of Medicine, Zagazig University. Twenty-four adult healthy male albino rats, weighing 170-185 gm were supplied from, Faculty of Medicine, Zagazig University, animal house. In the Physiology research laboratory lab, rats were kept, under hygienic conditions, in steel wire cages (4/cage) with free access to standard diet (mixed commercial rat laboratory chow) and water. Rats were maintained on a 12-hour light/dark cycle, were kept at room temperature (Bateman and Patisaul 2008), and were accommodated to laboratory conditions for one week before the experiments were done.

Experimental Design:

After acclimation, rats were randomly divided into three groups of 8 rats each as follows: 1st group (control) in which rats were fed a standard diet for 14 weeks; 2nd group (fatty liver untreated,NAFLD) in which rats were fed a high fat diet (HFD) which consisted of standard diet (72.8%), lard (25%), cholesterol (2%), and bile salts (0.2%) (Magdy, El-Kharashi et al. 2017) for 14 weeks; and, 3rd group (fatty liver metformin-treated, NAFLD+Metformin) in which rats were fed HFD for 14 weeks and were administered metformin daily by oral gavage from the start of the ninth week until the end of the 14th week. Commercial lard, cholesterol, bile salts and metformin hydrochloride were obtained from Sigma-Aldrich Chemicals, USA. Metformin was dissolved in distilled water and was given with oral gavage at a daily dose of 20 mg/kg (Awad, Mallah et al. 2016).  The rats of the 1st two groups were given distilled water by oral gavage (1 ml/day) from the start of the ninth week until the end of the 14th week. Diet and water were provided ad libitum and body weight was recorded once weekly from the start of experiment. At the end of the experimental period, body mass index (BMI) was calculated by dividing (weight in gm) on (length in cm)2 (Novelli, Diniz et al. 2007) and the rats were fasted overnight before being anaesthetized with ether and then they were killed by decapitation. Blood was collected and allowed to clot for 30 min at 25oC. Thereafter, it was centrifuged for 15 min at 3000 rpm for separation of serum. Serum was stored at -25oC (El, Sokar et al. 2017). Estimation of serum levels of both glucose and insulin using ELISA kits (MyBioSource.com), Catalogs No. (MBS7233226) and (MBS724709), respectively, was done. For assessing insulin resistance (IR), the homeostasis model assessment–IR (HOMA–IR) index was calculated by:

HOMA-IR =

fasting glucose (mg/dl) × fasting insulin (μIU/ml) 405  (Nayak, Hillemane et al. 2014).

There was a direct relation between insulin resistance and the value of HOMA–IR (Bonora, Targher et al. 2000). Serum levels of both total cholesterol (TC) and triglycerides (TG) were estimated using enzymatic colorimetric methods. Serum high density lipoprotein-cholesterol (HDL-C) was assayed using NS Biotec HDL-precipitating reagent. Serum low density lipoprotein-cholesterol (LDL-C) was calculated using Friedewald formula: LDL-C (mg/dl) = (TC) – [(HDL-C) + (

TG5)](Friedewald, Levy et al. 1972)

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and -glutamyltransferase (GGT) were determined using commercial kits (Sigma-Aldrich Chemicals, USA), Catalog No. (MAK052), (MAK055) and (MAK089), respectively. Serum levels of betatrophin were estimated by using rat betatrophin ELISA kits (Aviscera Bioscience, INC, 2348 Walsh Ave., Suite C, Santa Clara, CA 95051, USA), Catalog No. (SK00528-16). Serum C-Peptide levels were measured using rat ELISA kits (ALPCO), Catalog No. (80-CPTRT-E01). Serum interleulin-6 (IL-6) and tumor necrosis factor- (TNF- levels were estimated using rat ELISA kits (R&D Systems, Inc., 614 McKinley Place NE, Minneapolis, MN 55413, USA), Catalogs No. (SR6000B) and (SRTA00), respectively.

