In Vitro Nonalcoholic Fatty Liver Disease Model Elucidating the Effect of Immune Environment on Disease Progression and Alleviation.

Nonalcoholic fatty liver disease (NAFLD), which is a major cause of chronic liver disease, is characterized by fat accumulation in the liver. Existing models struggle to assess medication effects on liver function in the context of NAFLD's unique inflammatory environment. We address this by developing a 3D in vitro NAFLD model using HepG2 and THP-1 cells (mimicking liver and Kupffer cells) cocultured using transwell and hydrogel system. This mimics liver architecture and allows for manipulation of the immune environment. We demonstrate that the model recapitulates key NAFLD features: steatosis (induced by fatty acids), oxidative stress, inflammation, and impaired liver function embodying the interrelationship between NAFLD and the surrounding immune environment. This versatile model offers a valuable tool for preclinical NAFLD research by incorporating a disease-relevant immune environment.

Protective Role of Short-Chain Fatty Acids against Ang- II-Induced Mitochondrial Dysfunction in Brain Endothelial Cells: A Potential Role of Heme Oxygenase 2.

OBJECTIVES: Short-chain fatty acids (SCFAs), the main metabolites released from the gut microbiota, are altered during hypertension and obesity. SCFAs play a beneficial role in the cardiovascular system. However, the effect of SCFAs on cerebrovascular endothelial cells is yet to be uncovered. In this study, we use brain endothelial cells to investigate the in vitro effect of SCFAs on heme oxygenase 2 (HO-2) and mitochondrial function after angiotensin II (Ang-II) treatment. METHODS: Brain human microvascular endothelial cells were treated with Ang-II (500 nM for 24 h) in the presence and absence of an SCFAs cocktail (1 µM; acetate, propionate, and butyrate) and/or HO-2 inhibitor (SnPP 5 µM). At the end of the treatment, HO-2, endothelial markers (p-eNOS and NO production), inflammatory markers (TNFα, NFκB-p50, and -p65), calcium homeostasis, mitochondrial membrane potential, mitochondrial ROS and H2O2, and mitochondrial respiration were determined in all groups of treated cells. KEY RESULTS: Our data showed that SCFAs rescued HO-2 after Ang-II treatment. Additionally, SCFAs rescued Ang-II-induced eNOS reduction and mitochondrial membrane potential impairment and mitochondrial respiration damage. On the other hand, SCFAs reduced Ang-II-induced inflammation, calcium dysregulation, mitochondrial ROS, and H2O2. All of the beneficial effects of SCFAs on endothelial cells and mitochondrial function occurred through HO-2. CONCLUSIONS: SCFAs treatment restored endothelial cells and mitochondrial function following Ang-II-induced oxidative stress. SCFAs exert these beneficial effects by acting on HO-2. Our results are opening the door for more studies to investigate the effect the of SCFAs/HO-2 axis on hypertension and obesity-induced cerebrovascular diseases.

Selected coffee (Coffea arabica L.) extracts inhibit intestinal α-glucosidases activities in-vitro and postprandial hyperglycemia in SD Rats.

One of the therapeutic approaches for decreasing postprandial hyperglycemia is to retard absorption of glucose by the inhibition of carbohydrate hydrolyzing enzymes, α-amylase, and α-glucosidases, in the digestive organs. Coffee consumption has been reported to beneficial effects for controlling calorie and cardiovascular diseases, however, the clear efficacy and mode of action are yet to be proved well. Therefore, in this study we evaluated in- vitro rat intestinal α-glucosidases and porcine α-amylase inhibitory activities as well as in vivo (Sprague-Dawley rat model) blood glucose lowering effects of selected coffee extracts. The water extracted Sumatra coffee (SWE) showed strong α-glucosidase inhibitory activity (IC50, 4.39 mg/mL) in a dose-dependent manner followed by Ethiopian water extract (EWE) (IC50, 4.97) and Guatemala water extract (GWE) (IC50, 5.19). Excepted for GWE all the coffee types significantly reduced the plasma glucose level at 0.5 h after oral intake (0.5 g/kg-body weight) in sucrose and starch-loaded SD rats. In sucrose loading test SWE (p < 0.001) and EWE (p < 0.05) had significantly postprandial blood glucose reduction effect, when compared to control. The maximum blood glucose levels (Cmax) of EWE administration group were decreased by about 18% (from 222.3 ± 16.0 to 182.5 ± 15.4, p < 0.01) and 19% (from 236.2 ± 25.1 to 191.3 ± 13.2 h·mg/dL, p < 0.01) in sucrose and starch loading tests, respectively. These results indicate that selected coffee extract may improve exaggerated postprandial spikes in blood glucose via inhibition of intestinal sucrase and thus delays carbohydrate absorption. These in vitro and in vivo studies therefore could provide the biochemical rationale for the benefit of coffee-based dietary supplement and the basis for further clinical study.

