Synbiotic Goat Milk Ker Lowered Peroxisome Proliferator Activated Receptor Gamma (PPARγ) Gene Expression in Rat Adipose and Liver Tissue

Ker is a fermented milk product containing bacteria and yeast, whereas glucomannan from porang (Amorphophallus oncophyllus) tuber is known as a prebiotic in vivo. Diets with a high fat and high sugar will stimulate metabolic syndrome associated with changes in gene expression including peroxisome proliferator activated receptor gamma (PPARγ). The purpose of this study was to determine the effect of goat milk ker enriched with porang glucomannan (synbiotic ker) and goat milk ker without glucomannan (probiotic ker) on blood glucose, hemoglobin A1c (HbA1c), free fatty acid (FFA), tumor necrosis factor alpha (TNF-α), gene expression of peroxisome proliferator activated receptor gamma (PPARγ), and insulin-producing cells in rats fed a high-fat and high-fructose (HFHF) diet.


Introduction
Limited physical activity and enhanced exposure to unhealthy foods that are energy-dense ("obesogenic" environment) cause increased obesity. The prevalence of obesity in the last decade is becoming increasingly common and becoming a major nutritional problem throughout the world. The risk factors in development of obesity are also in uenced by genetic factors and physiological problems. Obesity may negatively affect on the development of insulin resistance, type-2 diabetes and metabolic syndrome.
Because of the limitations of obesity and metabolic syndrome therapy, prevention strategies are needed [1].
Prevention and treatment of metabolic syndrome can be performed both pharmacologically and nonpharmacologically. Functional food affecting health bene ts can be derived from animal or plant sources. Metabolic syndrome can be treated with various approaches, including targeting lipoproteins, blood pressure or anthropometric index. Peroxisome proliferator-activated receptors (PPARs) are play a role in the metabolic control of lipid and lipoprotein levels, i.e. triglycerides (TGs), blood glucose, and abdominal adiposity [2]. PPARγ is abundantly expressed in adipose tissue and, to a lesser extent, in macrophages and other cell types, and regulates adipogenesis, lipid storage, and glucose homeostasis [3]. PPARγ2 is speci c for adipose tissue, where it plays a pivotal role in adipogenesis and is an important mediator of insulin sensitivity [4] and a more potent transcription activator [5].
In reducing obesity in mice induced by a high fat diet, a probiotic ke r plays an important role in weight loss and reduce the epididymal fat layer and the diameter of adipocytes. The reduction in gene expression associated with adipogenesis and lipogenesis and also a lowered the levels of proin ammatory markers in epididymal fat has con rmed the role of ke r [6]. Recent studies show that properties of ke r and isolated microorganisms from it have the potential to be anti-atherosclerotic through an enhance in anti-in ammatory cytokines and reduce pro-in ammatory responses [7].
Porang (Amorphophallus oncophyllus) is a tuber locally that is often found in Indonesian forests, and it is being cultivated. Similar to Amorphophallus konjac, porang tuber contains glucomannan and has been shown to be a prebiotic in vivo [8], that selectively enhances the growth of probiotic bacteria such as lactobacilli and bi dobacterial [9]. Glucomannan is a water-soluble dietary ber that can improve blood sugar, blood fat concentration, and weight management and has other health bene ts. Subject with metabolic syndrome will be comfortable consuming glucomannan as a substitute for main carbohydrates, in the form of noodles given 4 weeks can reduce the risk of metabolic syndrome and reduce oxidative stress [10].
The purpose of this study was to evaluate the effect of synbiotic ke r (goat milk ke r with additional glucomannan from porang) and probiotic ke r ( goat milk ke r without porang glucomannan) on gene expression associated with the metabolic regulation of lipids and blood glucose, i.e. PPARγ in adipose and liver tissue in rats fed a high-fat and high-fructose diet.

Materials And Methods
Ke r preparation Synbiotic ke r made from a mixture of goat milk, porang glucomannan (as prebiotic), whey protein concentrate (WPC) and ke r grain. Glucomannan from porang tuber was obtained from the Faculty of Agrigultural Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia. Fresh goat milk was originated from Ettawah Crossbred goats in Yogyakarta, Indonesia. Whey protein concentrate (WPC) was obtained from the Sari Husada Milk industry in Yogyakarta, Indonesia. Ke r grain was purchased from a local supplier in Yogyakarta.
The methods of synbiotic ke r preparation was done according to [11] with slight modi cation. Goat milk, 0.1% whey protein concentrate (WPC), and 0.3% porang glucomannan were mixed, pasteurized at 75°C for 15 min, and cooled at room temperature. Ke r grains (2%) were inoculated into pasteurized milk and incubated at room temperature for 18 h. After incubation, the ke r was ltered to separate ke r grains. Probiotics ke r were prepared with goat milk, WPC and ke r grain without glucomannan.
Synbiotics ke r were prepared by addition of glucomannan into probiotics ke r.

