Metabolomics Signature in Prediabetes and Diabetes: Insights From Tandem Mass Spectrometry Analysis.

OBJECTIVE: This study investigates the metabolic differences between normal, prediabetic and diabetic patients with good and poor glycaemic control (GGC and PGC). DESIGN: In this study, 1102 individuals were included, and 50 metabolites were analysed using tandem mass spectrometry. The diabetes diagnosis and treatment standards of the American Diabetes Association (ADA) were used to classify patients. METHODS: The nearest neighbour method was used to match controls and cases in each group on the basis of age, sex and BMI. Factor analysis was used to reduce the number of variables and find influential underlying factors. Finally, Pearson's correlation coefficient was used to check the correlation between both glucose and HbAc1 as independent factors with binary classes. RESULTS: Amino acids such as glycine, serine and proline, and acylcarnitines (AcylCs) such as C16 and C18 showed significant differences between the prediabetes and normal groups. Additionally, several metabolites, including C0, C5, C8 and C16, showed significant differences between the diabetes and normal groups. Moreover, the study found that several metabolites significantly differed between the GGC and PGC diabetes groups, such as C2, C6, C10, C16 and C18. The correlation analysis revealed that glucose and HbA1c levels significantly correlated with several metabolites, including glycine, serine and C16, in both the prediabetes and diabetes groups. Additionally, the correlation analysis showed that HbA1c significantly correlated with several metabolites, such as C2, C5 and C18, in the controlled and uncontrolled diabetes groups. CONCLUSIONS: These findings could help identify new biomarkers or underlying markers for the early detection and management of diabetes.

Protective Effects of Alpha-lipoic Acid, Resveratrol, and Apigenin against Oxidative Damages, Histopathological Changes, and Mortality Induced by Lung Irradiation in Rats.

AIM: This study investigated the protective effects of three antioxidants on radiationinduced lung injury. BACKGROUND: Oxidative stress is one of the key outcomes of radiotherapy in normal tissues. It can induce severe injuries in lung tissue, which may lead to pneumonitis and fibrosis. Recently, interest in natural chemicals as possible radioprotectors has increased due to their reduced toxicity, cheaper price, and other advantages. OBJECTIVE: The present study was undertaken to evaluate the radioprotective effect of Alpha-lipoic Acid (LA), Resveratrol (RVT), and Apigenin (APG) against histopathological changes and oxidative damage and survival induced by ionizing radiation (IR) in the lung tissues of rats. METHODS: First, the lung tissue of 50 mature male Wistar rats underwent an 18 Gy gamma irradiation. Next, the rats were sacrificed and transverse sections were obtained from the lung tissues and stained with hematoxylin and eosin (H and E) and Mason trichrome (MTC) for histopathological evaluation. Then, the activity of Glutathione peroxidase (GPx), Superoxide Dismutase (SOD), and Malondialdehyde (MDA) was measured by an ELISA reader at 340, 405, and 550 nm. RESULTS: Based on the results of this study, IR led to a remarkable increase in morphological changes in the lung. However, APG, RVT, and LA could ameliorate the deleterious effects of IR in lung tissue. IR causes an increase in GPX level, and APG+IR administration causes a decrease in the level of GPX compared to the control group. Also, the results of this study showed that RVT has significant effects in reducing MDA levels in the short term. In addition, compared to the control group, IR and RVT+IR decrease the activity of SOD in the long term in the lung tissues of rats. Also, the analysis of results showed that weight changes in IR, LA+IR, APG+IR, and control groups were statistically significant. CONCLUSION: APG and RVT could prevent tissue damage induced by radiation effects in rat lung tissues. Hence, APG, LA, and RVT could provide a novel preventive action with their potential antioxidant anti-inflammatory properties, as well as their great safety characteristic.

Investigation of 3T3-L1 Cell Differentiation to Adipocyte, Affected by Aqueous Seed Extract of Phoenix Dactylifera L.

