ORIGINAL ARTICLE |
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1 Endocrine and Metabolic Unit, Nutrition, Metabolism and Cardiovascular Research Centre (NMCRC), Block C7, Level 3, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, Setia Alam, 40170, Shah Alam, Selangor, Malaysia;
2 Downstream Technology Division, CRAUN Research Sdn. Bhd., Lot 3147, Block 14, Jalan Sultan Tengah, 93050 Kuching, Sarawak, Malaysia
Corresponding Author: Ezarul Faradianna Lokman, Endocrine and Metabolic Unit, Nutrition, Metabolism and Cardiovascular Research Centre (NMCRC), Block C7, Level 3, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, Setia Alam, 40170, Shah Alam, Selangor, Malaysia. Tel: +60 33627824 / 7834.
Note: Ezarul Faradianna Lokman and Sal Hazreen Bugam contributed equally to the study and manuscript writing.
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ABSTRACT |
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INTRODUCTION |
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MATERIALS AND METHODS |
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RESULTS |
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DISCUSSION |
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ACKNOWLEDGEMENTS |
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FUNDING |
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CONFLICT OF INTEREST |
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AUTHORS CONTRIBUTIONS |
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REFERENCES |
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ABSTRACT
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Resistant starch (RS) Sago (Metroxylon sagu) intake has been linked with the improvement in postprandial hyperglycemia and diabetes management via several modes of action including delayed glucose absorption and inhibition of carbohydrate digestion in the gastrointestinal tract. However, to our knowledge, studies on local Malaysian sago RS associated with hepatic glucose production has not been reported elsewhere. Thus, this study was done to identify the underlying mechanisms of local Malaysian RS sago native and modified known as sago RS type 2 (sago RS2) and type 4 (sago RS4) respectively in glucose regulations by analyzing the targeted genes in hepatic glucose pathways. In this study, gene expression associated with Glucose and Glycogen Metabolism Pathways analysis in the liver of spontaneously type 2 diabetic rat, Goto kakizaki treated with water (control), Hi Maize (positive control), sago RS2 and RS4 was done using Rat Glucose Metabolism RT² Profiler PCR Array which consist of 84 genes. The results showed that several genes were significantly up- and down-regulated in the diabetic rats treated with Sago. Taldo1 was significantly upregulated whereas G6PC, Sdhb and Rplp1 genes were significantly downregulated in the rat liver treated with sago RS2. In the rat liver treated with sago RS4, Idh3g gene was significantly upregulated whereas G6pc, Pdk3, Eno3, Sdhb, Galm and Tkt genes were significantly downregulated. The gene expressions identified are associated in the blood glucose homeostasis involving the regulation and enzymatic pathways of glucose and glycogen metabolisms. In conclusion, the genes identified might be useful for therapeutic targets in glucose lowering effects by reducing hepatic glucose output indicating potential of our local sago in managing diabetes.
KEY WORDS: Type 2 diabetes; goto kakizaki; sago resistant starch; gene expression array; hepatic glucose regulation
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INTRODUCTION![]() |
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The estimated number of adults living with diabetes from age group 20-79 years is 463 million and the number will increase to 700 million by 2045 (1). The increasing prevalence of type 2 diabetes worldwide requires effective dietary approaches in managing blood glucose. Growing evidence indicates the beneficial effects of resistant starch (RS) associated with postprandial blood glucose regulation in diabetes management (2, 3). The higher resistant level in food will reduce digestion rate and lower Glycemic Index (GI) preventing blood glucose spike (4, 5). The low GI diet has been shown to be more effective in controlling glycated haemoglobin and fasting blood glucose in patients with type 2 diabetes (6). GI in RS is influenced by the amylose and amylopectin ratio and therefore starches with higher amylose content will have lower glycaemic indexes which is suitable for glycaemic control (7, 8).
In general, RS is resistant to degradation in the small intestine by α-amylase enzyme and passes to the large intestine to be fermented by microbiota hence, preventing glucose spike (5, 9). Foods containing RS generally give a low glycaemic response because RS is not digested in the small intestine (10). Recent investigations have focused on the possible associations between resistant starch and mechanisms mainly in the gastrointestinal such as delaying glucose absorption (11), inhibiting carbohydrate digestion in the gastrointestinal tract (12) and slowing down gastric emptying (13). Due to this fact, resistant starch has gained considerable attention for its health benefits in attenuating several diseases including diabetes and cardiovascular diseases.