Animal experiments
Male Sprague Dawley rats 8-12 weeks old were divided into 5 groups (each group consisted of 6 rats): 1) normal control (negative control rats) that received a standard diet only, 2) rats fed high-fat/high-fructose (HFHF) (positive control), 3) rats fed HFHF + probiotic ke r, 4) rats fed HFHF + synbiotic ke r, and 5) rats fed HFHF+ simvastatin. The dose of ke r was 3.6 mL/200 g body weight/day for 4 weeks. The dose of simvastatin was 0.72 mg/day.
The rats were adapted with standard diet AIN-93 for 1 week and then treated with a high fat and high fructose diet for 2 weeks. The rats were then divided into 5 groups as described above. A high-fat and high-fructose diet was administered until the end of the experiment (4 weeks). The composition of the standard diet and high-fat and high-fructose diet were prepared according to [12,13]

Gene expression analysis
PPARγ2 gene expression was analyzed through 4 stages: 1) isolation of RNA from white adipose tissue and liver tissue, 2) reverse transcription from RNA to cDNA using reverse transcriptase enzyme, 3) cDNA ampli cation by PCR and 4) quanti cation and detection of cDNA products with real-time PCR.
Total RNA was extracted from adipose tissue using TRIzol reagent, and mRNA levels were analyzed by real-time polymerase chain reaction (PCR). Reverse transcription of total RNA was performed using the First Strand cDNA Synthesis Kit (Roche) Transcriptor. Transcription reagents to produce cDNA. Real-time PCR was carried out in a mixture ( nal volume 20 µL) containing 2 µL cDNA (DNA template), 10 µL Evagreen, 1 µL GAPDH, 1 µL GAPDH, and 6 µL RNAse-free water. Likewise for Forword and Reverse PPARγ2 with additional reagents totaling 20 µL as well. The amount of mRNA was calculated as the ratio to the value of glyceraldehyde-3-phosphate dehydrogenase (GADPH) in each cDNA sample. The primary nucleotide sequences used to detect each mRNA were designed using Primary Express Software according to the sequences available in the GenBank database. The primary nucleotide sequences are shown in Table 2 [14]. Optimization of cDNA ampli cation products was performed using conventional PCR with a program at a temperature of 95°C for 5 minutes, 95°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute with 34x cycles. The temperature was maintained at 72°C for 5 minutes and 12°C for 5 minutes. The optimization program for real-time (RT)-PCR was at a temperature of 95°C for 5 minutes, 95°C for 1 minute, 60°C for 30 seconds and 72°C for 1 minute with 39x cycles. The melt curve was maintained at 65-95°C for 5 seconds, and then the plate was read. The average change in the level of gene expression (2 -Δ Δ CT ) PPARgamma-2 was analyzed according to [15].

Immunohistochemistry of insulin-producing cells
Mouse monoclonal insulin primary antibody (Abcam, [K36aC10] ab6995, Cambridge, USA) was used in this analysis. Pancreatic tissue slides were counterstained with hematoxylin, mounted with coverslips and observed under a light microscope. The number of Langerhans islets and insulin-producing cells were calculated using a colony counter and then documented by an Opti Lab (SOP No A-007) microscope.

Statistical analysis
Data from this analysis are presented as the mean±standard deviation. Data of blood plasma analysis before and after treatments including fasting blood glucose, HbA1c, FFA, and TNFα. The difference between the mean of blood plasma analysis before and after treatments was analyzed by pairedsamples t-test. One-way ANOVA followed by Duncan's multiple range test (DMRT) was used for statistical analyses of the gene expression of PPAR, total number of Langerhans islets and insulin-producing cells (p-values of less than 0.05 indicated signi cant differences). Statistical analyses were performed using SPSS version 17 software.

Blood glucose
The average fasting blood glucose levels in rats fed a high fructose and high fat diet before and after being treated with probiotic ke r, synbiotic ke r and simvastatin are shown in Table 3. Table 3 The average blood glucose in rats before and after treatments  Table 3 shows that in negative controls (normal rats, only receiving standard diet), blood glucose levels were still within the normal glucose range, and there was no difference before and after treatment. Rats that only received high-fructose and high-fat feed (positive control) showed higher glucose levels after being given the diet for 5 weeks (after treatment) compared to before treatment, although not signi cantly. Goat milk ke r enriched with porang glucomannan could reduce blood glucose levels, but the decrease was not signi cant, which was only 11.9 mg/dL. Simvastatin treatment signi cantly reduced blood glucose levels in rats fed a HFHF diet, which was approximately 139.02 mg/dL.