Figure-1: Experimental design.Rats were divided into 3 groups. 1st group was the control. Rats of the 2nd and the 3rd groups received HFD for 8 consecutive weeks. On the start of the 9th week, animals of the 3rd group (NAFLD+Metformin) received oral metformin along with HFD for more 6 weeks. Animals of the 1st group (control) and the 2nd group (NAFLD) received oral distilled water along with the corresponding diet as was taken in the first 8 weeks for more 6 weeks. On the day of termination (at the end of the 14th week), overnight (12hrs) fasted rats were sacrificed by decapitation under ether anesthesia.

Histopathology:

A histological study was performed following a midline laparotomy to remove the liver which was cut into sections of 5 m thickness and kept in 10% phosphate-buffered formalin for histopathological evaluation (El, Sokar et al. 2017). Masson’s trichrome and hematoxylin-eosin stains were used to evaluate fibrotic areas and necroinflammation activity (Ishak, Baptista et al. 1995).

Statistical Analysis:

The obtained data was expressed as mean ± standard error of the mean (SEM). For statistical significance, one-way ANOVA and Tukey HSD for Post hoc multiple comparisons were used to compare means. The software, IBM SPSS Statistics Version 24 Software for Windows, was used for that purpose. Significance was considered when P value ≤ 0.05.

Results

Table-1 showed BMI and serum biochemical changes among the different studied groups. Regarding BMI, there was a significant increase in its value in NAFLD group (0.55±0.016, P<0.01) in comparison to that in the control group (0.48±0.004), but, there was a significant decrease in its value (0.50±0.008, P<0.05) in the NAFLD+Metformin group in comparison to that in the NAFLD group (0.55±0.016). Also, there was no significant (P>0.05) changes in BMI in the NAFLD+Metformin group in comparison to that in the control group. On estimating serum glucose, serum insulin and HOMA-IR values, there was a significant (P<0.001) increase in their values in the NAFLD group (287.25±3.89) (5.17±0.18) & (3.67±0.16) in comparison to that in the control group (94.75±3.06), (2.31±0.11) & (0.54±0.04), respectively. Also, there was a significant increase in serum level of glucose in the NAFLD+Metformin group (110.75±5.51, P<0.05) in comparison to that in the control group (94.75±3.06). On the other hand, there was a significant (P<0.001) decrease in serum glucose, serum insulin and HOMA-IR values in the NAFLD+Metformin group (110.75±5.51) (2.03±0.15) & (0.56±0.06) in comparison to that in the NAFLD group (287.25±3.89) (5.17±0.18) & (3.67±0.16), respectively. Also, there was no significant (P>0.05) changes in serum insulin and HOMA-IR values in the NAFLD+Metformin group in comparison to that in the control group. On studying serum betatrophin level changes among different studied groups, there was a significant increase in its level in NAFLD group (167.45±4.3, P<0.001) in comparison to that in the control group (77.84±1.6). On the other hand, there was a significant decrease in serum betatrophin level in NAFLD+Metformin group (85.26±2.24, P<0.001)in comparison to that in NAFLD group(167.45±4.3). Also, there was no significant (P>0.05) changes in serum level of betatrophin in NAFLD+Metformin group in comparison to that in the control.

Table-1: BMI and serum biochemical changes among different groups:

Control NAFLD NAFLD+ Metformin
BMI (gm/cm2) 0.48±0.004 0.55±0.016a 0.50±0.008b
Glucose (mg/dl) 94.75±3.06 287.25±3.89c 110.75±5.51d&e
Insulin (IU/ml) 2.31±0.11 5.17±0.18c 2.03±0.15e
HOMA-IR 0.54±0.04 3.67±0.16c 0.56±0.06e
Betatrophin (ng/l) 77.84±1.6 167.45±4.3c 85.26±2.24e
C-peptide (ng/l) 121.79±1.14 142.93±1.55c 125.86±1.25e
IL-6 (pg/dl) 84.5±1.46 108.46±2.35c 87.87±1.19e
TNF- (pg/dl) 6±0.08 10.86±0.08c 6.33±0.13e
Total cholesterol (mg/dl) 90.3±0.96 152.14±2.72c 98.92±1.11a&e
Triglycerides (mg/dl) 71.81±1.78 136.83±1.88c 79.33±1.63d&e
HDL-C (mg/dl) 46.96±0.82 31.41±0.52c 41.84±0.51c&e
LDL-C (mg/dl) 28.98±1.41 93.37±2.59c 41.21±0.9c&e
ALT (U/L) 31.45±0.58 70.56±0.79c 34.78±0.6a&e
AST (U/L) 52.46±0.74 101.31±0.51c 65.89±1.35c&e
GGT (U/L) 21.44±0.77 75.58±1.1c 37.88±0.63c&e