Over the years, the potential antidiabetic effects of several resistant starch types have been tested in vitro via inhibitory activities of α-amylase (14–16) and α-glucosidase (17, 18)as well as in vivo experiments involving rodents models of diabetic (19–21) and non diabetic (3, 22). The promising in vitro and in vivo findings which showed beneficial effects of resistant starch justify extending research in clinical trials in healthy (23, 24) and diabetic subjects (25) focussing mainly on blood glucose and insulin secretion regulations as well as improving insulin resistance.
Besides postprandial glycaemia effects via the gastrointestinal tract, the effects of RS on the expression of hepatic glucose and glycogen metabolism-related genes might be of a great interest to explore. In principal, blood glucose homeostasis is achieved through insulin actions via several pathways including glycolysis, gluconeogenesis and glycogenesis (26). The liver has a wide range of functions in glucose homeostasis which include glucose absorption, glycogen storage and glucose production (27). Decreased insulin sensitivity in extra-hepatic tissues, such as muscle and adipose tissue, reduced glucose utilization and insulin resistance in liver increases glucose output. Therefore, hyperglycemia associated with impairments in the regulation of fasting glucose output and glucose tolerance may be improved by therapeutics which target insulin resistance in the liver (28).
In Malaysia, Sarawak has been one of the world’s biggest producers of starch since the 1970s’ (29) and the sago palms can be found primarily at the river side of Mukah and Betong divisions. Sago RS flour has been used in local industries to produce various types of commercialized food products including sago pearls, tabaloi (a traditional delicacy biscuit), keropok (shrimp crackers), jellies, puddings (29) and as food thickener in noodles and vermicelli (29). However, the health benefits of local RS particularly related to the glucose regulating effects and the mechanisms involved have not been further explored. In this study, we performed gene array analysis to determine the effect of our local sago (Metroxylon sagu) RS on gene regulations specifically associated with glucose regulations including glucose, glycogen and gluconeogenesis pathways in the liver.
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MATERIALS AND METHODS![]() |
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Sago Resistant Starch
Native sago starch (sago RS2) used in the study was obtained from local supplier in Sarawak. A portion of this sago RS2 was then subjected to further chemical modification to produce sago RS4 which was obtained from a separate project. The chemical modification involved dual processes of cross-linking and oxidizing the native starch (30). This starch modification was aimed at increasing the RS content of the native starch. The resistant corn starch is a commercial Hi Maize RS2 starch (LifeSource resistant corn Starch 260), while the regular corn starch was sourced from local grocery store. The assay for the RS content for these starches was conducted using Megazyme resistant starch assay kit (Megazyme, UK) adopting AOAC methods 2002.02. The percentage of resistant starch for sago RS2, sago RS4 and Hi Maize was 45.53 ± 0.24, 60.66 ± 0.39 and 41.64 ± 0.89 % respectively.
Animal treatment
Healthy male spontaneously type 2 diabetes Goto kakizaki (GK) rats aged 8 weeks with mean body weight 125.8 ± 8.8 g were purchased from Clea Japan, Inc. The rats were treated for one month with water (control), Hi Maize (0.07 g/1 kg of body weight), sago RS2 (0.4 g/1 kg of body weight) and sago RS4 (0.07g/1 kg of body weight). After a month treatment, rats were anesthetized using 5% isofluorene supplied with oxygen and blood was withdrawn by cardiac puncture method followed by immediate euthanize. Liver tissue was collected and kept in RNA later for gene expression analysis.
Animal Use Approval
Ethical approval for this animal study was obtained from the Animal Care and Use Committee, Ministry of Health Malaysia (ACUC/KKM/02(03/2018).
Tissue Homogenization
The liver tissues from different groups of diabetic Goto kakizaki rat were then homogenized using tissue ruptor (230V, 50-60Hz, UK) (Qiagen, USA) attached with disposable probes and then processed for RNA elute using RNA Extraction Kit (Qiagen, USA). The concentration and purity of RNA were determined by measuring the absorbance using Nanodrop (Thermo Fisher Scientific, USA). For RNA, pure nucleic acid yield at 260/280 absorbance ratio should be approximately between 1.8 to 2.0.
