Mechanisms of inhibition of advanced glycation end-products (AGEs) and α-glucosidase by Heliotropium bacciferum: Spectroscopic and molecular docking analysis.

Diabetes mellitus is characterized by hyperglycemia that makes insulin more prone to glycation and form advanced glycation end products (AGEs). Here, we report the effect of glyoxal (GO) on the formation of AGEs using human insulin as model protein and their structural modifications. The present investigation also reports the anti-AGE potential of Heliotropium bacciferum (Leaf) extracts. The phytochemical analysis of H. bacciferum revealed that free phenolic extract contains higher amount of total phenolic (3901.58 ± 17.06 mg GAE/100 g) and total flavonoid content (30.41 ± 0.32 mg QE/100 g) when compared to bound phenolic extract. Naringin and caffeic acid were identified as the major phenolic ingredients by UPLC-PAD method. Furthermore, bound phenolics extract showed significantly higher DPPH and superoxide radicals scavenging activity (IC50 17.53 ± 0.36 µg/mL and 0.306 ± 0.038 mg/ mL, respectively) (p ≤ 0.05). Besides, the bound phenolics extract also showed significant (p ≤ 0.05) chelating power (IC50 0.063) compared to free phenolic extract. In addition, bound phenolic extract could efficiently trap GO under physiological conditions. Spectroscopic investigation of GO-modified insulin illustrated changes in the tertiary structure of insulin and formation of AGEs. On the other hand, no significant alteration in secondary structure was observed by far UV-CD measurement. Furthermore, H. bacciferum extract inhibited α-glucosidase activity and AGEs formation implicated in diabetes. Molecular docking analysis depicted that GO bind with human insulin in both chains and forms a stable complex with TYR A: 14, LEU A:13, ASN B:3, SER A:12 amino acid residues with binding energy of - 2.53 kcal/mol. However, caffeic acid binds to ASN A:18 and GLU A:17 residues of insulin with lower binding energy of -4.67 kcal/mol, suggesting its higher affinity towards human insulin compared to GO. Our finding showed promising activity of H. bacciferum against AGEs and its complications. The major phenolics like caffeic acid, naringin and their derivatives could be exploited for the drug development for management of AGEs in diabetes.

Genetic Variants of HNF4A, WFS1, DUSP9, FTO, and ZFAND6 Genes Are Associated with Prediabetes Susceptibility and Inflammatory Markers in the Saudi Arabian Population.

Prediabetes is a reversible, intermediate stage of type 2 diabetes mellitus (T2DM). Lifestyle changes that include healthy diet and exercise can substantially reduce progression to T2DM. The present study explored the association of 37 T2DM- and obesity-linked single nucleotide polymorphisms (SNPs) with prediabetes risk in a homogenous Saudi Arabian population. A total of 1129 Saudi adults [332 with prediabetes (29%) and 797 normoglycemic controls] were randomly selected and genotyped using the KASPar SNP genotyping method. Anthropometric and various serological parameters were measured following standard procedures. Heterozygous GA of HNF4A-rs4812829 (0.64; 95% CI 0.47-0.86; p < 0.01), heterozygous TC of WFS1-rs1801214 (0.60; 95% confidence interval (CI) 0.44-0.80; p < 0.01), heterozygous GA of DUSP9-rs5945326 (0.60; 95% CI 0.39-0.92; p = 0.01), heterozygous GA of ZFAND6-rs11634397 (0.75; 95% CI 0.56-1.01; p = 0.05), and homozygous AA of FTO-rs11642841 (1.50; 95% CI 0.8-1.45; p = 0.03) were significantly associated with prediabetes, independent of age and body mass index (BMI). Additionally, C-reactive protein (CRP) levels in rs11634397 (AA) with a median of 5389.0 (2767.4-7412.8) were significantly higher than in the heterozygous GA genotype with a median of 1736.3 (1024.4-4452.0) (p < 0.01). In conclusion, only five of the 37 genetic variants previously linked to T2DM and obesity in the Saudi Arabian population [HNF4A-rs4812829, WFS1-rs1801214, DUSP9-rs5945326, ZFAND6-rs11634397, FTO-rs11642841] were associated with prediabetes susceptibility. Prospective studies are needed to confirm the potential clinical value of the studied genetic variants of interest.

Agitation does not induce fibrillation in reduced hen egg-white lysozyme at physiological temperature and pH.