BACKGROUND: Obesity, often associated with insulin resistance and type 2 diabetes, is a metabolic disease that can result in dyslipidemia and hyperglycemia. Many reports describe the hypoglycemic and hypolipidemic properties of the Phoenix dactylifera L. seed extract in STZ-induced diabetic rat models, however, its anti-diabetic effects in other diabetic models are less characterized in the literature. This study set out to determine the possible effects of the Phoenix dactylifera L. seed extract on adipogenesis and glucose homeostasis. METHODS: 3T3-L1 cells were cultured in adipocyte differentiation media with or without varying doses of Phoenix dactylifera L. extract (0.312-1 mg/ml). Assays were performed on days 5, 8, and 12 after induced differentiation. RESULTS: Our results demonstrate that the triglyceride content in treated groups was significantly lower compared to controls. Further, treating 3T3-L1 cells with Phoenix dactylifera L. seed extract reduced adipogenesis through the downregulation of PPAR-γ and CEBP-α, and adipocyte-specific genes involved in fatty acid metabolism including ap2, ACACA, and FAS. CONCLUSION: Phoenix dactylifera L. seeds have the potential to inhibit adipogenesis and obesity. Overall, this study explored the inhibitory effects of Phoenix dactylifera L. seed extract on adipogenesis in 3T3-L1 cells on the molecular level.

Aqueous Cichorium intybus L. seed extract may protect against acute palmitate-induced impairment in cultured human umbilical vein endothelial cells by adjusting the Akt/eNOS pathway, ROS: NO ratio and ET-1 concentration.

BACKGROUND: Endothelial dysfunction, which is a vascular response to oxidative stress and inflammation, involves a cascade of downstream events that lead to decreased synthesis of insulin-mediated vasodilator nitric oxide (NO) and increased production of vasoconstrictor protein endothelin-1 (ET-1). NO, and ET-1 production by endothelial cells is regulated by phosphatidylinositol 3-kinase (PI3K)-Akt-eNOS axis and mitogen-activated protein kinase (MAPK) axis of the insulin signaling pathway, respectively. METHODS: After treating the human umbilical vein endothelial cells (HUVECs) with either palmitate complexed with bovine serum albumin (BSA) (abbreviated as PA) or the aqueous Cichorium intybus L. (chicory) seed extract (chicory seed extract, abbreviated as CSE) alone, and simultaneously together (PA + CSE), for 3, 12, and 24 h, we evaluated the capacity of CSE to reestablish the PA-induced imbalance between PI3K/Akt/eNOS and MAPK signaling pathways. The level of oxidative stress was determined by fluorimeter. Insulin-induced levels of NO and ET-1 were measured by Griess and ELISA methods, respectively. Western blotting was used to determine the extent of Akt and eNOS phosphorylation. RESULTS: Contrary to PA that caused an increase in the reactive oxygen species (ROS) levels and attenuated NO production, CSE readjusted the NO/ROS ratio within 12 h. CSE improved the metabolic arm of the insulin signaling pathway by up-regulating the insulin-stimulated phospho-eNOS Ser1177/total eNOS and phospho-Akt Thr308/total Akt ratios and decreased ET-1 levels. CONCLUSIONS: CSE ameliorated the PA-induced endothelial dysfunction not only by its anti-ROS property but also by selectively enhancing the protective arm and diminishing the injurious arm of insulin signaling pathways.

Evaluation of the Radioprotective Effects of Melatonin Against Ionizing Radiation-Induced Muscle Tissue Injury.

BACKGROUND: Radiotherapy (RT) is a treatment method for cancer using ionizing radiation (IR). The interaction between IR with tissues produces free radicals that cause biological damages.As the largest organ in the human body, the skeletal muscles may be affected by detrimental effects of ionizing radiation. To eliminate these side effects, we used melatonin, a major product secreted by the pineal gland in mammals, as a radioprotective agent. MATERIALS AND METHODS: For this study, a total of sixty male Wistar rats were used. They were allotted to 4 groups: control (C), melatonin (M), radiation (R) and melatonin + radiation (MR). Rats' right hind legs were irradiated with 30 Gy single dose of gamma radiation, while 100 mg/kg of melatonin was given to them 30 minutes before irradiation and 5 mg/ kg once daily afternoon for 30 days. Five rats in each group were sacrificed 4, 12 and 20 weeks after irradiation for histological and biochemical examinations. RESULTS: Our results showed radiation-induced biochemical, histological and electrophysiological changes in normal rats' gastrocnemius muscle tissues. Biochemical analysis showed that malondialdehyde (MDA) levels significantly elevated in R group (P<0.001) and reduced significantly in M and MR groups after 4, 12, and 20 weeks (P<0.001), However, the activity of catalase (CAT) and superoxide dismutase(SOD)decreased in the R group and increased in M and MR groups for the same periods of time compared with the C group (P<0.001), while melatonin administration inverted these effects( P<0.001).Histopathological examination showed significant differences between R group for different parameters compared with other groups (P<0.001). However, the administration of melatonin prevented these effects(P<0.001). Electromyography (EMG) examination showed that the compound action potential (CMAP) value in the R group was significantly reduced compared to the effects in the C and M groups after 12 and 20 weeks (P<0.001). The administration of melatonin also reversed these effects (P<0.001). CONCLUSION: Melatonin can improve biochemical, electrophysiological and morphological features of irradiated gastrocnemius muscle tissues.Our recommendation is that melatonin should be administered in optimal dose. For effective protection of muscle tissues, and increased therapeutic ratio of radiation therapy, this should be done within a long period of time.