Hemoglobin (Hb)A1c
Based on Table 4, HbA1c levels in rats after treatment with synbiotic ke r were lower (p<0.05) than those before treatment. However, other groups of rats, including those who received probiotic ke r treatment, did not show any signi cant difference before and after treatment. Free fatty acid (FFA) Table 5 shows that the average plasma FFA levels in rats after various treatments were higher (p<0.05) in all groups of rats than before treatment, although the increase in FFA after ke r treatment was not signi cant. Tumor necrosis factor alpha (TNFα) Based on Table 4, there was no decrease in TNFα levels in rats after various treatments, except in rats treated with synbiotic ke r. PPARγ-2 gene expression The average change in the level of PPARγ2 gene expression (2 -Δ Δ CT ) in white adipose tissue from HFHF rats treated with ke r with or without glucomannan was not signi cantly different from that of rats treated with simvastatin. The rats treated with ke r had a lower change in PPAR γ2 gene expression than HFHF rats without ke r (p<0.05) ( Table 7). Normal control rats had an average Δ CT of -3.07, an average Δ Δ CT of 0.00 and an average of changes in the gene expression of PPAR γ2 (2 -Δ Δ CT ) of 1.00.
Based on Table 8, the pattern of the changes in the expression of PPARγ2 genes in liver tissue was similar to that of white adipose tissue, in which rats treated with ke r added or without glucomannan showed lower changes in gene expression (p <0.05) compared to rats without ke r treatment. Likewise, the simvastatin-treated rats also had lower changes in PPARγ2 gene expression (p <0.05) than the HFHF rats. Normal control rats had an average Δ CT of 6.29, Δ Δ CT of 0.00 and an average of changes in gene expression of PPAR γ2 (2 -Δ Δ CT ) of 1.00. Table 7 and Table 8 showed that the change in PPARγ2 gene expression in liver tissue was higher than that in adipose tissue. In the rats fed a high-fructose and high-fat (HFHF) diet without ke r supplementation, the highest changes in PPAR gene expression (p <0.05) were observed in both adipose and liver tissue.

Immunohistochemical (IHC) of ß-cells
The average number of Langerhans islets and insulin-producing beta cells with various treatments is shown in Table 9. The average number of Langerhans islets and insulin-producing beta cells in rats fed high fat and high fructose without ke r supplementation showed the lowest number, although not signi cantly different (Table 9).
Immunohistochemical (IHC) staining of pancreatic tissue showed that insulin-producing beta cells showed a brown color when using rat anti-insulin antibodies (Fig.1). Figure 7 shows that HFHF rats were rarely observed on Langerhans islets and had very weak intensity of IHC staining on insulin-producing beta cells, and there were few insulin-producing beta cells (Fig. 1), although the number of Langerhans islets and beta cells in all treatment groups was not signi cantly different ( Table 9). Rats that received ke r treatment showed intense beta cell staining intensity as in negative control rats (normal normal rats). The HFHF rats given simvastatin showed less strong IHC staining intensity compared to ke r treatment.