Several proteins and peptides tend to form an amyloid fibril, causing a range of unrelated diseases, from neurodegenerative to certain types of cancer. In the native state, these proteins are folded and soluble. However, these proteins acquired ß-sheet amyloid fibril due to unfolding and aggregation. The conversion mechanism from well-folded soluble into amorphous or amyloid fibril is not well understood yet. Here, we induced unfolding and aggregation of hen egg-white lysozyme (HEWL) by reducing agent dithiothreitol and applied mechanical sheering force by constant shaking (1000 rpm) on the thermostat for 7 days. Our turbidity results showed that reduced HEWL rapidly formed aggregates, and a plateau was attained in nearly 5 h of incubation in both shaking and non-shaking conditions. The turbidity was lower in the shaking condition than in the non-shaking condition. The thioflavin T binding and transmission electron micrographs showed that reduced HEWL formed amorphous aggregates in both conditions. Far-UV circular dichroism results showed that reduced HEWL lost nearly all alpha-helical structure, and ß-sheet secondary structure was not formed in both conditions. All the spectroscopic and microscopic results showed that reduced HEWL formed amorphous aggregates under both conditions.

Elucidating the binding and inhibitory potential of p-coumaric acid against amyloid fibrillation and their cytotoxicity: Biophysical and docking analysis.

P-Coumaric acid (p-CA) is a plant metabolite with anti-inflammatory and antioxidant effects. Due to its therapeutic potential, p-CA has attracted much attention from the scientific community lately. Oxidative stress, amyloid formation, and impaired proteasomal degradation are hallmarks of neurodegenerative diseases like Alzheimer's (AD) and are targets for developing therapeutics against such conditions. Here, we have investigated the anti-amyloidogenic properties of p-coumaric acid on hen egg white lysozyme (HEWL). Heat, pH, and agitation (55 °C, pH 2.0, 600 rpm) stress were used to induce amyloid formation in lysozyme. The aggregates characterization was done by turbidity, Rayleigh light scattering (RLS), and thioflavin-T (ThT) assays. Moreover, ANS (1-anilino naphthalene sulphate) binding assay and circular dichroism (CD) were employed to unveil protein hydrophobicity and secondary structure perturbation, respectively. Lysozyme demonstrated increased hydrophobicity and transition of α-helix to ß-sheet under aggregating conditions. Moreover, co-incubation of lysozyme with p-coumaric acid attenuates the process of amyloid in a concentration dependent manner. At 50 and 200 µM concentrations of p-coumaric acid, lysozyme retained its native-like folded structure. Cytotoxicity protection on human SK-N-SH neuroblastoma cell line was also observed using MTT assay and phase contrast microscopy. In addition, transmission electron microscopy (TEM) reaffirms the fibrillar nature of lysozyme aggregates and their attenuation by p-coumaric acid. The steady state fluorescence revealed that the mode of fluorescence quenching for the HEWL-p-coumaric acid interaction is static rather than dynamic. Moderate strength of binding in order of 104 M-1 exists between HEWL and p-coumaric acid. Thermodynamic parameters (∆H and ∆S) obtained from van't Hoff plot suggested spontaneous reaction with hydrophobic interaction. A slight micro-environmental change in HEWL around Tyr residue was observed during the binding process with the help of synchronous fluorescence. Molecular docking analysis reported the involvement of amino acid residues (TRP63, LEU75, ASP101, LYS97) to form a complex between HEWL-p-coumaric acid. The observed anti-amyloidogenic and inherent antioxidative properties of p-coumaric acid could be helpful to design a neuroprotective agent.

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease.

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.

Associations of zinc-α-2-glycoprotein with metabolic syndrome and its components among adult Arabs.

Epidemiological studies suggest that the Zinc-α-2-glycoprotein (ZAG) plays significant physiological roles. In this study we investigate whether ZAG could be considered as a clinical biomarker in the diagnosis and prognosis of metabolic syndrome (MetS) in Saudi population. As such insights urgently required for management of MetS. Thus, we have determined serum levels of ZAG in patients with MetS and normal individuals. We have also assessed the correlation between ZAG and different components of MetS. In this case-control study, clinical information of 200 Saudi male and female subjects (age range 30-65) with MetS (n = 100) and healthy controls (n = 100) were extracted from the database of the Chair of Biomarkers of Chronic Disease (CBCD) in King Saud University (KSU), Riyadh, Saudi Arabia. MetS was screened according to NCEP ATP III criteria (National Cholesterol Education Program Adult Treatment Panel III). Fasting glucose and lipid profile levels were measured using Konelab. Serum TNF-α, IL- 6, CRP and ZAG levels were measured using commercially available assays. There was an age-dependent significant increase in ZAG level among MetS subjects than controls (43.8 ± 19.5 vs 48.1 ± 14.8; P = 0.04). A significant inverse correlation between ZAG and serum HDL-cholesterol (r = - 0.20, P < 0.05) was observed. Whereas, triglycerides (r = 0.25, P < 0.01), waist circumference (WHR) (r = 0.17, P < 0.05) and CRP (r = 0.24, P < 0.01) were all significantly and positively associated with ZAG. Circulating ZAG is associated with MetS in an age-dependent manner. Serum ZAG is a potential biomarker for MetS.

