Journal of Pharmaceutical Sciences & Emerging Drugs ISSN: 2380-9477

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Research Article, J Pharm Sci Emerg Drugs Vol: 4 Issue: 2

Quercetin Ameliorates Chronic Type 2 Diabetes Induced by Long-Term High-Fat, High-Sucrose Diet via Modifying Expression of Genes Involved in Energy Homeostasis and Inflammation

Almass A Abuzaid1, Mohamed A Osman2* and Abdalla O Elkhawad3
1Department of Public Health, University of Medical Sciences and Technology, Khartoum, Sudan
2Academy of Sciences, the Human Nutrition Section, Kirkwood Regional Center at the University of Iowa, Coralville, IA, USA
3Faculty of Pharmacy, Department of Pharmacology, University of Medical Sciences and Technology, Khartoum, Sudan
Corresponding author : Mohamed A. Osman
Associate Professor of Nutritional Sciences and immunobiology, Kirkwood Regional Center at the University of Iowa, 2301 Oakdale Blvd., Coralville, IA 52241, Room 320, USA
Tel: (319) 400-8308
E-mail: [email protected]
Received: December 05, 2016 Accepted: December 13, 2016 Published: December 21, 2016
Citation: Abuzaid AA, Osman MA, Elkhawad AO (2016) Quercetin Ameliorates Chronic Type 2 Diabetes Induced by Long-Term High-Fat, High-Sucrose Diet via Modifying Expression of Genes Involved in Energy Homeostasis and Inflammation. J Pharm Sci Emerg Drugs 4:2 doi:10.4172/2380-9477.1000117

Abstract

Type 2 diabetes (T2D) is a metabolic health disorder that induces aberrant gene expression thought to maintain the disease persistent in absence of the initial triggers. The current T2D therapeutics pose adverse side effects, some of which are hypoglycemia and weight gain-exacerbated insulin resistance. We studied whether regulation of adiponectin (ADIPOQ), glucose transporter -2 (GLUT-2) and C-reactive protein (CRP) genes by quercetin would completely cure T2D induced by longterm consumption of high-fat, high-sucrose diet (HFHSD) in male Wistar rats. Thus, sixty male Wistar rats were randomized into three experimental groups (n=20): normal control (C) fed chow diet,diabetic control (D) fed the HFHSD and diabetic quercetin-treated(Q) fed the HFHSD and gavaged with quercetin at 80 mg.kg-1bw.day-1 starting on Day 0 through Day 120. On Days 0 and 120, rats were euthanized and blood samples were used to study the ensuinghyperglycemia, hyperinsulinemia, insulin resistance and plasma CRP concentration. The effect of quercetin on T2D-associated injury of islet of Langerhans and the relative splenic weight (RSPW%) was determined.Gene expression of ADIPOQ in epididymal fat and GLUT-2 and CRP in liver was assessed using RT-PCR. Quercetin attenuated the hyperglycemia, hyperinsulinemia, insulin resistance score and plasma CRP concentration on Day 120 in the Q rats. The number of islets of Langerhans was enhanced on Day 120 and the RSPW (%). decreased by quercetin on Day 120 in the Q rats. Quercetin also upregulated ADIPOQ and GLUT-2 genes and downregulated CRP gene in the Q rats on Day 120. In conclusion, quercetin ameliorated T2D induced by long-term HFHSD via regulating genes involved in energy homeostasis and inflammation leading to attenuation of the hyperglycemia and insulin resistance, the hallmark of T2D pathogenesis. Unlike currently in use anti-T2D therapy, adverse side effects were not triggered by quercetin.