Data was expressed as Mean±SEM. a P value <0.01 when compared with the control group. b P value <0.05 when compared with the NAFLD group.c P<0.001 in comparison to the control group. d P<0.05 in comparison to the control group. e P<0.001 in comparison to the NAFLD group.

Regarding serum C-peptide level changes among different groups, there was a significant increase in its level in NAFLD group (142.93±1.55, P<0.001) in comparison to that in the control group (121.79±1.14). On the other hand, there was a significant decrease in serum C-peptide level in the NAFLD+Metformin group (125.86±1.25, P<0.001) in comparison to that in the NAFLD group (142.93±1.55). Also, there was no significant (P>0.05) changes in serum level of C-peptide in the NAFLD+Metformin group in comparison to that in the control group. On studying changes in serum levels of both IL-6 and TNF-there was a significant (P<0.001) increase in their levels in the NAFLD group (108.46±2.35) and (10.86±0.08) in comparison to that in the control group (84.5±1.46) and (6±0.08), respectively. On the other hand, there was a significant (P<0.001) decrease in serum levels of both IL-6 and TNF- in the NAFLD+Metformin group (87.87±1.19) and (6.33±0.13) in comparison to that in NAFLD group (108.46±2.35) and (10.86±0.08), respectively.

Figure-2: Correlations between serum betatrophin levels and: BMI & HOMA-IR in the NAFLD group (A); ALT & AST in the NAFLD group (B); TC, TG & HDL-C in the NAFLD group (C); IL-6 & TNF- in the NAFLD group (D); TC & IL-6 in the NAFLD+Metformin group (E). r is the correlation coefficient.

Also, there was no significant (P>0.05) changes in serum levels of both IL-6 and TNF- in NAFLD+Metformin group in comparison to that in the control. Regarding changes in serum levels of total cholesterol, triglycerides and LDL-Cthere was a significant (P<0.001) increase in their serum levels in the NAFLD group (152.14±2.72), (136.83±1.88) and (93.37±2.59) in comparison to that in the control group (90.3±0.96), (71.81±1.78) and (28.98±1.41), respectively. Also, there was a significant increase in serum levels of total cholesterol, triglycerides and LDL-C in the NAFLD+Metformin group (98.92±1.11, P<0.01), (79.33±1.63, P<0.05) and (41.21±0.9, P<0.001) in comparison to that in the control group (90.3±0.96), (71.81±1.78) and (28.98±1.41), respectively On the other hand, there was a significant (P<0.001) decrease in serum levels of total cholesterol, triglycerides and LDL-C in the NAFLD+Metformin group (98.92±1.11), (79.33±1.63) and (41.21±0.9) in comparison to that in the NAFLD group (152.14±2.72), (136.83±1.88) and (93.37±2.59), respectively

Figure-3: Photomicrographs of cut sections in the liver of: control rats (A), NAFLD rats (B, C, D & E) and NAFLD metformin treated rats (F, G & H). Photomicrograph A showed normal classic hepatic lobule with normal hepatocytes and central vein (blue arrow) and portal veins (yellow arrows), hematoxylin and Eosin 100x. Photomicrograph B showed swelling and Mallory-Denk bodies in fatty liver. Swollen hepatocytes with rarefied cytoplasm (yellow arrow). Mallory-Denk bodies are eosinophilic irregular shaped aggregates in the cytoplasm of hepatocytes marked hepatocyte note hepatic architectures are distributed and other hepatocytes are filled with fat. hematoxylin and eosin 630 X.Photomicrograph C showed dense infiltration of inflammatory cells around portal tracts (green star), scattered inflammatory cells (yellow arrows) also present in Perisinusoidal space with congested sinusoids and blood vessels the hepatocytes in zone 3 appear bright arterial branch is congested (blue line), hematoxylin and eosin staining 630 X. Photomicrograph D showed hepatocytes in zone 3 appeared bright with fatty drops inside (green arrow). The Von Kupffer cells are prominent (blue arrow), hematoxylin and eosin staining 630 X.Photomicrograph E showed dense fibrosis around portal tract and perisinusoidal fibrosis of fatty liver, massons trichrome x 100.Photomicrograph F showed little cellular infiltration around portal tract and classic hepatic lobule preserved most of hepatocytes are normal. Some fat drops still present inside hepatocytes with little Von Kupffer cellshematoxylin and eosin staining 630 X. Photomicrograph G showed classic hepatic lobule hepatocytes that are regular with congested sinusoids little infiltration around portal tract, hematoxylin and eosin staining100 X. Photomicrograph H showed little amount of connective tissue around sinusoids, Masson trichrome x100.