Effects of 3-methyladenine, an autophagy inhibitor, on the elevated blood pressure and arterial dysfunction of angiotensin II-induced hypertensive mice.

Autophagy is an intracellular degradation system that disassembles cytoplasmic components through autophagosomes fused with lysosomes. Recently, it has been reported that autophagy is associated with cardiovascular diseases, including pulmonary hypertension, atherosclerosis, and myocardial ischemia. However, the involvement of autophagy in hypertension is not well understood. In the present study, we hypothesized that excessive autophagy contributes to the dysfunction of mesenteric arteries in angiotensin II (Ang II)-induced hypertensive mice. Treatment of an autophagy inhibitor, 3-methyladenine (3-MA), reduced the elevated blood pressure and wall thickness, and improved endothelium-dependent relaxation in mesenteric arteries of Ang II-treated mice. The expression levels of autophagy markers, beclin1 and LC3 II, were significantly increased by Ang II infusion, which was reduced by treatment of 3-MA. Furthermore, treatment of 3-MA induced vasodilation in the mesenteric resistance arteries pre-contracted with U46619 or phenylephrine, which was dependent on endothelium. Interestingly, nitric oxide production and phosphorylated endothelial nitric oxide synthase (p-eNOS) at S1177 in the mesenteric arteries of Ang II-treated mice were increased by treatment with 3-MA. In HUVECs, p-eNOS was reduced by Ang II, which was increased by treatment of 3-MA. 3-MA had direct vasodilatory effect on the pre-contracted mesenteric arteries. In cultured vascular smooth muscle cells (VSMCs), Ang II induced increase in beclin1 and LC3 II and decrease in p62, which was reversed by treatment of 3-MA. These results suggest that autophagy inhibition exerts beneficial effects on the dysfunction of mesenteric arteries in hypertension.

Anti-Obesity and Anti-Adipogenic Effects of Administration of Arginyl-Fructose-Enriched Jeju Barley (Hordeum vulgare L.) Extract in C57BL/6 Mice and in 3T3-L1 Preadipocytes Models.

In our previous study, we reported that arginyl-fructose (AF), one of the Amadori rearrangement compounds (ARCs) produced by the heat processing of Korean ginseng can reduce carbohydrate absorption by inhibiting intestinal carbohydrate hydrolyzing enzymes in both in vitro and in vivo animal models. This reduced absorption of carbohydrate might be helpful to control body weight gain due to excessive carbohydrate consumption and support induced calorie restriction. However, the weight management effect, except for the effect due to anti-hyperglycemic action, along with the potential mechanism of action have not yet been determined. Therefore, the efforts of this study are to investigate and understand the possible weight management effect and mechanism action of AF-enriched barley extracts (BEE). More specifically, the effect of BEE on lipid accumulation and adipogenic gene expression, body weight gain, body weight, plasma lipids, body fat mass, and lipid deposition were evaluated using C57BL/6 mice and 3T3-L1 preadipocytes models. The formation of lipid droplets in the 3T3-L1 treated with BEE (500 and 750 µg/mL) was significantly blocked (p < 0.05 and p < 0.01, respectively). Male C57BL/6 mice were fed a high-fat diet (30% fat) for 8 weeks with BEE (0.3 g/kg-body weight). Compared to the high fat diet control (HFD) group, the cells treated with BEE significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBP/α), the mRNA expression of downstream lipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1c (SREBP-1c). Supplementation of BEE effectively lowered the body weight gain, visceral fat accumulation, and plasma lipid concentrations. Compared to the HFD group, BEE significantly suppressed body weight gain (16.06 ± 2.44 g vs. 9.40 ± 1.39 g, p < 0.01) and increased serum adiponectin levels, significantly, 1.6-folder higher than the control group. These results indicate that AF-enriched barley extracts may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

The microsomal triglyceride transfer protein inhibitor lomitapide improves vascular function in mice with obesity.