Keywords: Long-term HFHSD; Type 2 diabetes; Quercetin; Adiponectin; Glucose transporte-2, Inflammation; Insulin resistance;Hyperglycemia

Keywords

Long-term HFHSD; Type 2 diabetes; Quercetin; Adiponectin; Glucose transporte-2, Inflammation; Insulin resistance; Hyperglycemia

Introduction

T2D is increasingly emerging as a global public health concern [1,2]. In 2013, the prevalence of diabetes was estimated to be 8.4%, with 382 million people afflicted and over 5 million diabetes-related mortality [1,2]. T2D accounts for nearly 90% of diabetes cases worldwide, and has been linked mainly to obesity and physical inactivity [3]. Studies in diabetic animals and humans have shown alteration of several genes and metabolites that may be fundamental in mediating structural and functional deficits that manifest as serious chronic complications [3].
Several genetic variants are implicated in the pathogenesis of T2D include adiponectin gene (ADIPOQ), glucose transporter-2 (GLUT- 2), C-reactive protein (CRP) transcription factor 7 like 2 (TCF7L2), peroxisome proliferator activator-activated receptor-γ (PPAR-γ), fatmass and obesity-associated gene (FTO), potassium voltage-gated channel subfamily J member 11(KCNJ11), neurogenic-locus-notchhomolog- protein-2 (NOTCH2), wolframin ER transmembrane glycoprotein (WFS1), regulatory subunit associated protein-1 like-1(CDKAL1), insulin like growth factor 2 mRNA-binding protein (2IGF2BP2), solute carrier family 30 member 8 (SLC30A8), hematopoietically expressed homeobox (HHEX) and zinc finger 1 (JAZF1) [4-13] suggesting that modification of these genes must be fundamental for T2D treatment alongside the cardinal metabolic disorders.
The currently in use antidiabetic drugs carry adverse side effects and mitigate T2D without curing the underlying etiology [14,15]. Meglitinides induce weight gain, which might exacerbate insulin resistance leading to increased drug dose. Sulfonylureas (glyburide, glipizide and glimepiride) cause hypoglycemia and age-related renal dysfunction [16,17]. Also, thiazolidinediones (pioglitazone) cause hypoglycemia, edema and increased bone fracture risk [18]. Thus, there have been intense interests in identifying phytochemicals that cure the T2D via favorable gene modification leading to β-cell regeneration and restoring physiological glucose homeostasis.
CRP is an acute-phase protein produced in responses to numeral pathophysiological conditions including tissue damage, infection and inflammation [19,20]. Plasma CRP concentration correlates positively with BMI, suggesting that CRP is a useful biomarker for obesity-linked chronic inflammatory conditions, such as T2D [21]. Moreover, CRP has been suggested an independent strong predictor of hyperglycemia, insulin resistance and overt T2D [22].
GLUT-2, encoded by SLC2A2, is predominantly expressed on hepatocytes, renal tubular cells, intestinal microvilli and pancreatic β-cells [23,24]. GLUT-2 is involved in glucose-sensing in pancreatic β-cells where it induces the glucose-triggered insulin secretion. GLUT-2 is also expressed on hepatocytes and hypothalamus [25,26]. Recent evidence suggests that GLUT-2 could be involved in the pathogenesis of diabetes. In diabetic mouse models, GLUT-2 gene expression is down-regulated in pancreatic β-cells [27], but upregulated on hepatocytes [28]. Modulation of GLUT-2 gene expression by nutraceutical flavonoids has been documented in experimental animals with altered glucose homeostasis [29-31].
Adiponectin is an adipokine protein consists of 244 amino acids and secreted by the white adipose tissue [32]. Adiponectin synthesis and secretion are regulated by several molecules including insulin [33]. Importantly, plasma adiponectin concentration significantly decreases in T2D individuals exhibiting obesity, dyslipidemia and insulin resistance [33].
Quercetin is a flavonoid abundant in various plant species. Growing body of evidence proves a positive association between consuming flavonoid-rich foods and T2D prevention [29-31]. Quercetin modifies various molecular targets and regulates different signaling pathways in pancreatic β-cells, hepatocytes, adipocytes, and myocytes [29-31]. Quercetin also promotes proliferation of β-cells leading to enhanced insulin secretion and decreased hyperglycemia, a hallmark of T2D [29]. In addition, attenuation of insulin resistance and inflammation by quercetin has been clearly established [29]. While the effect of quercetin on short-term induced T2D is well studied [29-31], effect of quercetin on long-term T2D induced by HFHSD consumption [31] is poorly understood and awaits further clarification.
We hypothesized that T2D induced by long-term HFHSD would induce aberrant expression of ADIPOQ, GLUT-2, and CRP genes leading to persistence of T2D and quercetin administration would favorably modify expression of these genes causing regression of T2D. Thus, our objective was to unveil whether regulation of these genes by quercetin would completely cure T2D induced by longterm consumption of HFHSD in male Wistar rats. The experimental protocol was approved by the Institutional Review Board, Faculty of Pharmacy, University of Medical Sciences and Technology.