On comparing serum levels of HDL-C among different groups, there was a significant (P<0.001) decrease in its level in both NAFLD (31.41±0.52) and NAFLD+Metformin (41.84±0.51) groups in comparison to that in the control group (46.96±0.82). On the other hand, there was a significant (P<0.001) increase in serum levels of HDL-C in the NAFLD+Metformin group (41.84±0.51) in comparison to that in the NAFLD group (31.41±0.52). Regarding changes in serum levels of ALT, AST and GGTthere was a significant (P<0.001) increase in their serum levels in the NAFLD group (70.56±0.79), (101.31±0.51) and (75.58±1.1) in comparison to that in the control group (31.45±0.58), (52.46±0.74) and (21.44±0.77), respectively. On the other hand, there was a significant (P<0.001) decrease in serum levels of ALT, AST and GGT in the NAFLD+Metformin group (34.78±0.6), (65.89±1.35) and (37.88±0.63) in comparison to that in the NAFLD group (70.56±0.79), (101.31±0.51) and (75.58±1.1), respectively. Figure-2A showed a significant positive correlation between serum betatrophin levels and that of BMI (r=0.95, P<0.001) & HOMA-IR (r=0.83, P<0.05) in the NAFLD group. Figure-2B showed a significant positive correlation between serum betatrophin levels and that of ALT (r=0.75, P<0.05) & AST (r=0.81, P<0.05) in the NAFLD group. Figure-2C showed a significant positive correlation between serum betatrophin levels and that of TC (r=0.98, P<0.001) & TG (r=0.99, P<0.001), but, a significant negative correlation between serum betatrophin levels and that of HDL-C (r=-0.97, P<0.001) in the NAFLD group. Figure-2D showed a significant positive correlation between serum betatrophin levels and that of IL-6 (r=0.93, P<0.001) & TNF- (r=0.96, P<0.001) in the NAFLD group. Figure-2E showed a significant positive correlation between serum betatrophin levels and that of TC (r=0.75, P<0.05) & IL-6 (r=0.74, P<0.05) in the NAFLD+Metformin group. Figure-3 showed photomicrographs of cut sections in the liver of: control rats (A), NAFLD rats (B, C, D & E) and NAFLD metformin treated rats (F, G & H). Photomicrograph 3A showed normal classic hepatic lobule with normal hepatocytes and central vein and portal veins. Photomicrograph 3B showed swelling and Mallory-Denk bodies in fatty liver. Swollen hepatocytes with rarefied cytoplasm. Mallory-Denk bodies are eosinophilic irregular shaped aggregates in the cytoplasm of hepatocytes marked hepatocyte note hepatic architectures are distributed and other hepatocytes are filled with fat. Photomicrograph 3C showed dense infiltration of inflammatory cells around portal tracts, scattered inflammatory cells also present in Perisinusoidal space with congested sinusoids and blood vessels the hepatocytes in zone 3 appear bright arterial branch is congested. Photomicrograph 3D showed hepatocytes in zone 3 appeared bright with fatty drops inside. The Von Kupffer cells are prominent. Photomicrograph 3E showed dense fibrosis around portal tract and perisinusoidal fibrosis of fatty liver. Photomicrograph 3F showed little cellular infiltration around portal tract and classic hepatic lobule preserved most of hepatocytes are normal. Some fat drops still present inside hepatocytes with little Von Kupffer cells. Photomicrograph 3G showed classic hepatic lobule hepatocytes that are regular with congested sinusoids little infiltration around portal tract. Photomicrograph 3H showed little amount of connective tissue around sinusoids.