Induction of transient cell cycle arrest by H2 O2 via modulation of ultradian oscillations of Hes1, Socs3, and p-Stat3 in fibroblast cells.

Biological clocks, time-keeping systems, enable the living organisms to synchronize their biochemical processes with their environment. Among these molecular oscillators, ultradian oscillators have been identified with volatility less than 24 h. Transcription factor Hes1, a member of the basic Helix-loop-Helix (bHLH) protein family, has an oscillation duration of 2 h in vertebrates. Due to the pivotal role of oxidative stress in many human diseases, we evaluated the effect(s) of oxidative stress on Hes1 oscillator, its upstream regulators, and its downstream cell cycle regulators. NIH/3T3 mouse fibroblast cells were treated with sublethal (250 µM) and lethal (1000 µM) doses of H2 O2 for 30 min. H2 O2 generated a delay in p-Stat3 and Socs3 mRNAs followed by suppression of Hes1 protein. These events were accompanied by simultaneous upregulation of p21 and downregulation of cyclinD1, resulting in a temporary arrest of the cell cycle. In conclusion, the elimination of Hes1 protein oscillation by H2 O2 may represent a defense mechanism against oxidative stress in fibroblast cells.

Effect of Cichorium intybus L. seed extract on renal parameters in experimentally induced early and late diabetes type 2 in rats.

Blood and urine biochemistry screening tests are important for initial detection of diabetes, determination of severity of its complications, and monitoring of therapy. We evaluated the effects of aqueous chicory seed extract (CSE), on renal biochemical parameters, histology, and Na+/glucose cotansporters, SGLT1 and SGLT2 expression levels using metformin, and aspirin as controls. Late stage type 2 diabetes (LT2D; FBS, >300 mg/dl) and early stage type 2 diabetes (ET2D; FBS, 140-220 mg/dl) were induced in rats by streptozotocin (STZ group) and a combination of STZ and niacinamide (NIA/STZ group), respectively. A non-diabetic group was included as control. Treatment included daily intraperitoneal injections of either CSE (125 mg/kg b.w.) or metformin (100 mg/kg b.w.) and oral aspirin (120 mg/kg b.w.) for 21 days. At the end, blood and 24 h urine samples were collected; and kidneys were saved at -80 ËšC. CSE reduced urinary α1-microgobulin excretion in ET2D (p = .043), and serum uric acid (p = .045), and glomerular diameter (p < .01) in LT2D. Metformin appeared to be more effective in LT2D with respect to serum uric acid, urea, and BUN (< .05). Both CSE and metformin improved histology. Aspirin improved several blood and urine variables, but appeared to aggravate morphological damages to the kidney tissue. The absolute values of albumin, α1-microglobulin or total protein in urine rather than their creatinine ratios seemed more useful in the detection of early kidney damage; CSE was able to repair the kidney damage and α1-microglobulin was sensitive enough to allow monitoring of the improvements caused by the treatment.

Effect of Cichorium intybus L. on the expression of hepatic NF-κB and IKKß and serum TNF-α in STZ- and STZ+ niacinamide-induced diabetes in rats.