Materials and Methods

Kits and reagents
Quercetin was purchased from the Sigma Aldrich USA. The C-reactive protein (CRP) US kit was purchased from APTEC Diagnostics nv 9100 St-Niklaas, Belgium. Target gene expression kits and molecular biology reagents were purchased from Invitrogen, Carlsbad, CA, USA. Other solvents and materials for histopathology works were purchased from local venders in Khartoum.
Animals
Sixty, healthy male Wistar rats, six-week old, with 210 g average body weight, were used for this study. Initially, each two rats were housed in a separate cage in the Restricted Animal Facility of the Faculty of Pharmacy, University of Khartoum. All rats were adapted for two weeks on stranded chow diet then randomized into three treatment groups (n=20): Normal Control (C) fed the standard chow diet, Diabetic Control (D) fed high-fat, high-sucrose diet (HFHSD) and Diabetic Quercetin-Treated (Q) fed the HFHSD and gavaged with quercetin at 80 mg. Kg bw-1.day-1 until the end of the study. Diets were fed ad libitum and rats were given free access to drinking water. The environment within the room was maintained to provide a temperature of 22-25ºC, a relative humidity of 35- 45%, and 12/12 h light/dark cycle.
Induction of obesity, T2D and insulin resistance
To induce long-term obesity, T2D and insulin resistance, the rats in the D and Q Groups were fed the HFHSD ad libitum (adapted from HT.88137; Table 1) for 12 months. By the end of the 12th month (Day 0), the rats in the different groups were housed individually, and the rats in the Q Group were gavaged quercetin at 80 mg.kg-1 bw.day-1 until the end of the study (Day 120).
Table 1: The macro- and micronutrients constituents and energy of the obesogenic, diabetogenic HFHSD fed to the D and Q groups.
Samples collection and analyses
Fasting blood samples and tissues collection: On days 0 and 120, 10 rats from each treatment group were euthanized by decapitation under light halothane® anesthesia. Blood samples were withdrawn by cardiac puncture and plasma was separated by centrifugation at 5,000ᵡg for 15 min in a refrigerating centrifuge and stored at -80ºC until used for quantification of plasma glucose, insulin and CRP concentrations. Pancreas, spleen, liver and epididymal fat were dissected aseptically in a biosafety cabinet and processed for molecular, morphometric and histomorphometrical analyses.
Determination of plasma glucose, insulin and CRP concentrations
Plasma glucose and CRP concentrations were determined spectrophotometrically by using commercial kits (Aptec Diagnostics NV, Belgium). Plasma insulin was analyzed by using the immuneenzymatic assay TOSOH AIA-360 Chemistry Analyzer (Japan). Insulin resistance was calculated by using the homeostasis model assessment of insulin resistance (HOMA-IR = insulin, μIU /mL × glucose, mg/dL) /405.
RT-PCR analysis and determination of gene expression