Gene expression analysis associated with glucose and glycogen metabolism
Gene expression analysis was performed using Rat Glucose Metabolism RT² Profiler PCR Array, Catalog Number PARN-006ZA (Qiagen, USA) consisting of 84 key genes attached on the plates which are involved primarily in the regulation and enzymatic pathways of glucose and glycogen metabolisms. For glucose metabolism, the genes involved in each pathway include Glycolysis (Aldoa, Aldob, Aldoc, Bpgm, Eno1, Eno2, Eno3, Galm, Gapdh, Gapdhs, Gck, Gpi, Hk2, Hk3, Pfkl, Pgam2, Pgk1, Pgk2, Pgm1, Pgm2, Pgm3, Pklr, Tpi1); Gluconeogenesis (Fbp1, Fbp2, G6pc, G6pc3, Pc ,Pck1, Pck2); Regulation of Glucose Metabolism (Pdk1, Pdk2, Pdk3, Pdk4, Pdp2, Pdpr); Tricarboxylic Acid Cycle (TCA) Cycle (Acly, Aco1, Aco2, Cs, Dlat, Dld, Dlst, Fh, Idh1, Idh2, Idh3a, Idh3b, Idh3g, Mdh1, Mdh1b, Mdh2, Ogdhl, Pdha2, Pdhb, Pdhx, Sdha, Sdhb, Sdhc, Sdhd, Sucla2, Suclg1, Suclg2) and Pentose Phosphate Pathway (G6pd, H6pd, Pgls, Prps1, Prps1l1, Rbks, Rpia, Taldo1, Tkt). For glycogen metabolism, the genes involved in each pathway include Glycogen Synthesis (Gys1, Gys2, Ugp2); Glycogen Degradation (Agl, Pgm1, Pgm2, Pgm3, Pygl, Pygm) and Regulation of Glycogen Metabolism (Gsk3a, Gsk3b, Phka1, Phkb, Phkg1, Phkg2).
For RT² Profiler PCR Array analysis, a total of 500 ng RNA elute obtained from each liver treated sample was converted to cDNA using RT2 PreAMP cDNA Synthesis Kit. cDNA was then added to RT2 qPCR Master Mix and aliquoted 100 µl into each PCR array well. The samples were analyzed using Real Time PCR Instrument (StepOnePlus, ABI Biosystem, USA). RT2 Profiler PCR Data Analysis Software was used for data analysis. In the data analysis step, data normalization step was performed with selected Housekeeping Genes (B2M, Hprt1, ldha, Rplp1). Fold-regulation represents fold-change results in a biologically meaningful way. Fold-change values greater than one indicates a positive- or an up-regulation, and the fold-regulation is equal to the fold-change. Fold-change values less than one indicate a negative or down-regulation, and the fold-regulation is the negative inverse of the fold-change. The p values are calculated based on a Student’s t-test of the replicate 2∧ (- Delta CT) values for each gene in the control group and treatment groups. The cut-off points for gene array analysis were >2 fold change with p-value <0.05.
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RESULTS![]() |
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Table 1 depicts list of genes involved in Glucose and Glycogen Pathways in the spontaneously diabetic, Goto kakizaki rat treated with Hi Maize, sago RS2 and RS4. The array results showed the up- and down regulations of several genes gives an overview on how the homeostasis is achieved involving the glucose and glycogen metabolisms by the liver of diabetic Goto kakizaki rat in response to Sago treatment. In the Hi maize group, Bpgm was found to be significantly upregulated as compared to the control group. G6pc which is the key enzyme for glycogenolysis and gluconeogenesis, which are crucial for converting glycogen to glucose was found to be significantly downregulated in both sago RS2 and RS4 treated groups compared to the control group. Other genes involved in the sago RS2 group include Sdhb, Taldo1 and Rplp1.
In the sago RS4 treated group, other than G6pc, the most highly downregulated genes involved was Pdk3. Other genes being down- and up regulated in response to sago RS4 include Eno3, Galm, Idh3g, Pdk3, Sdhb, Tkt. These genes play an important role in regulating blood glucose level.
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DISCUSSION![]() |
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This current study focused on gene expression analysis to identify the role of genes in both glucose and glycogen metabolism pathways in an animal model of type 2 diabetic, Goto kakizaki (GK) rat in response to resistant starch, which might be associated with the inhibition of glucose production from the liver. The results indicate that sago RS2 and RS4 may have effects in the liver tissue by modulating the expression of multiple genes involved in glucose and glycogen metabolisms. The GK rat develops hyperglycemia post-natally and maintains moderately increased plasma glucose levels throughout its lifetime due to major impaired in beta cell function and insulin resistance in muscle and liver (31).
Postprandial glycaemia is influenced by the degree to which hepatic glucose production is suppressed in response to the glucose-containing carbohydrate digestion (32). In this study, several genes responsible for different gene functions were found to be up- and down regulated in response to sago RS treatment. The study showed that G6pc, which is important for glucose homeostasis (33) was significantly downregulated in the liver treated with both sago RS2 and RS4. The gene has been shown to be associated with the blood glucose regulations involving the glycogenolysis and gluconeogenesis pathways which might contribute to the glucose lowering effects of sago RS2 and sago RS4. Wang et al., 2015 showed that G6pc was upregulated in liver tissues of streptozotocin-induced diabetic rats in response to RS treatment using oligonucleotide microarray technique (34).
