Adiponectin biochemical and histopathological effects on obesity / type-II diabetes mellitus and pancreatic ß-cell dysfunction in experimental rats

1 Pathology Department, Faculty of Medicine and Applied Medicinal Science, Northern Borders University, KSA. 2 Physiology Department, Faculty of Medicine, Northern Borders University, KSA. 3 Histology Department, Faculty of Medicine, Northern Borders University, KSA. 4 Biochemistry Department, Faculty of Medicine, Northern Borders University, KSA. 5 Histology Department, Faculty of Applied Medicinal Science, Northern Borders University, KSA.


INTRODUCTION
Adiponectin is an adipocyte hormone which involves in glucose and lipid metabolism.It makes up about 0.01% of total human plasma proteins with varied concentrations.This concentration is valued 1000 times greater than other hormones, like leptin and insulin (Haluzik and Parizkova, 2004).Adiponectin is expressed and secreted only from white and brown adipose tissues (Ukkola and Santaniemi, 2002).Adiponectin has been proposed as a factor that may be considered as a link between the insulin resistance, excess obesity and beta cell dysfunction in the case of diabetes mellitus type-II (Kahn and Flier, 2000).It plays an important role in hyperglycemia, dyslipidemia and other inflammatory mechanisms (Shulman, 2000).It has been identified as a potential therapeutic target for the treatment of diabetes (Wild et al., 2004) for its anti-diabetic, anti-atherogenic and anti-inflammatory roles.Its production is inhibited by obesity beside diabetes mellitus type-II (DM-II) (Zhang et al., 2010).
Obesity is a complex multifactor disease characterized by an excessive accumulation of adipose tissue that may impair health.It is a major risk factor for the development of DM-II and is thought to confer increased risk for type-II diabetes through obesity-associated insulin resistance (Nathan et al., 2009).Expression and plasma levels of adiponectin decrease when insulin resistance and obesity are increased and increase when insulin sensitivity is improved and weight is lost (Wilson and Gyi, 2010).The fact that obesity is the state of adiponectin deficiency makes this hormone a target for possible therapeutic interventions, which focusing on the possibility of adiponectin treatment may improve obesity-related insulin resistance.Low adiponectin levels, in addition to insulin resistance, have been shown in DM-II, since adiponectin has been demonstrated to play an important role in response to insulin as its expression leads to decrease glucose levels (Butler et al., 2003).Consistent with the low circulating levels of adiponectin observed in DM-II, the concentration is inversely related to insulin resistance as its levels are decreased in DM-II (Retnakaran et al., 2005).Insulin resistance is the major characteristic of DM-II.Hypoadiponectinemia has a closer relationship with the degree of insulin resistance and hyperinsulinemia (Combs et al., 2001).In the Kingdom of Saudi Arabia, the rise in the prevalence of DM-II has been stated to gain attention years after rapid industrialization, which took place in the country (Alzaid, 1997).
In the pathogenesis of DM-II, progressive deterioration of ß-cell function leads to an inability to secrete sufficient insulin to compensate for insulin resistance (Hotta et al., 2000).In rodent models of obesity/without diabetes, there is an adaptive increase in ß-cells mass to meet metabolic demands (Milan et al., 2002).Obese humans with diabetes had a respective 40 and 63% deficit in relative ß-cells volume.The decreased ß-cells volume in patients with DM-II was due to a reduced number of ß-cells (Souza et al., 2005).Type-II diabetes is accompanied by chronic insulin resistance and a progressive decline in ßcell function (Fruebis et al., 2005).Dysfunction of ß-cells or increased rates of ß-cells death (apoptosis) would result in reduced capacity to produce insulin (Reinehr et al., 2005).ß-cells mass is regulated by a balance of their replication and apoptosis, as development of new islets from exocrine pancreatic ducts (neogenesis).Disruption of any of the pathways of ß-cells formation or increased rates of ß-cells death would result in decreased ß-cell mass and thus reduce the capacity to produce insulin (Stefan et al., 2002).The amount of ß-cells in mammalian adults is tightly regulated and maintained at about 1% of the weight of the pancreas (Weyer et al., 2001).Thus, the purpose of this study was to evaluate the association of adiponectin level with the incident DM-II; with/without obesity.The hypothesis that the high glucose  concentrations adversely affect the survival and cause apoptosis of pancreatic islets ß-cells was tested.To further insight, the differences of adiponectin levels accompanied by the baseline of lipid profile, glucose and insulin levels were investigated.Besides that, histopathological and immunohistochemistry studies were done on the pancreatic ß-cells.