Epididymal adipose tissue and liver tissues were dissected in biosafety cabinet in 1.0 mL of One Step RNA reagent (Bio Basic Inc., Ontario, Canada), flash frozen in liquid nitrogen and subsequently stored at -80ºC. Frozen samples (~100 mg) were homogenized using Tissue Homogenizer (Bioneer Inc., model KA-7030). Total RNA was extracted using chloroform and precipitated with isopropanol as per the manufacturer’s instructions (Bio Basic Inc., Ontario, Canada). The pellets were washed with 75% ethanol and resuspended in RNasefree water. Nucleic acid concentration and purity were determined using the Nanodrop®. For cDNA synthesis, RNA (1 μg) was treated at 70ºC for 5 min and reverse-transcribed using 100 units of Moloney murine leukemia virus reverse transcriptase (Gibco), 50 pmol of poly (dT) primer and 20 nmol of dNTPs in a total volume of 11 μL at 37ºC for 1 h. After heating at 94ºC for 5 min, PCR amplification was performed with 2.5 units Taq polymerase (Perkin-Elmer, Foster City, CA, USA), 3 mM MgCl2 and 50 pmol of forward and reverse primers specific for respective genes in a total volume of 25 μL (Table 2). The housekeeping gene transcript glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as internal standard. The PCR products were visualized under UV lamp by electrophoresis in 1.5% agarose gel stained with ethidium bromide. Intensities of PCR bands were determined by using the Image J analyzer (NIH, USA) (http:// rsb.info.nih.gov/nih-image/).
Table 2: The PCR protocols and forward and reverse primers of GLUT-2, ADIPOQ and CRP genes assay on Day120.
Morphometric and histomorphometric analyses
The RSPW (%) of dissected spleens was determined after precise excision of the attached adipose tissue. The spleens weight was accurately determined in grams using a sensitive digital scale and RSPW was determined relative to the body weight ((spleen weight, g/ body weight, g)×100).
Pancreatic tissues intended for histopathological works were rapidly fixed in 10% formalin in PBS, dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin wax blocks. Pancreatic sections (5 μm thick) were stained with hematoxylin and eosin (HE). Detection of the number of islets of Langerhans was carried out on an Olympus research microscope (Olympus BX51, Japan) with an ocular horizontal micrometer (PYSNE101B19201, GT Vision, Suffolk, UK) and an area-measuring ocular grid (PYSNE10- 01B19207, GT Vision Ltd) inserted into the eyepiece. The ocular micrometer and the ocular grid were calibrated with a 1 mm stage micrometer (Graticles ID5151 GXM GRAT1 1mm stage, GT Vision, UK). Thirty histological stained sections (ten from each group) were used for morphometric analysis to determine the number of islets in each section of the pancreas. The histopathological images were captured by using the SPOT ideaTM CMOS | 5.0 Mp digital hooked a SPOT ideaTM CMOS software (Diagnostic Instruments, Inc, MI, USA).
Statistical analysis
The data were analyzed by using the graphic and statistical software GraphPad Prism 5® (GraphPad Software Inc., La Jolla, CA, USA). The gene expression, hyperinsulinemia, insulin resistance score and islets of Langerhans data were analyzed by using the One-way ANOVA followed by Tukey pairwise multiple comparison tests for the variables means. Plasma glucose and CRP and the RSPW data were analyzed by using the Two-way ANOVA followed by Bonferroni test for pairwise comparison of the means with significance set at α=0.05 and 95% confidence intervals. Data were expressed as the means ± SEM. Statistical significance was declared when P<0.05. (*) stands for P<0.05, (**) for P<0.01, (***) for P<0.001 and (****) for P<0.0001.