Regulation of insulin secretion and homeostasis state were also controlled by Sdhb (35) which was significantly downregulated in response to sago RS2 treatment whereas Taldo1 which is involved in Pentose Phosphate Pathway associated with glucose metabolism pathway (36) was significantly upregulated. In sago RS4, Pdk3 which was significantly downregulated plays a role in glucose homeostasis in maintaining normal blood glucose (37).
Liver plays an important role to regulate the glucose supply and other metabolic fuels as a source of energy to other tissues. Homeostasis is achieved through balancing glucose output and storage in liver and kidney, and regulating its utilization in peripheral tissues. In the fasting state, glucose is produced from the liver through glycogenolysis involving the breakdown of liver glycogen stores. With prolonged energy deprivation, the primary glucose source is via gluconeogenesis, where glucose is synthesized from non-carbohydrate precursors such as glycerol, lactate and the amino acid alanine (38).
The gene expression level in the liver tissue might be influenced by the percentage of resistant starch for sago RS2 and RS4 affecting the glucose and glycogen metabolisms. The high resistant starch level effects the GI and prevent glucose spike which is important in diabetes management (4). Wijanarka et al., 2020 in their study reported that intervention of high RS modified gayam starch fed to Sprague dawley rats induced streptozotocin-nicotinamide decreased blood glucose level and increased the short chain fatty acid (20). Sun et al., 2018 in the study showed that treatment with RS2 in type 2 diabetes, Goto kakizaki rat induced better regulation of lipid in plasma and liver, fructosamine, oral glucose tolerance test, insulin, glucose metabolism and pancreatic damage (19). RS2 showed antidiabetic effect by altering the expression of genes and proteins levels related to hepatic gluconeogenesis and glycogen synthesis. Wahjuningsih, 2018 in their study reported that Wistar rat which consumed Menthik Wangi rice, sago analog rice, and Sagu Kidney Bean Analog Rice showed higher insulin sensitivity was compared to the control group receiving only standard diet. Therefore, the insulin response is increased in the target tissue including the liver (3).
Our study has identified several genes using quick and reliable gene expression analysis specifically for rat glucose metabolism from the liver of diabetic rats treated with RS for a month. However, it would be more interesting to find out the effects on the gene regulations with an extended treatment duration. Furthermore, glucose and glycogen metabolism enzymatic pathways may be further explored using other tissues such as skeletal muscle.
In conclusions, the results suggested that our local Sago RS may have an important role in diabetes management through the alteration of the expression levels of the targeted genes related to hepatic glucose and glycogen metabolism pathways. To further confirm this, in vivo experiments such as hepatic glucose output, hepatic glycogen content and hepatic glycogen synthase activity (28) can be carried out to validate the mechanism behind the improved hepatic insulin sensitivity.
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ACKNOWLEDGEMENTS![]() |
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The authors gratefully acknowledge the Director General of Health, Malaysia, Director of the Institute for Medical Research, Ministry of Health Malaysia, the Sarawak State Government and the Management of CRAUN Research Sdn. Bhd., for permission to publish the findings of this study. The skilled technical assistance of Khairul Mirza Mohamad and Rabizah Md Lazim is acknowledged.
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FUNDING![]() |
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This study was supported by both CRAUN Research Sdn Bhd, under project number DTD-1.2.2 (59%) and Ministry of Health Malaysia Research Grant (NMRR-18-3353-45113; 19-013) (41%).
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CONFLICT OF INTEREST![]() |
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The authors declare no conflict of interest. CRAUN Research is an agency entrusted by Sarawak State Government to spearhead the Sago Research in Sarawak. The animal work in this project is a joint-collaboration with the Institute for Medical Research (IMR) of which has assumed full control on the technical conduct of the study.
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AUTHORS CONTRIBUTIONS![]() |
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Ezarul Faradianna Lokman.; Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing, Siti Mastura Abdul Aziz.; Formal Analysis, Investigation, Resources, Writing – Review & Editing, Aina Shafiza Ibrahim.; Formal Analysis, Investigation, Resources, Nurleyna Yunus.; Funding Acquisition, Writing – Review & Editing, Awang Zulfikar Rizal Awang Seruji.; Formal Analysis, Investigation, Resources, Sal Hazreen Bugam.; Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing.
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REFERENCES![]() |
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