Animals
The experiments were carried out on male and female Wistar rats (aged: 75 to 90 days old) weighting about 250 to 300 g obtained from the Faculty of Pharmacy, King Saud University, Kingdom of Saudi Arabia.During the experiment, the animals were housed in clean properly ventilated cages under constant controlled climatic conditions: temperature (23°C) and lighting conditions (12 h light /12 h dark).Rat food and filtered tap water were provided ad libitum to all animals.They were acclimatized to their environment at least two weeks before starting the experiment.The practical part of the study was done in Northern Borders University, Faculty of Medicine, Arar and King Abd-Elaziz University, Faculty of Medicine, Jeddah.

Streptozotocin diabetic rat model
Adult Wistar rats weighting about 250 to 300 g (75 to 90 days old) were used for inducing diabetes.The animals were injected by streptozotocin at dose of 60 mg/kg of body weight intravenously.Streptozotocin induced diabetes within 3 days by destroying ß-cell (Yamauchi et al., 2003a).Animals received a standard rodent chow diet containing (by weight): 19.80% protein, 39.25% carbohydrate, 4.41% fat, 13.25% fibre, and 2.76 kcal/g of metabolizable energy, given to groups (3, 4, 7, and 8).

Obese rat model
Adult Wistar rats weighting about 250 to 300 g (75 to 90 days old) received a high-fat diet (20% fat content).Hypercaloric chow was used to make a mixture with 131.01 g of sucrose, 84.77 g of soy oil, 12.33 g of cholesterol and 1.23 g of cholic acid with 1000 g of a previously triturated standard chow.The dietary ingredients were homogenized in 60°C warm distilled water and homogenate was used to prepare the pellets.This continued for 10 to 14 weeks (Hug et al., 2004).Hypercaloric chow containing: 15.25% protein, 43.34% carbohydrate, 11.86% fat, 10.20% fibre (by weight) and 3.41 kcal/g of metabolizable energy were given to groups (2, 4, 5, and 8).

Body mass index determinations
The animals were anaesthetized (0.1 ml i.p. of 1% sodium barbiturate) for measurement of body length (nose-to-anus, or nose-anal length) (Pannacciulli et al., 2003).The body weight and body length were used to determine body mass index (g./cm 2 ) once a week.

Biochemical studies
The animals were anaesthetized (0.1 ml i.p. of 1% sodium barbiturate) and blood was collected intravenously (i.v.) of rats tails using capillary tubes containing heparin.The blood samples were collected at same time of measurements day and that was repeated weekly.The food was withdrawn 10 h before blood collection.The blood samples were stored in ethylenediaminetetraacetic acid (EDTA)-coated tubes.

Histopathological and Immunohistochemical studies
By the end of the experiment, the rats were anaesthetized by ether and sacrificed by decapitation.The pancreas was extracted.The specimens were immersed in 10% formalin neutral buffered for 24 h, washed, dehydrated, cleared and embedded in paraffin.Then, specimens were processed into 5 µm sections for light microscopic examination routine hematoxylin and eosin stain (H&E) (luiz and Jose, 2010).Immunohistochemical staining occurred by applying anti-insulin antibodies stain method; which stain ß-cells (endocrine secretary portion in pancreatic islets of Langerhans) as follow: Ab-7842 added at 1/1000 dilution.The sections were fixed with formaldehyde and blocked with 5% serum prior for incubation with primary antibody for 12 h.A Cy2 conjugated goat polyclonal antibody was used as secondary antibody.ß-cells, unit of insulin secretion, were stained in green or reddish brown (Fujita et al., 2008).

Statistical analysis
The results were presented as means ± standard deviations (SD).Analysis of variance (ANOVA) for two variables (Two Way-ANOVA) was used together with student t-test.Significant analysis of variance results were subjected to post hoc.Statistical significance was set at P<0.05 and high significance was set at P≤ 0.01 (Fisher, 1970).

Body mass index determinations
Table 1 summarizes body mass index (g/cm 2 ) during the study period (14-weeks) for normocaloric groups (ND) which include (groups 1, 3, 5, and 7) and hypercaloric diet (HD) groups which include (groups 2, 4, 6, and 8).ND and HD groups started with a similar body weight at week 0.There was significant separation of body weight between ND and HD groups at week 5 till week 9; but a high significant separation was observed till study end (week 14).

Determination of diabetes
Animals were injected with streptozotocin and became markedly diabetic with type-II as manifested by fasting serum glucose levels (Table 2).Baseline values of glucose were relatively similar in day 0 in all studied groups.Fasting serum glucose levels were significantly high in diabetic groups when compared with non-diabetic groups/with or without obesity and without adiponectin injection and also high from those with adiponectin injection.There was no significant variation in serum glucose levels between adiponectin injection groups (Tables 2).After adiponectin treatment, a high significant decrease in serum glucose levels was noticed when compared with those without adiponectin treatment in diabetic/with or without obesity groups.