Results

Effects of quercetin on the energy homeostasis
Effect of quercetin on adiponectin (ADIPOQ) gene expression in the epididymal adipose tissue: The RT-PCR assay of the adiponectin gene revealed a significant decrease (P<0.001) in the ADIPOQ gene expression in the D rats under the effects of HFHSD consumption compared with the C rats consumed the chow diet (Figure 1A). In contrast, ADIPOQ gene expression was significantly increased (P<0.001) in the Q rats under effect of quercetin relative to that of the D rats on Day 120 (Figure 1A). ADIPOQ expression, however, was not normalized (P<0.001) by quercetin relative to the normal control C rats (Figure 1A).
Figure 1: ADIPOQ gene expression in epididymal adipose tissues, hyperinsulinemia, insulin resistance score and number of islet of Langerhans per pancreatic section of male Wistar rats (n=10/group) fed the standard chow diet (C), the HFHSD (D) or the HFHSD and gavaged with quercetin (Q) at 80 mg.kg-1 bw.day-1 on Day 0 and/or 120.
Effect of quercetin on hyperinsulinemia, insulin resistance (HOMA-IR) and hyperglycemia: As seen in Figure 1B, induction of T2D by the HFHSD significantly increased (P<0.001) the hyperinsulinemia in the D rats compared to the C and Q rats on Day 120 (Figure 1B). Administration of quercetin ameliorated (P<0.001) the hyperinsulinemia in the Q rats relative to the D rats, but failed to normalize it (P<0.01) for the comparison with C rats; Figure 1B). Consequently, the diabetic D rats demonstrated high (P<0.001; Figure 1C) insulin resistance score compared with the C and Q rats on Day 120. Quercetin administration decreased (P<0.001) the insulin resistance score in the Q rats compared to the D rats. The insulin resistance score in the Q rats, however, remained elevated (P<0.01) relative to the control C rats (Figure 1C).
As illustrated in Figure 1D, on Day 0, under effect of the HFHSD, the D and Q rats developed severe (P<0.0001) hyperglycemia relative to the C rats. But, on Day 120, quercetin administration to T2D Q rats significantly (P<0.0001) decreased their plasma glucose concentration in comparison with the diabetic D rats. Noteworthy, the Q rats remained to be hyperglycemic (P<0.001) relative to the C rats on Day 120 (Figure 1D).
In Figure 1, Panel A. RT-PCR analysis of the ADIPOQ gene in epididymal adipose tissue. RNA was extracted and reversetranscribed (1 mg). RT-PCR assay was carried out for the ADIPOQ gene as described in the materials and methods. G3PDH was used as an internal standard. ADIPOQ gene expression was significantly downregulated (P<0.001) in the D rats fed the HFHSD relative to the C rats fed the chow diet (0.044 ± 0.01 vs. 1.0 ± 0.09 units, respectively). Conversely, quercetin upregulated (P<0.001) ADIIPOQ in the Q rats relative to D rats (0.334 ± 0.041 vs. 1.0 ± 0.09 units, respectively), yet, lower (P<0.001) compared to the C on Day 120. Panel B. Hyperinsulinemia in μIU/mL. Hyperinsulinemia was measured via immune-enzymatic assay as described in the materials and methods section. Quercetin ameliorated (P<0.001) the hyperinsulinemia in the Q rats relative to the D rats (18.63 ± 1.47 vs. 28.3 ± 3.71 μIU/ mL, respectively), but failed to normalize it (P < 0.01) in comparison with the C rats (18.63 ± 1.47 vs. 14.15 ± 1.34 μIU/mL, respectively). Panel C. The insulin resistance scores. The insulin resistance scores were calculated by using the equation HOMA-IR = insulin, μIU / mL × glucose, mg/dL)/405 as shown in the materials and methods section. The diabetic D rats demonstrated high (P<0.001) insulin resistance score compared with the C and Q rats on Day 120 (20.1 ± 0.5 vs. 3.01 ± 0.50 and 6.42 ± 1.65 units, respectively). Quercetin decreased (P<0.001) the insulin resistance score in the Q rats compared to the D rats (6.42 ± 1.65 vs. 20.1 ± 0.5 units, respectively). But, normalization of the insulin resistance was not (P<0.01) achieved in the Q rats relative to the C rats (6.42 ± 1.65 vs. 3.01 ± 0.50 units). Panel D. Plasma glucose concentration in mg/dL on Day 120. Plasma glucose concentration was determined spectrophotometrically as