Discussion
All rats fed a high fat and high fructose diets demonstrated a risk factor for metabolic syndrome with fasting blood glucose > 100 mg/dL [2]. After ke r treatments (probiotic and synbiotic ke r) in this study, there were no decrease in blood glucose in rats fed HFHF diet, except for simvastatin treatment. However, a konjac-derived glucomannan supplement (3.6 g/day) administered for 28 days reduced blood lipid and glucose levels by enhancing fecal excretion of neutral sterol and bile acid and alleviated the elevated glucose levels in hyperglycemic diabetic subjects [16]. In contrast to a previous study by [17], skim milk ke r given at a dose of 3.6 ml/day for 4 weeks could signi cantly reduce blood glucose levels by 111.00 mg/dL. In the present study, the decrease in low blood glucose was possibly because synbiotic ke r was still not enough to play a role in reducing blood glucose in rats that consumed HFHF diets during the experiment. In the study by [17], diabetic rats were not fed a HFHF diet. The low dose of glucomannan in ke r and the difference in the conditions of the subjects may not cause a signi cant reduction in blood glucose levels.
The decrease in blood glucose by simvastatin treatment in this study is in accordance with a previous study by [18], in which mice fed a high-fat diet and treated with rosuvastatin showed lower blood glucose, which might be due to improved glucose uptake, but beta cell activity is inhibited through lowered insulin levels and inhibited Ca 2+ signaling in beta cells, resulting in lowered insulin secretion. Duoble effects on glucose homeostasis by rosuvastatin are due to increased insulin sensitivity, while beta cell activity is inhibited. In another study by [19], glucose uptake in adipose tissue was upregulated in pravastatintreated mice fed a high fat/high sucrose diet and db/db mice. In contrast to studies by [20,21], simvastatins can increase the risk of T2DM, particularly in prediabetic subjects, due to hyperglycemia by impairing the function of islet β cells and have a negative effect on glucose homeostasis, especially on fasting blood glucose levels. Atorvastatin at a high dose causes worsening of glycemic control in patients with DM [22]. According to [23], individual types of statins may have different effects on glucose metabolism. Based on the results of these studies, the possible effect of statins on blood glucose levels depends on the dose and type of statin and the condition of the subject used for the study.
Porang glucomannan added to ke r could improve glucose metabolism to reduce glycosylated hemoglobin. According to [24], the synergistic effects of these two components, probiotics and prebiotics, make it a more effective supplement than probiotics or prebiotics separately. In another study by [25], the fructose diet was rapidly metabolized by the liver, causing changes in carbohydrate and lipid metabolism as well as hepatic in ammation, which led to the development of hyperglycemia, insulin resistance, hyperinsulinemia, and hypertriglyceridemia as major risk factors for diabetes complications. The administration of a high fructose diet (68.35%) over a long period of time can induce complications related to type 2 diabetes, namely, high blood glucose, glycosylated HbA1c, cholesterol, triglycerides and oxidative stress [26]. However, the results indicate that the administration of fermented milk containing the probiotic Lactobacillus rhamnosus GG (150 g/kg standard diet) can reduce the increase in glycosylated hemoglobin (HbA1c) in rats induced by diabetes by feeding high fructose feeds [26]. Additionally, the 24 individuals with T2DM had signi cantly decreased HbA1c by 7.7% after glucomannan noodle intervention [10].
Probiotic and synbiotic ke r in the present study could maintain plasma FFA levels in HFHF rats. In a previous study, konjac-glucomannan supplementation (5%) in baboons resulted in lower than baseline values for triglycerides and circulating free fatty acids after 9 weeks [27]. The lower dose of glucomannan from porang tuber in the present study compared to the previous study by [27] resulted in no decrease in plasma FFA. According to [28,27], increased levels of circulating FFAs can stimulate brinogen synthesis in the liver. Elevated plasma brinogen is characteristic of insulin resistance in the liver (insulin may regulate the synthesis of bribinogen). Glucomannan from konjac, which is fermented in the colon, can decrease FFA production, including propionate, leading to a decrease in brinogen synthesis. Therefore, colonie production and absorption of SCFAs (propionate) from soluble ber may contribute to this ber's metabolic effects [27]. The various physiological processes, including the control of lipolysis and lipogenesis in adipose tissue, in ammation, endocrine signaling and the composition and characteristics of cellular membranes may be affected by each kind of FFA. The progress of insulin resistance and coagulatory damage may result from the physiological changes caused by changed plasma FFA levels or pro les [29].
In the present study, porang glucomannan added to ke r can play a role in reducing the occurrence of in ammation through decreased production of pro-in ammatory cytokines in rats fed high-fat high fructose. The effect of soluble ber in porang glucomannan on the improvement of metabolic disorders is in accordance with a previous study using chitosan ber [30], which is given to rats with metabolic disorders (induced by diabetes), can improve insulin resistance and chronic in ammation through decreased lipid absorption and slowed absorption of glucose in the small intestine after eating, resulting in a decrease in hepatic lipids and weight of adipose tissue, and reduced plasma adipocytokine levels including leptin, TNFα and plasminogen activator inhibitor-1 (PAI-1).