Discussion

This study was conducted to evaluate the changes in serum betatrophin levels in a rat model of NAFLD, the possible role of betatrophin in the pathogenesis of NAFLD, the effect of metformin treatment of NAFLD on serum betatrophin levels and the potential use of estimation of serum betatrophin in the follow up of NAFLD treatment. In this study, we utilized HFD to induce NAFLD in male albino rats. The results of this study showed a significant increase in BMI in NAFLD group in comparison to that in the control group. These results were supported by Browning, Szczepaniak et al. (2004), Kumashiro, Erion et al. (2011) and Magdy, El-Kharashi et al. (2017) who stated that HFD caused obesity, hyperlipemia and insulin resistance, which were risk factors for NAFLD development. Also, Zheng, Hoos et al. (2008) and Zhu, Zhang et al. (2011) found that BMI was positively correlated with high fat diet. Moreover, Wannamethee, Shaper et al. (2005) declared that increased BMI was used as a measure of excess body fat which increased the risk of developing type 2 diabetes and NAFLD. On the other hand, in the NAFLD+Metformin group, there was a significant decrease in BMI in comparison to that in the NAFLD group. This was supported by Magdy, El-Kharashi et al. (2017) who found a significant decrease in body weight on using metformin in obese rats. On the contrary, Shin, Lee et al. (2014) demonstrated that metformin did not change body weight in mice fed the HFD. This discrepancy may be referred to species difference. Also, the results of this study declared that there was a significant increase in serum glucose, serum insulin and HOMA-IR in the NAFLD group in comparison to that in the control group indicating incidence of insulin resistance. These results were supported by Marchesini, Bugianesi et al. (2003) and Abdelmalek and Diehl (2007) who declared that insulin resistance occurred in cases of NAFLD. On the other hand, there was a significant decrease in serum glucose, serum insulin and HOMA-IR values in the NAFLD+Metformin group in comparison to that in the NAFLD group indicating increased insulin sensitivity by using metformin. These results were supported by Nathan, Buse et al. (2009), Park, Kinra et al. (2009) and Shaw (2013) who confirmed that metformin was effective in treatment of obese diabetic patients, and they confirmed that it increased insulin sensitivity. Also, results of this study were supported by Stephenne, Foretz et al. (2011) and Musso, Cassader et al. (2012) who declared that metformin increased muscle glucose uptake, but, decreased hepatic gluconeogenesis and they attributed this effect to AMP-activated protein kinase (AMPK) activation. Moreover, Apaijai, Pintana et al. (2012) confirmed that metformin improved insulin resistance and oxidative stress caused by HFD consumption by decreasing serum insulin, cholesterol and HOMA-IR index. Moreover, the results of the current study showed that there was a significant increase in serum betatrophin level in NAFLD group in comparison to that in the control group. Also, there was a significant positive correlation between serum betatrophin levels and that of BMI and HOMA-IR in the NAFLD group. These results were in agree with Arias-Loste, García-Unzueta et al. (2015), Lee, Lee et al. (2016) and Hu, Shao et al. (2017) who declared that serum betatrophin increased in NAFLD patients and they declared that serum betatrophin may be a potential non-invasive marker for identification of NAFLD. This was also supported by Yi, Park et al. (2013) who stated that insulin resistance is accompanied by an increase in betatrophin level. This was further confirmed by Chen, Lu et al. (2014), Fu, Berhane et al. (2014), Hu, Sun et al. (2014), and Abu-Farha, Abubaker et al. (2015) who found increased serum betatrophin levels in type 2 diabetes and NAFLD. Moreover, Fenzl, Itariu et al. (2014),  Chen, Chen et al. (2015), Tokumoto, Hamamoto et al. (2015) and Yamada, Saito et al. (2015) reported a positive correlation between serum levels of betatrophin and both obesity and HOMA-IR. On the contrary, Gómez-Ambrosi, Pascual et al. (2014) reported decreased serum betatrophin levels in both obesity and obesity-associated insulin resistance. Also, Cahová, Habart et al. (2017) stated that hepatic expression of betatrophin was unaffected by insulin resistance. This discrepancy may be related to species difference. On the other hand, there was a significant decrease in serum