BACKGROUND: Inflammation is an early event in the development of diabetes type 2 (T2D). Cichorium intybus L. (chicory) possesses anti-inflammatory action. We compared the anti-inflammatory aspect of aqueous chicory seed extract (CSE) in early and late stage T2D in rats. METHODS: Wistar albino rats were divided into nine final groups (n = 6). Three main groups consisted of non-diabetic (Control), early stage diabetes (ET2D; niacinamide/streptozotocin, i.e., NIA/STZ), and late stage diabetes (LT2D; STZ). Within each main group, a subgroup was treated with CSE (125 mg/kg; i.p.); within each diabetic group (STZ and NIA/STZ) a subgroup received metformin (100 mg/kg; i.p.); another subgroup in STZ group received aspirin (120 mg/kg; oral). After 21 days, fasting blood glucose (FBS), insulin, and TNF-α level were measured in serum; IKKß and NF-κB (p65) mRNA and protein expression were evaluated by real time PCR and Western blotting; p65 DNA binding activity was determined by ELISA, in liver tissue. RESULTS: The mRNA and protein expression levels of IKKß, and P65 genes increased in both stages of T2D (p < 0.01); CSE decreased their expression (p < 0.001, mRNAs; p < 0.05, proteins). The increased DNA-binding capacity of NF-κB (p < 0.0001) in diabetes was lowered by CSE (p < 0.001). The effect of CSE was limited to ET2D requiring insulin. CONCLUSIONS: The anti-inflammatory action of CSE is due to a direct modulation of cytokine expression. The dependency of chicory action on the presence of insulin indicates its usefulness in the early stages of diabetes and for the purpose of preventing and delaying diabetes onset.

Amelioration by chicory seed extract of diabetes- and oleic acid-induced non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) via modulation of PPARα and SREBP-1.

We evaluated the effect of chicory (Cichorium intybus L.) seed extract (CI) on hepatic steatosis caused by early and late stage diabetes in rats (in vivo), and induced in HepG2 cells (in vitro) by BSA-oleic acid complex (OA). Different dosages of CI (1.25, 2.5 and 5 mg/ml) were applied along with OA (1 mM) to HepG2 cells, simultaneously and non-simultaneously; and without OA to ordinary non-steatotic cells. Cellular lipid accumulation and glycerol release, and hepatic triglyceride (TG) content were measured. The expression levels of sterol regulatory element-binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor alpha (PPARα) were determined. Liver samples were stained with hematoxylin and eosin (H&E). Significant histological damage (steatosis-inflammation-fibrosis) to the cells and tissues and down-regulation of SREBP-1c and PPARα genes that followed steatosis induction were prevented by CI in simultaneous treatment. In non-simultaneous treatment, CI up-regulated the expression of both genes and restored the normal levels of the corresponding proteins; with a greater stimulating effect on PPARα, CI acted as a PPARα agonist. CI released glycerol from HepG2 cells, and targeted the first and the second hit phases of hepatic steatosis. A preliminary attempt to characterize CI showed caffeic acid, chlorogenic acid, and chicoric acid, among the constituents.

Effect of chicory seed extract on glucose tolerance test (GTT) and metabolic profile in early and late stage diabetic rats.

BACKGROUND AND PURPOSE OF THE STUDY: The goal was to evaluate and compare the effects of aqueous extract of the seeds of chicory, Cichorium intybus L., on glucose tolerance test (GTT) and blood biochemical indices of experimentally-induced hyperglycemic rats. METHODS: Late stage and early stage of Type 2 diabetes mellitus (T2DM) were induced in rats by streptozotocin (STZ) and a combination of STZ and niacinamide (NIA/STZ), respectively. Within each group, one subgroup received daily i. p. injections of chicory extract (125 mg/kg body weight, for 28 days). Body weight and fasting blood sugar (FBS) were measured weekly. Blood was analyzed for glycosylated hemoglobin (HbA1c) and sera for alanine aminotransferase (ALT), aspartate aminotransferase (AST), nitric oxide (NO), triacylglycerol (TG), total cholesterol (TC), total protein, and insulin on days 10 and 28 after treatment. Intraperitoneal glucose tolerance test (IPGTT) along with insulin determination was performed on a different set of rats in which the chicory-treated groups received the extract for 10 days. RESULTS: During 4 weeks of treatment, chicory prevented body-weight loss and decreased FBS. ALT activities and levels of TG, TC and HbA1c decreased, and concentration of NO increased in the chicory treated groups (p < 0.05). Unlike late-stage diabetes, fasting serum insulin concentrations were higher and GTT pattern approximated to normal in chicory-treated early-stage diabetic rats. CONCLUSIONS: Chicory appeared to have short-term (about 2 hours, as far as GTT is concerned) and long-term (28 days, in this study) effects on diabetes. Chicory may be useful as a natural dietary supplement for slowing down the pace of diabetes progress, and delaying the development of its complications.

Cytotoxicity of subtoxic AgNP in human hepatoma cell line (HepG2) after long-term exposure.