Favorable Changes in Fasting Glucose in a 6-month Self-Monitored Lifestyle Modification Programme Inversely Affects Spexin Levels in Females with Prediabetes.

Spexin (SPX) is a novel peptide thought to have a role in various metabolic regulations. Given its presumed body-weight regulatory functions, we aimed to determine whether lifestyle intervention programs on weight loss and fasting glucose (FG) improvement among people with impaired glucose regulation also alter levels of circulating SPX. A total of 160 Saudi adult males and females with prediabetes were randomly selected from a larger cohort (N = 294) who underwent a 6-month lifestyle modification program to improve their glycemic status. Participants were split into two groups based on differences in glucose levels post-intervention, with the first 50% (improved group) having the most significant reduction in FG. SPX was measured at baseline and after 6 months. Changes in SPX was significant only in the improved group [baseline: median (Q1-Q3) of 164 pg/ml (136-227) vs follow-up: 176 pg/ml (146-285); p < 0.01]. When stratified by sex, the significant increase was observed only in females [159 pg/ml (127-252) vs 182.5 (152,369.1); p < 0.01]. Furthermore, SPX levels showed a significant inverse association with FG (ß = -0.22, p = 0.003) even after adjustment with age and BMI, again only in females. Circulating SPX levels increase over time in people with prediabetes, particularly women who responded favorably in a 6-month lifestyle intervention program. Whether an unknown mechanism regulating the sexual disparity seen in SPX levels post-intervention exists should be further investigated using a larger sample size.

Circulating spexin levels are influenced by the presence or absence of gestational diabetes.

The study aimed to determine whether circulating spexin (SPX) is modified during the course of pregnancy and whether it is affected by the presence of glucose intolerance, i.e., Gestational Diabetes Mellitus (GDM). This prospective study included 102 pregnant women (63 non-GDM and 39 GDM; mean age 29.4 ±â€¯5.1 years; mean BMI 28.0 ±â€¯6.1 kg/m2). Anthropometrics, glycemic and lipid profiles, as well measurements of circulating adipocytokines and SPX were measured at baseline and after 3 and 6 months. In GDM patients, SPX levels increased significantly after 6-months, in parallel with a borderline significant increase in glucose (p = 0.07). In non-GDM patients, however, median SPX level decreased from baseline to 6-months (p < 0.01), and this change was not associated with changes in glucose levels. Change in glucose from baseline to 6-months was positively associated with change in SPX in GDM patients only (R = 0.37; p < 0.05). SPX levels are positively influenced by glucose intolerance in pregnant women with GDM, while they decrease in control women without GDM.

Synaptosomal Protein of 25 kDa (Snap25) Polymorphisms Associated with Glycemic Parameters in Type 2 Diabetes Patients.

A possible role of Snap25 polymorphisms in type 2 diabetes mellitus (T2DM) was evaluated by analyzing three SNPs within intron 1 in a region known to affect the gene expression in vitro. Genomic DNA from 1019 Saudi individuals (489 confirmed T2DM and 530 controls) was genotyped for SNPs rs363039, rs363043, and rs363050 in Snap25 using the TaqMan Genotyping Assay. Significantly higher levels of fasting glucose and HbA1c were detected in T2DM patients carrying the rs363050 (AG/GG) genotypes compared to the (AA) genotype (f = 4.41, df = 1, and p = 0.03 and f = 5.31, df = 1, and p = 0.03, resp.). In these same patients, insulin levels were significantly decreased compared to the (AA) individuals (f = 7.29, df = 1, and p = 0.009). Significant associations were detected between rs363050 (AG/GG) genotypes and increasing fasting glucose levels (p = 0.01 and OR: 1.05), HbA1c levels (OR: 5.06 and p = 0.02), and lower insulinemia (p = 0.03 and OR: 0.95) in T2DM patients. The minor Snap25 rs363050 (G) allele, which results in a reduced expression of Snap25, is associated with altered glycemic parameters in T2DM possibly because of reduced functionality in the exocytotic machinery leading to suboptimal release of insulin.