betatrophin level in NAFLD+Metformin group in comparison to that in NAFLD group. This result was supported by Guo, Yu et al. (2016) who owed this decrease to weight reduction and improved fasting plasma glucose and insulin secretion. Also, the results of this study were supported by Tang, Li et al. (2010), Ren, Kim et al. (2012) and Zhang (2012) who found that betatrophin knockout mice have decreased serum levels of triglycerides, whereas betatrophin overexpression has been shown to markedly elevate serum triglycerides. These findings were in agree with Wang, Quagliarini et al. (2013) who evidenced that betatrophin inhibited lipoprotein lipase activity. Moreover, this was supported by Angulo (2002) who stated that insulin resistance played an essential role in NAFLD development and he said that this could be a therapeutic target in the prevention of the progression of NAFLD. On the contrary, Fenzl, Itariu et al. (2014) found that serum betatrophin level was unchanged in type 2 diabetes, while, Gómez-Ambrosi, Pascual et al. (2014) found it decreased. This discrepancy might be due to the use of different kits or relatively small samples. The results of this study showed a significant increase in serum levels of total cholesterol, triglycerides and LDL-C in the NAFLD group in comparison to that in the control group. These results were supported by Liu, Huang et al. (1995),  Kim, Lee et al. (2008) and İyileştirmektedir (2009) who found that feeding of HFD resulted in excess hepatic triglycerides accumulation due to increased synthesis and decreased secretion of triglycerides and increased de novo lipogenesis. Ashakumary, Rouyer et al. (1999) stated that the liver has an antihyperlipidemic mechanism by which it suppressed the synthesis of fatty acids and activated their oxidation, through certain genes regulation. Yang, Li et al. (2008) suggested that the changes induced by HFD was partly due to oxidative damage as antioxidants had a protective effect on the pathways leading to transcription of these genes. Also, these results were in agreement with Olorunnisola, Bradley et al. (2012) and Kumar, Bhandari et al. (2014) who reported that dyslipidemia induced by ingestion of HFD was the primary cause of lipid peroxidation and the decrease in the strength of the antioxidative defenses. Moreover, these results were supported by Simonen, Kotronen et al. (2011) who stated that, in the fatty liver, the ability of insulin to stop the production of both glucose and very low-density lipoprotein cholesterol is impaired which causes hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, which, in turn, lead to lower HDL-C concentration. The results of this study declared that there was a significant positive correlation between serum betatrophin levels and that of TC and TG in the NAFLD group. This was supported by Hu, Shao et al. (2017) found that serum betatrophin was positively correlated to TG even after adjusting a lot of risk factors for NAFLD, which indicated that serum betatrophin had a role in the pathogenesis of NAFLD, and, they confirmed that serum betatrophin was an independent risk factor for NAFLD and a potential non-invasive marker for its progression. Also, serum betatrophin may be helpful for the early diagnosis of NAFLD and improvement of its prognosis. Also, the results of this study showed a significant decrease in serum levels of total cholesterol, triglycerides and LDL-C in the NAFLD+Metformin group in comparison to that in the NAFLD groupThese results were in agreement with Adaramoye, Akintayo et al. (2008) who explained this decrease by the reduction in biosynthesis of hepatic triglyceride and cholesterol redistribution between lipoprotein molecules. These results were supported by Fullerton, Galic et al. (2013), Arner and Langin (2014) and Wang, Zhang et al. (2016) who stated that metformin significantly improved lipid profile in patients with fatty liver. On the other hand, the results of this study showed a significant decrease in serum levels of HDL-C in NAFLD group in comparison to that in the control group, but, there was a significant increase in its serum levels in the NAFLD+Metformin group in comparison to that in the NAFLD group. Also, there was a significant negative correlation between serum betatrophin levels and that of HDL-C in the NAFLD group. Moreover, the results of this study showed a significant increase in serum C-peptide level in NAFLD group in comparison to that in the control