described in the material and methods. On Day 0, the hyperglycemia was significant (P<0.0001) in the D and Q rats compared to the C rats (240.7 ± 21.1 and 242 ± 18.7 vs. 85 ± 8.4 mg/dL, respectively). On Day 120, quercetin administration to T2D Q rats significantly (P<0.0001) decreased their hyperglycemia relative to the diabetic D rats. Noteworthy, the Q rats remained to be hyperglycemic (P<0.001) compared to the C rats on Day 120. Values are means ± SEM. Statistical significance was declared when P<0.05 and means with different superscripts or (*) differ significantly at P<0.05. (*) stands for P<0.05, (**) for P<0.01, (***) for P<0.001 and (****) for P<0.0001.
Effect of quercetin on T2D-associated inflammation
Effect of quercetin on C-reactive protein (CRP) gene expression in the hepatic tissues: Shown in Figure 2A, quercetin administration to T2D Q rats induced significant (P<0.001; Figure 2A) decrease in their hepatic CRP gene expression in comparison with the diabetic D rats on Day 120. Quercetin administration, failed to normalize CRP gene expression as it remained to significantly higher than that of the C rats (P<0.05; Figure 2A). Under the effects of the HFHSD, CRP gene expression measured more than two-fold greater (P=0.001) in the diabetic D rats compared with the C rats on Day 120.
Figure 2: Plasma CRP concentration (mg/dL), CRP gene expression relative to the control and the RSPW (%) on Days 0 and/or 120 of male Wistar rats (n=10/group) fed the standard chow diet (C), HFHSD (D) or the HFHSD and gavaged with quercetin (Q) at 80 mg.kg-1 bw.day-1.
Effect of quercetin on plasma C-reactive protein (CRP) concentration: In an attempt to examine the proinflammatory effects of consuming a high-calorie diet by male Wistar rats, we quantified the inflammatory marker CRP in their plasma. On Day 0, feeding the HSHFD to the D and Q rats remarkably (P<0.0001; Figure 2B) increased their plasma CRP concentration higher than the C rats. The intensity of the inflammatory state was similar between the D and Q rats on Day 0 as revealed by no difference (P>0.05) between their plasma CRP concentrations (Figure 2B).
As the D and Q rats advanced to Day 120, however, administration of quercetin significantly
(P˂0.0001) decreased plasma CRP concentration in the Q rats in comparison with the D Group (Figure 2B). But, plasma CRP concentration in Q rats was yet significantly higher (P˂0.01) than that of the control C rats (Figure 2B).
Effect of quercetin on the number of islets of Langerhans: The combined effect of long-term HFHSD feeding and inactivity substantially affected the histological architecture of the pancreases. The number of islets of Langerhans was significantly (P<0.001) decreased in the diabetic D relative to the C and Q rats on Day 120. Driven by quercetin, the islet number increased significantly (P<0.001) in the Q rats relative to the D rats, yet lower (P< 0.001) compared to the nondiabetic C rats on Day 120 (Figure 2C).
Effect of quercetin on the relative splenic weight (RSPW%): On Day 0, the RSPW (%) of the D and Q rats was significantly (P ≤ 0.001) heavier compared with the C rats (Figure 2D). Despite this, the RSPW (%) of the Q and D rats did not differ (P>0.05). On Day 120, we found that administration of quercetin attenuated the RSPW (%) of the Q rats to be not significantly different (P>0.05) relative to the C rats, but significantly (P<0.05) lower compared with the diabetic D rats. On the contrary, the RSPW (%) of the D rats continued to be significantly (P<0.0001) higher relative to the control C rats on both days (Figure 2D).