In other study, supplementation with a combination of ber (konjac glucomannan) and bacterial cellulose in high-fat diet-induced obesity in mice had a more positive effect on obesity-associated hepatic in ammation by reducing the levels of TNFα and IL-6 and suppressing the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf-2) in comparison with supplementation with bacterial cellulose or konjac glucomannan alone [31]. In addition, glucomannan and spirulina combination blocks detrimental effects promoted by hypercholesterolemic diets in Zucker rats, one of which could decrease plasma TNFα as one of an in ammation biomarkers [32].
Normally, PPARγ2 is most abundantly expressed in adipocytes and plays major adipogenic and lipogenic roles in the tissue [33]. Because the rats in the present study received a high-fat and high-fructose diet, it was possible to cause fatty liver. According to [34,35], in non-alcoholic fatty liver disease (NAFLD) patients and experimental animals there was an increase in the expression of PPARγ in the liver. In addition, in mice fed a high-fat diet showed a high PPARγ expression in the liver [36].
In the present study, the change in gene expression was the lowest in rat tissue that was treated with synbiotic ke r, although this difference was not signi cant compared to probiotic ke r treatment. It is possible that ke r-containing probiotics synergize with the prebiotic glucomannan and play a role in the downregulation of PPARγ2 expression in white adipose and hepatic tissue. The result in the present study was similar to that of a previous study [37], in which mice fed a high-fat diet supplemented with 0.2% ke r powder for 8 weeks lowered PPARγ gene expression in the epididymal fat. In another study, mice fed a high-fat diet and 1 × 10 7 or 1 × 10 9 CFU /mice probiotic L. plantarum LG42 supplementation daily for 12 weeks reduced PPARγ expression in adipose tissue [38]. Decreased levels of PPAR-γ and GLUT4 mRNA after high fructose treatment were also enhanced by Lactobacillus reuteri GMNL-263 administration [39]. It was further emphasized by [31], besides reducing PPARγ expression, the mixed bacterial cellulose and glucomannan from konjac also lowered the protein expression of PPARγ by reducing the size of cells in the adipose tissue of high-fat diet-fed mice.
Consumption of dietary bers, especially mixed bacterial cellulose/konjac glucomannan, resulted in an improved antioxidant defense system and reduced lipid peroxidation in the liver by increasing the activity of antioxidant enzymes and reducing the formation of malondialdehyde (MDA) in the liver. Moreover, supplementation with these bers regulated the levels of leptin and adiponectin and inhibited the protein expression of PPARγ by reducing the size of cells in the adipose tissue of high-fat diet-fed mice [31] The highest changes in PPAR γ2 gene expression in both adipose and liver tissue of rats treated HFHF without ke r in the present study were in accordance with the results in a previous study [40], who found that the PPARγ expression level was signi cantly higher in rats fed a high-fat diet than in rats fed a normal diet, which is mainly related to fat formation. PPARγ2 is also expressed in the liver, speci cally in hepatocytes, and its expression level positively correlates with fat accumulation induced by pathological conditions such as obesity and diabetes [33] There was no change in the number of Langerhans islets and insulin-producing beta cells in all treatments, indicating that a high-fructose high-fat diet received during the experiment did not cause βcell damage. This was also evidenced by unchanged average fasting blood glucose levels in HFHF rats before and after being treated with ke r ( Table 2). According to [41], individuals with type 2 diabetes have decreased β-cell mass compared to nondiabetic individuals, and fasting blood glucose will increase if the volume (mass) of cells is less than the 1.1% threshold [42]. If it is below this threshold value, changes in insulin sensitivity and functional damage in insulin secretion will have a major impact on blood glucose.
A high fat and high fructose diets in the present study had not yet led to diabetes but only caused prediabetes because blood glucose levels ranged from 100 mg/dL to 125 mg/dL, which is at risk of becoming diabetic (≥ 126 mg/dL), whereas normal blood glucose was < 100 mg/dL [43,44].
Immunohistochemical staining showed that rats fed HFHF diet without ke r addition had the weakest color intensity. However, rats fed HFHF diet with probiotic or synbiotic ke r showed a strong color intensity as in normal rats (Fig. 1). These result indicate that the probiotic microorganisms in ke r have an important role in improving insulin-producing β-cells. This was supported by a previous studies on diabetic rats treated with konjac extract (containing glucomannan) alone showed less strong in improving insulin-producing β-cells than the rats treated with combination of konjac and inulin extract [45].

Conclusions
The metabolic syndrome caused by the habit of consuming high-fat and high-fructose diets can be improved by consuming synbiotic ke r, through decreasing HbA1c, TNFα, and gene expression of PPARγ2 and preventing the increase in FFA. Therefore, synbiotic ke r containing porang glucomannan is expected to be a suggestion for the food industry to develop synbiotic-based functional foods that have the potential to improve metabolic syndrome.

Data Availability
The original data used to present results of this study are available upon request.

Con ict of Interest
We certify that there is no con ict of interest with any nancial, personal, or other relationships with other individu or organization associated with the material discussed in the manuscript.

Authors' Contributions
EH had a great contribution on research planning, discuss and review paper. S and N had a contribution to overall planning, preparing and performing this research.