UNLABELLED: We aimed at evaluating the toxicity effects of low (subtoxic) concentrations of silver nanoparticles nanoparticles (AgNP, 5-10 nm) in human hepatoblastoma (HepG2) cell line after and during a period of about one month. METHODS: XTT and MTT assays were used to draw a dose-response curve; IC50 (half maximal inhibitory concentration) value of the AgNP on HepG2 cells was calculated to be 2.75-3.0 mg/l. The cells were exposed to concentrations of 0% (control), 1%, 4% and 8% IC50 of AgNP (corresponding to 0.00, 0.03, 0.12 and 0.24 mg/l of AgNP, respectively) for four consecutive passages. The treated cells were compared to the control group with respect to morphology and proliferation at the end of the period. RESULTS: The biochemical studies revealed significant increases of lactate dehydrogenase and alanine aminotransferase enzyme activity in the culture media of cells receiving 4% and 8% IC50; the increases in the aspartate aminotransferase enzyme activity and nitric oxide concentration became significant at 8% IC50. In the cell extracts, the average total protein and activity of glutathione peroxidase enzyme remained unchanged; the decrease in the average content of glutathione (GSH) and superoxide dismutase (SOD) activity became significant at 4% and 8% IC50. There were increases in lipid peroxidation (significant at 4% and 8% IC50) and cytochrome c content (significant at 8% IC50). The accumulations of the effects, during the experiment from one generation to the next, were not statistically remarkable except in cases of GSH and SOD. The results indicate clearly the involvement of oxidative changes in the cells after exposure to low doses of AgNP. CONCLUSION: The results might help specify a safer amount of AgNP for use in different applications.

G1 phase arrest and apoptosis induction in human thyroid cancer cell line Thr.C1.PI33 by 3-hydrogenkwadaphnin isolated from Dendrostellera lessertii.

BACKGROUND: Dendrostellera lessertii (Thymelaeaceae) is a toxic plant that grows in parts of Iran. The anti-proliferative properties of its crude methanol extract and one of its active components, 3-hydrogenkwadaphnin (3-HK), have been established using several cancer cell lines. METHODS: In a further attempt to determine the mode of action, two groups of synchronously growing cells were treated with a single dose of 3-HK (3.5 nM) and/or a single dose of the crude extract (equivalent to 0.36 mg plant powder). Every 8 hours, the percentages of cells within G1, S, and G2-M phases were determined by flow cytometric (FCM) analysis; electron microscopic pictures were taken after fixation with 2% glutaraldehyde. RESULTS: Twelve hours after treatments, apoptotic cell death was confirmed by the observation of marked morphological changes of the plasma membrane as microvillar disappearance and the appearance of apoptotic bodies in the treated cells. FCM analyses revealed that the G1 phase arrest was under the influence of the pure substance. CONCLUSION: The results confirmed the previously drawn conclusion that the raw material and the pure substance from D. lessertii exert their anti-tumor effects through cell cycle arrest at G1 phase and diversion of cell fate toward programmed cell death.

Alkaline phosphatase retained in HepG2 hepatocarcinoma cells vs. alkaline phosphatase released to culture medium: difference of aberrant glycosylation.

Liver tissue is the source of 90% of serum alkaline phosphatase (AP). The serum levels and structures of tumor marker proteins change under many disease conditions as well as cancer. The study was aimed at determining the type of alkaline phosphatase (AP) present in HepG2 hepatocellular carcinoma cell line. Alkaline phosphatase rich extracts of healthy human liver, HepG2 hepatocarcinoma cells, as well as the condition medium of HepG2 cells were prepared by extraction with 40% n-butanol and 30-50% acetone precipitation, and subjected to various chromatographic procedures. Lectin affinity chromatography of the samples with concanavalin A-Sepharose 4B showed considerable differences in the elution patterns. Non-denaturing polyacrylamide gel electrophoresis of the culture medium yielded a relatively slow migrating band of activity that coincided with none of the three bands of activity produced by the normal liver extract, nor with the bands of the cell pellet extract. Inhibition patterns were established by measuring the enzyme activities in the presence of varying concentrations of L-phenylalanine, L-leucine, L-homoarginine, and levamisole. The APs from the cell line were neuraminidase sensitive. According to the results the main AP produced and released to the medium by HepG2 cell line is an aberrantly glycosylated tissue non-specific AP. In addition, the differences between the cell-pellet AP and the culture medium AP seemed to stem from different sugar moieties in their structures.