In Figure 2, Panel A. CRP gene expression in hepatocytes. As described in the materials and methods, total RNA was extracted from liver tissues and reverse-transcribed (1 μg). The RT-PCR assay was conducted for the CRP gene using thermo cycler conditions and primers shown in Table 2. G3PDH was used as an internal standard. HFHSD consumption significantly (P<0.001) increased CRP gene expression more than two-fold greater in the diabetic D rats compared with the C rats on Day 120 (2.47 ± 0.49 vs. 1.0 ± 0.1 units, respectively). Quercetin failed to normalize CRP gene expression as it remained significantly (P<0.05) higher in contrast with the C rats (1.62 ± 0.49 vs.1.0 ± 0.1 units, respectively) although it decreased it relative to the D rats (1.62 ± 0.49 vs. 2.47 ± 0.49 units respectively). Panel B. Plasma CRP concentrations in mg/dL. As described in the materials and methods, plasm CRP concentrations were assayed spectrophotometrically. On Day 0, the HFHSD equally increased plasma CRP concentration in the D and Q rats to be higher (P<0.0001) than the C rats (9.58 ± 2.4 and 11.03 ± 2.07 vs. 2.81 ± 0.54 mg/dL, respectively). In contrast, on Day 120, administration of quercetin significantly (P˂0.0001) decreased plasma CRP concentration in the Q rats compared with the D rats. But, plasma CRP in the Q rats remained higher (P˂0.01) compared with the C rats (5.44 ± 1.61 vs. 11.13 ± 1.99 and 2.65 ± 0.86 mg/dL, respectively). Panel C. The number of islet of Langerhans per section of pancrease. Pancreatic sections were processed and stained with H&E as described in the materials and methods. The number of the iselts was determined by a blind Pathologist using power 10 of the light microscope. On Day 120, the number of islets of Langerhans significantly (P < 0.001) decreased in the diabetic D rats relative to the C and Q rats (4.68 ± 1.6 vs. 16.67 ± 1.9 and 9.33 ± 2.2 islets, respectively). Nonetheless, the islet number significantly (P<0.001) enhanced by quercetin administration to the Q rats relative to the D rats, but remained lower (P<0.001) compared to the nondiabetic C rats (9.33 ± 2.2 vs. 4.68 ± 1.6 and 16.67 ± 1.9 islets, respectively). Panel D. The RSPW (%). As described in the materials and methods, the RSPW was calculated by the equation ((spleen weigh, g/body weight, g) × 100). On Day 0, the RSPW (%) of the D and Q rats was equally significantly (P ≤ 0.001) heavier compared with the C rats (0.43 ± 0.09 and 0.42 ± 0.03 vs. 0.29 ± 0.03%, respectively). By Day 120, affected by quercetin, the RSPW (%) of the Q rats significantly (P<0.05) decreased relative to the D rats and was completely normalized relative to the C rats (P>0.05 for the difference; 0.33 ± 0.02 vs. 0.41 ± 0.09 and 0.26 0.03 %, respectively). On the contrary, the RSPW (%) of the D rats continued to be significantly (P<0.0001) higher relative to the control C rats on both days. Values are means ± SEM. Statistical significance was declared when P<0.05. Means with different superscripts or (*) differ significantly at P<0.05. (*) stands for P<0.05, (**) for P<0.01, (***) for P<0.001 and (****) for P<0.0001.
Effects of quercetin on GLUT-2 gene expression in the liver tissue
Induction of T2D by long-term HFHSD caused significant (P=0.001) inhibition of GLUT-2 gene expression in the hepatic tissues of the diabetic D rats in contrast to the C rats consumed the standard chow diet (Figure 3). On the contrary, administration of quercetin increased (P=0.001) GLUT-2 gene expression in the Q rats in comparison to that of the D rats (Figure 3), but still lower (P<0.05) relative to the control C rats.
Figure 3: GLUT-2 gene expression in hepatocytes. As described in the materials and methods, total RNA was extracted from liver tissues and reverse-transcribed (1 μg). The RT-PCR assay was carried out for the CRP gene using thermocycler protocol and primers shown in Table 2. G3PDH was used as an internal standard. T2D induction caused significant (P<0.001) attenuation of GLUT-2 gene expression in the diabetic D rats hepatocytes in contrast to the control C rats (0.40 ± 0.03 vs. 1.0 ± 0.03 units, respectively). On the contrary, administration of quercetin increased (P<0.001) GLUT-2 gene expression in the Q rats in comparison to the D rats, but still lower (P<0.05) relative to the control C rats (0.80 ± 0.04 vs. 0.40 ± 0.03 and 1.0 ± 0.03 units, respectively). Values are means ± SEM. Statistical significance was declared when P<0.05. Means with different superscripts or (*) differ significantly at P<0.05. (*) stands for P<0.05, (**) for P<0.01, (***) for P<0.001 and (****) for P<0.0001.