group. These results were supported by Toffolo, De Grandi et al. (1995) and Chan, Tong et al. (2004) who found that serum C-peptide levels were increased in obese type 2 diabetic patients and they stated that C-peptide was considered as a marker of -cell function. Also, Jones and Hattersley (2013) stated that C-peptide was produced in pancreatic -cells by cleavage of proinsulin to insulin and C-peptide in equal amounts and thus, it can be used to assess the endogenous secretion of insulin. Moreover, Thunander, Törn et al. (2012) stated that obese insulin-resistant patients had high serum levels of C-peptide. Furthermore, Jones and Hattersley (2013) confirmed that on assessing pancreatic -cell function, measurement of serum C-peptide was used in preference to that of insulin.  But, Li, Meng et al. (2014) found that C-peptide exerted proinflammatory effects in different body tissues, thus, C-peptide may share in the pathogenesis of NAFLD. On the other hand, there was a significant decrease in serum C-peptide level in the NAFLD+Metformin group in comparison to that in the NAFLD group which confirmed the important role of metformin in improving NAFLD. Also, the results of this study showed that serum levels of both IL-6 and TNF- were significantly increased in the NAFLD group in comparison to that in the control group. Also, there was a significant positive correlation between serum betatrophin levels and that of IL-6 and TNF- in the NAFLD group. These results were supported by Reid (2001), Armutcu, Coskun et al. (2005), Miller and Adeli (2008) and Cohen, Horton et al. (2011) who stated that as fat accumulated in the liver, there was a continuous hepatic proinflammatory cytokines generation from the Kupffer cells, causing a vicious cycle of insulin resistance and steatohepatitis. Also, this was supported by Wieckowska, Papouchado et al. (2008) and Hendy, Elsabaawy et al. (2017) who stated that production of IL-6 and TNF occurred early in liver injury. Moreover, Tilg and Hotamisligil (2006) and Polyzos, Kountouras et al. (2009) stated that TNF-α had a proinflammatory action, increased insulin resistance, and caused NAFLD through suppression of secretion and action of adiponectin which normally reduced insulin resistance and had anti-steatotic and anti-inflammatory effects. Furthermore, Wullaert, van Loo et al. (2007) declared that as TNF-α increased with expansion of adipose tissue, the incidence of insulin resistance, fibrogenesis and apoptosis of the hepatocytes increased. In addition, Van Greevenbroek, Schalkwijk et al. (2013) stated that obesity caused a low-grade inflammation showing higher levels of circulating proinflammatory cytokines which can interfere with normal insulin function and, thereby, induced insulin resistance and were also implicated in -cell dysfunction. The results of this study were also in agreement with Fu, Xie et al. (2009) who stated that oxidative stress was essential in the pathogenesis of NAFLD and led to membrane lipid peroxidation and production of pro-inflammatory cytokines. Also, Wang, Wang et al. (2008) found that HFD induced apoptosis that increased hepatic expression of TNF. Moreover, Feingold, Marshall et al. (1994) explained the incidence of insulin resistance in HFD by the increase in TNF-which increased adipose tissue lipolysis that increased the plasma free fatty acid. Furthermore, Ahima and Flier (2000) declared that TNF-increased leptin production that reduced insulin secretion and increased insulin resistance. On the other hand, there was a significant decrease in serum levels of both IL-6 and TNF- in the NAFLD+Metformin group in comparison to that in NAFLD group. These results were supported by Djouder, Tuerk et al. (2010) and Chen, Brooks et al. (2017) who stated that insulin sensitivity improvement and IL-6 and TNF- levels reduction may be the mechanisms by which metformin improved NAFLD. Also,  Woo, Xu et al. (2014) confirmed that metformin inhibited HFD induced liver inflammatory responses, hepatic steatosis and insulin resistance. Moreover, the results of this study showed that there was a significant increase in serum levels of ALT, AST and GGT in the NAFLD group in comparison to that in the control group. Also, there was a significant positive correlation between serum betatrophin levels and that of ALT and AST in the NAFLD group. These results were supported by Reid (2001), George, Pera et al. (2003), Zheng, Hoos et al. (2008), Prasad (2010), Hamed (2011) and Magdy, El-Kharashi et al. (2017) who found a significant rise in serum AST and ALT in rats with NAFLD. Hamed (2011) attributed this increase in liver enzymes activity to the increase in free radicals and lipid peroxidation process that affected hepatocyte membrane permeability and led to leakage of enzymes into the circulation. On the other hand, Demori, Voci et al. (2006) declared that the increased activities of hepatic enzymes in serum occurred in response to HFD-induced oxidative stress. This was supported by (Cui, Liu et al. 2011) who suggested that excessive HFD caused overproduction of free radicals that affected the liver. On the other hand, results of this study showed a significant decrease in serum levels of ALT, AST and GGT in the NAFLD+Metformin group in comparison to that in the NAFLD group. These results were supported by Chen, Brooks et al. (2017) and Magdy, El-Kharashi et al. (2017) who found that metformin improved liver function. The results of histopathology of this study showed signs of fatty liver in NAFLD in the form of swelling of hepatic cells with rarefied cytoplasm and presence of Mallory-Denk bodies. Mallory-Denk bodies were eosinophilic irregular shaped aggregates in the cytoplasm of hepatocytes (Takahashi and Fukusato 2014). Also, there was dense infiltration of inflammatory cells around portal tracts, scattered inflammatory cells were also present in Perisinusoidal space with congested sinusoids. Moreover, hepatocytes appeared bright with fatty drops inside and the Von Kupffer cells were prominent with dense fibrosis around portal tract and perisinusoidal fibrosis of fatty liver. These results were in agreement with Farrell and Larter (2006), Zheng, Hoos et al. (2008) and Bailey, Mantena et al. (2009), who observed similar hepatic architecture changes after a high fat diet. This was attributed to the presence of fatty liver, as a result of hypercholesterolemia and accumulation of vacuoles of fat. This was also supported by Wu, Zhao et al. (2010) who found that, rats fed high fat diet showed severe histopathological NASH lesions including steatosis, ballooning degeneration and inflammation. This is further supported by Brunt and Tiniakos (2010) and Tandra, Yeh et al. (2011) who stated that NAFLD showed over accumulation of fat in the form of well-defined droplets in hepatocytes. Also, Chalasani, Wilson et al. (2008) and Takahashi and Fukusato (2014) reported that lobular inflammation was positively associated with NAFLD. Moreover, Brunt (2010) explained the cause of hepatocellular ballooning that occurred in NAFLD by change of the intermediate filament cytoskeleton as cytokeratins 8 and 18 were disrupted and dispersed to the periphery of cytoplasm instead of equal distribution throughout the cytoplasm. These results were supported by Shinohara, Hanawa et al. (2008) who referred the histopathological changes in NASH to oxidative stress and mitochondrial dysfunction. On the other hand, the results of this study showed that the histopathology was slightly improved in NAFLD+Metformin group as there was a little cellular infiltration around portal tract and classic hepatic lobule preserved, and, most of hepatocytes were normal, some fat drops still present inside hepatocytes with little Von Kupffer cells. These results were supported by Apaijai, Pintana et al. (2012) and Shaw (2013) who confirmed that metformin reduced hepatic steatosis and they referred such improvement to the finding that metformin-induced AMPK activation caused suppression of acetyl-coA carboxylase, resulting in a decrease in hepatic lipid accumulation and an increase in insulin sensitivity. These results were further supported by Magdy, El-Kharashi et al. (2017) who found that metformin produced significant improvement in all histological features of NASH.

Conclusion

In NAFLD, betatrophin levels were increased. This could be an important mechanism to explain the glucose intolerance during the course of NAFLD. It could be also considered as a mechanism by which NAFLD occurred. Also, they were significantly reduced after metformin treatment which indicated that it may be used as an indicator of the improvement of NAFLD. Thus, estimation of serum level of betatrophin can be considered as a biomarker that reflected the early occurrence of NAFLD and to follow up the effect of treatment. Further studies are requested to confirm these results.



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