Lipid profile
Serum levels of triacylglycerol increased in HD groups than ND groups (Table 3).HDL was lower in HD rats than ND rats (Table 4).LDL was increased in HD rats than ND rats (Table 5).Triacylglycerol, HDL and LDL levels do not vary among diabetic/without obesity groups.There were significances for triacylglycerol, HDL and LDL levels in 5th week till 9th week, but there was a high significant difference till 14th week.After adiponectin induction, a high significant decrease was observed in serum LDL and triacylglycerol levels and a high significant increase in serum HDL levels when compared with those without adiponectin treatment in obesity groups.

Hormonal profile
Baseline values of adiponectin and insulin levels were similar among the all groups in day 0. Serum adiponectin levels were highly decreased significantly and insulin levels were highly increased in diabetic groups/without adiponectin treatment significantly when compared with other non-diabetic groups/without adiponectin induction (Tables 6 and 7).Adiponectin levels decreased significantly in obese group without adiponectin treatment in the 5th week till week 9, with high significance till week 14.After adiponectin induction, a high significant decrease was observed in serum insulin levels and a high significant increase in adiponectin levels in groups with adiponectin treatment when compared with those without adiponectin treatment to be in the demanded balance (relatively similar to control group) (Tables 6 and  7).There was no significant variation in the serum adiponectin and insulin levels between adiponectin injection groups.

Histopathological studies
For histopathological studies of pancreatic tissue sections by routine hematoxylin and eosin stain (H&E) (Figure 1), the comparison between these sections showed pancreatic Langerhans islets of diabetic rats' degeneration with necrotic changes.There was a severe vaculation of pancreatic islets with an extensive fibrosis and loss of architecture.Also, there were hyaline and amyloidal droplets which were due to extracellular proteinaceous materials deposition.These of all alterations were less than those of the obese cases.On the other hand, after adiponectin treatment, there were regeneration for pancreatic Langerhans islet cells and a reduction in vaculation and fibrosis, also hyaline and amyloidal droplets were lightly observed.

Immunohistochemical studies
For a comparison of insulin content, anti-insulin antibody stain was applied.Strongly positive immune-reaction was observed in the control and adiponectin groups.After adiponectin administration, diabetic/with or without obesity groups showed moderate positive immunereaction of insulin in pancreatic β-cells; as adiponectin helped in regeneration of pancreatic β-cells, then insulin secretion became in the demanded balance (Figure 2).On the other hand, at diabetic/with or without obesity groups, but without adiponectin induction, there was a light to null immune-reaction of insulin in pancreatic βcells.