Discussion

This study describes the ability of quercetin to regulate genes associated with chronic T2D induced by long-term (12 mo) HFHSD consumption, risk factor for human T2D [32-35].
Induction of T2D by the HFHSD in the D rats was accompanied by significant attenuation of ADIPOQ gene expression in epididymal adipose tissue, hyperinsulinemia, insulin resistance and hyperglycemia (Figures 1A, B, C and D) on Day 120. HFD consumption lowers adiponectin gene expression and propagates insulin resistance in rodents [36,37] and impaired adiponectin signaling induces T2D in humans [38].
In this study, administration of quercetin to T2D rats upregulated adiponectin gene expression (Figure 1A) and decreased hyperinsulinemia, insulin resistance and hyperglycemia on Day 120 (Figures 1B,C and D). It has been reported that adiponectin decreases in human affected with T2D [38] and adiponectin gene therapy alleviates the HFHSD-mediated impairments in insulin sensitivity in mice [32] Here, effects of quercetin on adiponectin gene expression and hyperglycemia mimicked action of the anti- T2D therapy, rosiglitazone®, which promotes PPAR-γ leading to increased adiponectin gene expression [39,40]. It is well established that modulation of adipocytes function leading to upregulation of adiponectin gene expression, is a putative mechanism via which the anti-diabetic thiazolidinediones improve insulin action in T2D individuals [39-41]. Our findings in Figures 1A, B, C and D support earlier findings that quercetin induces cellular glucose uptake and metabolism via increasing expression of ADIPOQ and GLUT-4 genes in muscles of T2D rats to [29-31] leading to euglycemia and enhanced insulin sensitivity.
Curiously enough, induction of adiponectin gene expression in the Q rats by quercetin was accompanied by significant decrease in their visceral fat and BMI (authors’ manuscript under preparation). In agreement, plasma adiponectin concentration inversely correlates with the BMI in healthy humans and decreases in T2D humans [42].
As the inflammatory biomarker CRP gene expression and plasma concentration increased by HFHSD in the D and Q rats on Day 0, we observed a parallel increase their RSPW relative to C rats indicating development of state of general inflammation (Figure 2 A, B and C). The proinflammatory effect of HFHSD has been earlier described in Wistar rats [43] and humans [44].
In agreement with previous reports in mice [29,45], as plasma CRP concentration and gene expression in hepatocytes of the Q rats attenuated under effect of quercetin on Day 120, we observed tendency for normalization in their RSPW (Figure 2 A, B and C). It has been shown that quercetin decreases spleen mass in mice affected by HFD-induced inflammation [45]. Serum CRP concentration inversely correlates with the dietary intake of quercetin in humans [46]. Quercetin suppresses CRP gene expression and secretion by molecular mechanism involves modulation of IL-6 and IFN-γ, proinflammatory molecules proposed to stimulate CRP synthesis in mice [45,47]. Dai and colleagues (2013) showed that quercetin exerts its anti-inflammatory to prevent β-cell injury via preventing NF-κB translocation and signaling [29]. In line with Dai et al, we observed that the number of islets of Langerhans decreased by the proinflammatory effect of the HFHSD in the D rats while in the Q rats administration of quercetin enhanced the number of islet of Langerhans proving the potent anti-inflammatory property of quercetin on Day 120 (Figure 1D).
Furthermore, GLUT-2 gene expression dramatically attenuated by HFHSD in the hepatocytes of the D rats (Figure 3). We observed a significant increase in GLUT-2 gene expression after quercetin administration in the Q rats on Day 120. GLUT-2 is a transmembrane carrier protein that permits passive transport of glucose across hepatocyte plasma membrane [26]. GLUT-2 also regulates renal glucose reabsorption [24]. Thus, it appears that quercetin lowers hyperglycemia via two opposing mechanisms; up-regulation of GLUT- 2 and GLUT-4 [30] gene expression on hepatocytes and myocytes to increase glucose uptake [30] and downregulation of GLUT-2 gene expression on the intestinal microvilli to inhibit absorption [48]. It has been our observation that most metabolic parameters examined in this study were significantly attenuated, but not normalized, by quercetin. Lack of normalization might be ascribed to HFHSD residual effects. We report that the anti-T2D effect of quercetin mimicked that of thiazolidinediones as both promote ADIPQ gene. Quercetin, however, did not induce hypoglycemia or weight gain in the Q rats, adverse side effects attributed to thiazolidinediones use in T2D individuals [18].
In conclusion, quercetin substantially ameliorated T2D induced by long-term HFHSD consumption via regulating expression of ADIPO, GLUT-2 and CRP genes involved in induction of hyperglycemia, hyperinsulinemia and insulin resistance, the hallmark of T2D pathogenesis.

Acknowledgements

The authors thank the Faculty of Pharmacy, University of Khartoum for providing housing of the Wistar rats at their Restricted Animal Facility. Our appreciation is extended to Professor Hamid S. Abdulla, Faculty of Veterinary Medicine, University of Khartoum for assisting with the animal works. The authors also thank the Department of Statistics, University of Iowa, Iowa City, USA for helping with the statistical analysis.

Conflict of interest

Authors declare no conflict of interest.

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