Association between promoter region genetic variants of PTH SNPs and serum 25(OH)-vitamin D level.

Parathyroid hormone (PTH) plays a crucial role in calcium metabolism and skeletal development via altering vitamin D level. Besides, hypersecretion of PTH is implicated in the etiology of osteoporosis. In this study, we analyzed association between promoter region sequence variants of PTH gene and circulating 25-hydroxy-vitamin D (25(OH)D) level. Genotypes of PTH SNPs rs1459015, rs10500783 and rs10500784 and circulating serum 25(OH)D level of healthy adults (N=386) of different nationalities living in Riyadh were determined and relation between the different PTH allelic variants and corresponding mean 25(OH)D values were obtained using Analysis of Variance (ANOVA) and Bonferroni post-hoc test for multiple comparisons. We observed a high prevalence of vitamin D deficiency (<50 nmol/l) among all nationals which ranged from 59% among Indians to 82% among Yemeni. Comparison of the means of 25(OH)D levels corresponding to different genotypes of PTH SNPs indicated that the T allele of SNP rs1459015 was associated with higher 25(OH)D level in the Sudanese (P=0.03), while the T allele of SNP rs10500783 was associated with higher 25(OH)D level in Saudis (P=0.03). Analysis of results also indicated that the Sudanese carriers of the CC genotype of SNP rs1459015 had a higher risk of suffering from vitamin D deficiency (P=0.02). In conclusion, our study indicated significant association between specific PTH gene promoter region variants and altered levels of 25(OH)D and vitamin D deficiency among specific nationals.

Association of Type 2 Diabetes Mellitus related SNP genotypes with altered serum adipokine levels and metabolic syndrome phenotypes.

The pathogenesis of T2DM involves secretion of several pro-inflammatory molecules by the dramatically increased adipocytes, both by number and size, and associated macrophages of adipose tissue. Since T2DM is usually preceded by obesity and chronic systemic inflammation, the objective of this study was to explore for any association between genetic variants of previously established 36 T2DM-associated SNPs and altered serum adipocytokine levels and metabolic syndrome phenotypes. Study consisted of 566 subjects (284 males and 282 females) of whom 147 were T2DM patients and 419 healthy controls. Study subjects were genotyped for 36 T2DM-linked single nucleotide polymorphisms (SNPs) using the KASPar SNP Genotyping System and grouped into different genotypes for each SNP. Various anthropometric and biochemical parameters were measured following standard procedures. The mean values of serum levels of individual adipocytokines and the presence/absence of metabolic syndrome phenotypes corresponding to various genotypes were compared by determining the odds ratios. Genotypic variants of five and seven of the 36 T2DM-related SNPs were significantly associated with altered serum levels of adiponectin and aPAI, respectively. Six variants of the 36 SNPs were associated with metabolic syndrome manifestations. This study identified positive associations between genotypic variants of five and seven of the 36 T2DM related SNPs and altered serum levels of adiponectin and aPAI, respectively. Six of 36 SNPs were also associated with metabolic syndrome in the studied population. The relation between specific SNPs and individual phenotypic traits may be useful in explaining the causal mechanisms of hereditary component of T2DM.

Association between type 2 diabetes mellitus-related SNP variants and obesity traits in a Saudi population.

Obesity, commonly measured as body mass index (BMI), has been on a rapid rise around the world and is an underlying cause of several chronic non-communicable diseases, including type 2 diabetes mellitus (T2DM). In addition to the environmental factors, genetic factors may also contribute to the ongoing obesity epidemic in Saudi Arabia. This study investigated the association between variants of 36 previously established T2DM SNPs and obesity phenotypes in a population of Saudi subjects. Study subjects consisted of 975 obese (BMI: ≥30), 825 overweight (25-30) and 423 lean controls (18-25) and of these 927 had a history of T2DM. Subjects were genotyped for 36 SNPs, which have been previously proved to be T2DM linked, using the KASPar method and the means of BMI and waist circumference (WC) corresponding to each of the genotypes were compared by additive, recessive and dominant genetic models. Five and seven of 36 T2DM-related SNPs were significantly associated with the BMI and WC, respectively. Variants of SNPs rs7903146, rs1552224 and rs11642841 in the control group and rs7903146 in T2DM group showed significant association with both BMI and WC. Variant of SNP rs10440833 was significantly associated with BMI in T2DM group of both males [OR = 1.8 (1.0, 3.3); P = 0.04] and females [OR = 2.0 (1.0, 3.9); P = 0.04]. Genetic risk scores explained 19 and 14% of WC and hip size variance in this population. Variants of a number of established T2DM related SNPs were associated with obesity phenotypes and may be significant hereditary factors in the pathogenesis of T2DM.