DISCUSSION
Adiponectin may provide a link between obesity and diabetes.It exerts profound anti-diabetic, anti-atherogenic and anti-inflammatory roles (Fasshauer et al., 2002).This study demonstrates that serum adiponectin levels were altered after the induction of DM-II and/or hyperlipidemia.Serum adiponectin levels were decreased in diabetic rats when compared with their non-diabetic counterparts.This finding agreed with previous studies showing that adiponectin levels decrease in DM-II (Mao et al., 2006).
Streptozotocin-induced diabetes resembles human DM-II and reduces adiponectin, due to decreased insulinmediated glucose metabolism in adipose tissue (Fasshauer and Paschke, 2003).The present results showed that serum adiponectin levels were negatively associated with the body mass index, so obesity is characterized by hypoadiponectinemia (Trayhurn and Beattie, 2001) and were decreased in DM-II (Yamauchi et al., 2003b).This study results agreed with the result of Abbasi et al. (2004).Insulin resistance is a major feature in the etiology of obesity and type-II diabetes, and the relationship between insulin and adiponectin was studied.There are some conflicting reports concerning the influence of insulin on the adiponectin gene expression (Matsushita et al., 2007).This study found decreased adiponectin levels in hyperlipidemic rats, in DM-II animals, as well as in those ones having both DM-II and hyperlipidemia.The reason may be the lack of an immune mechanism that leads to β-cell damage and insulin deficiency in our model of streptozotocin-induced diabetes (Dubiel et al., 2012).In this study, although animals were fed with highfat diet presented with hypercholesterolemia, little difference was observed in adiponectin levels among non diabetic groups/with obesity induction as there is difference in serum lipid profile.This finding is in agreement with weak correlation between adiponectin hormone and levels of total cholesterol (Kern et al., 2003).
In this study, LDL levels increased, HDL levels decrease and triglyceride levels increased, because saturated fat resulted due to blood cholesterol increase.These results resemble the results of Ouchi et al. (2003) which stated that eating too much fat may cause hyperlipidemia with increase in the total triglyceride and LDL and decrease in HDL.The increase in total cholesterol is associated with the decrease in HDL as cholesterol is present in all tissues and lipoproteins plasma and free cholesterol is expelled from tissues by HDL (Andreelli et al., 2006).HDL cholesterol is often called "good" because it is a lipoprotein that transports lipids from the periphery (extra-hepatic) to the liver.However, LDL is a lipoprotein that transports lipids from liver to peripheral (extra-hepatic) and is often called "bad"cholesterol.The state of hypercholesterolemia is characterized     by increasing blood cholesterol levels above normal; then obesity is caused (Stefan et al., 2002).On the other hand, after adiponectin treatment for obese and diabetic groups, the levels of adiponectin increased and insulin levels were in balance.Adiponectin is a possible insulinsensitizing agent (Kim et al., 2006).Recently, it has been reported that adiponectin levels in diabetic rats were suppressed when compared with non-diabetic rats (Kahn, 2003).Plasma adiponectin levels were shown to be a marker of glucose metabolism and obesity.After recombinant adiponectin treatment, reduced serum glucose was observed in diabetic rodents stimulating insulin secretion, which was in agreement with the study of Bergman et al. (2002).Recently, adiponectin treatment has been shown to improve insulin resistance with attenuation of body weight gain (Okamoto et al., 2003).Another potential mechanism for adiponectin protective effect is improved insulin secretion and action, which has been shown recently (Matsuzawa et al., 2002).
Adipocyte-derived mediator has been proposed as a factor that link insulin resistance, excess obesity and ß-cell dysfunction in type-II diabetes (Meyerhardt et al., 2008).Type-II diabetes is characterized by a combination of insulin resistance and alterations in ß-cell function (Shargorodsky, 2009).The latter may be ascribed to a certain extent as deleterious effect of chronic hyperglycemia is referred to glucotoxicity (Wolpin et al., 2008).This evidence for a link between hyperglycemia and dysfunction in pancreatic islets of Langerhans comes from vivo and in vitro studies in animal models (Oda, 2008).
The main features of apoptosis are architecture of the islet not maintained (Shargorodsky, 2009).A novel animal model of type 2 diabetes, showed that there was hyperglycemia development which was associated with pancreatic ß-cell failure (Yan, 2008) and this was shown in the histopathological and immunohistochemical studies on the diabetic rats in this study.However, adiponectin injection helped in neogenesis of the pancreatic ß-cells and regeneration of the pancreatic islets of Langerhans.Several studies have shown that chronic elevation of blood glucose concentrations in both humans and experimental animal models leads to ß-cell dysfunction in terms of insulin secretion and insulin synthesis (Waki et al., 2003), despite some evidence for glucose toxicity leading to cell death (Pollak, 2007).

Figure 1 .
Figure 1.The histopathology of pancreatic islet of Langerhans.Normal appearance in control group (a).Normal appearance in adiponectin/without diabetes nor obesity group (b).Diabetic rats/without adiponectin treatment with a severity of vaculation of pancreatic Langerhans islets and extensive fibrosis.There was severe degenerative and necrotic changes with obvious amyloid and hyaline droplets (e), (g).The degeneration and necrotic changes in Langerhans islet were less than those in obese rats/without adiponectin treatment (c).Islets of Langerhans with light to null degenerative changes and light vacuoles in rats after adiponetin treatment (d), (f), (h).(H&E x400).

Figure 2 .
Figure 2. Immunohistochemicaal studies of pancreatic islet of Langerhans.Strongly stained β-cells with antiinsulin antibody immune reactive stain in control group (a) and in adiponectin/without diabetes nor obesity group (b).Moderate staining β-cells in Langerhans islets in adiponectin treatment groups (obesity/diabetic rats) (d), (f) and (h) as there was increased number of insulin immune-reactive granules in β-cells.There is light to null staining β-cells in Langerhans islets in the groups without adiponectin treatment (diabetic rats) (e), (g) (obesity rats) (c).(Anti-insuline antibody stain x400).

Table 1 .
Body mass index (gm/cm).Values were expressed as means ± standard deviation.

Table 2 .
Mean of serum glucose levels (mg/dl), values expressed as means ± standard deviation.

Table 6 .
Mean of serum adiponectin (ng/ml) levels.Values expressed as means ± standard deviation.