Assessing the contribution of 38 genetic loci to the risk of type 2 diabetes in the Saudi Arabian Population.

BACKGROUND: Previous genome-wide association studies have identified multiple type 2 diabetes (T2D) genetic risk loci in many populations. However, the contribution of these loci to T2D in the Middle Eastern populations with high T2D prevalence is unknown. METHODS: Here, we investigated the association of 38 T2D risk loci in the Saudi Arabian population (1166 patients with T2D and 1235 healthy controls), which has one of the world's highest prevalence of T2D. RESULTS: Eight common genetic variants showed a significant association with T2D in our study population. The effect sizes of these loci were comparable to those previously identified in other populations with the exception of HNF4A, which showed a trend for larger effect size in our study population (OR = 1·27) compared to that reported in South Asian populations (OR = 1·09; I(2) = 65·9). Analysis of risk allele scores (RASs) defined by the 8 loci showed that subjects in the top RAS quintile (n = 480) had 2·5-fold increase in disease risk compared to those in the bottom quintile (n = 480; P = 9·5 × 10(-12)). RASs were also associated with fasting glucose level (ß = 0·12; P = 2·2 × 10(-9)), but not with BMI (P = 0·19). Analysis of a subgroup of subjects with BMI≤30 resulted in two additional loci (SLC30A8; P = 0·03, HMG20A; P = 0·02) showing significant association with T2D. CONCLUSIONS: We have shown for the first time that variants at WFS1, JAZF1, SLC30A8, CDKN2A/B, TCF7L2, KCNQ1, HMG20A, HNF4A and DUSP9 are associated with T2D in the Saudi population. Our findings also suggest substantial overlap of T2D risk loci across many ethnic groups regardless of disease prevalence.

Metabolic syndrome biomarkers and early breast cancer in Saudi women: evidence for the presence of a systemic stress response and/or a pre-existing metabolic syndrome-related neoplasia risk?

BACKGROUND: Obesity has been linked to many adverse health consequences, including breast cancer. This study aims to determine adipocytokine and other biological changes in recently diagnosed breast cancer patients before therapy is started. METHODS: A total of 109 female Saudi subjects [56 newly diagnosed, treatment-naïve, histologically-confirmed breast cancer cases and 53 age- and BMI-matched controls] were enrolled in this study. Anthropometric data were collected. Serum insulin, adipocytokines and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured using a customized multiplex Luminex assay. Hypersensitive C-Reactive Protein (CRP), tumor necrosis factor-alpha (TNF-α), and angiotensin II (ANG II) were measured using ELISA. RESULTS: A few days in the diagnosis, breast cancer subjects had significantly higher systolic blood pressure (p = 0.03), glucose (p = 0.01), triglycerides (p = 0.001), leptin (p = 0.044), resistin (p = 0.04), ANG II (p = 0.02), TNF-α (p = 0.045), and CRP (p = 0.04) than the controls. On the other hand, HDL (p = 0.01) and adiponectin (p = 0.02) were significantly lower in cancer subjects than controls. A significant association was found between elevated triglycerides (TG) and breast cancer [OR (95% CI), 6.1(1.8, 15.6), p = 0.004], as well as elevated ANG II [OR (95% CI), 5.2(1.2, 14.3), p = 0.03]. On the other hand, aPAI and HDL correlated negatively with breast cancer [OR (95% CI), 0.076(0.01, 0.34), p = 0.001; 0.30(0.09, 0.95), p 0.04, respectively]. CONCLUSION: Circulating ANGII and triglycerides were positively associated with early breast cancer. In contrast, HDL-cholesterol correlated negatively with ANG II and aPAI in these patients. This suggests that patients with recently diagnosed breast cancer have biochemical changes consistent with an activated stress response and/or that patients with metabolic syndrome manifestations have a higher risk of developing this disease.