OBJECTIVE: In this study, the effect of lomitapide, a microsomal triglyceride transfer protein inhibitor, on the cardiovascular function in obesity was investigated. METHODS: Eight-week-old C57BL/6 mice were fed with high-fat diet for 12 weeks in the presence and absence of lomitapide. Lomitapide was administered by gavage (1 mg/kg/d) during the last 2 weeks of high-fat feeding. Body weight, blood glucose, body composition, and lipid profile were determined. Vascular function and endothelial function markers were studied in the aorta and mesenteric resistance arteries. RESULTS: Lomitapide treatment reduced body weight in mice with obesity. Blood glucose, percentage of fat mass, total cholesterol, and low-density lipoprotein levels were significantly reduced, and the percentage of lean mass was significantly increased after lomitapide treatment. The vascular response to sodium nitroprusside in the aorta and mesenteric arteries was similar among groups. However, the vascular response to acetylcholine was improved in the treated group. This was associated with decreased levels of vascular endoplasmic reticulum stress, inflammation, and oxidative stress. CONCLUSIONS: Treatment with lomitapide attenuated the increase in body weight in mice with obesity and restored the lipid profile and vascular function. These effects were accompanied by a decrease in inflammation and oxidative stress.

Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models.

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

Vasodilatory Effect of Phellinus linteus Extract in Rat Mesenteric Arteries.

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IKCa) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IKCa channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K+ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K+ channel blockers: iberiotoxin (BKca channel blocker), apamin (SKca channel blocker), and charybdotoxin (IKca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC20).

AdipoRon, adiponectin receptor agonist, improves vascular function in the mesenteric arteries of type 2 diabetic mice.

BACKGROUND: An orally active synthetic adiponectin receptor agonist, AdipoRon has been suggested to ameliorate insulin resistance, and glucose tolerance. However, the chronic effect of AdipoRon in the vascular dysfunction in type 2 diabetes has not been studied yet. Thus, in this study, we examined whether AdipoRon improves vascular function in type 2 diabetes. METHODS: Type 2 diabetic (db-/db-) mice were treated with AdipoRon (10 mg/kg/everyday, by oral gavage) for 2 weeks. Body weight and blood glucose levels were recorded every other day during the experimental period. Diameter of mesenteric arteries was measured. And western blot analysis was performed with mesenteric arteries. RESULTS: Pressure-induced myogenic response was significantly increased while endothelium-dependent relaxation was reduced in the mesenteric arteries of db-/db- mice. Treatment of AdipoRon normalized potentiated myogenic response, whereas endothelium-dependent relaxation was not affected by treatment of AdipoRon. The expression levels of AdiR1, AdiR2, APPL1, and APPL 2 were increased in the mesenteric arteries of db-/db- mice and treatment of AdipoRon did not affect them. Interestingly, AdipoRon treatment increased the phospho-AMPK and decreased MYPT1 phosphorylation in db-/db- mice while there was no change in the level of eNOS phosphorylation. CONCLUSION: The treatment of AdipoRon improves vascular function in the mesenteric arteries of db-/db- mice through endothelium-independent mechanism. We suggest that MLCP activation through reduced phosphorylation of MYPT1 might be the dominant mechanism in the AdipoRon-induced vascular effect.

Understanding the immunopathogenesis of autoimmune diseases by animal studies using gene modulation: A comprehensive review.

Autoimmune diseases are clinical syndromes that result from pathogenic inflammatory responses driven by inadequate immune activation by T- and B-cells. Although the exact mechanisms of autoimmune diseases are still elusive, genetic factors also play an important role in the pathogenesis. Recently, with the advancement of understanding of the immunological and molecular basis of autoimmune diseases, gene modulation has become a potential approach for the tailored treatment of autoimmune disorders. Gene modulation can be applied to regulate the levels of interleukins (IL), tumor necrosis factor (TNF), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), interferon-γ and other inflammatory cytokines by inhibiting these cytokine expressions using short interfering ribonucleic acid (siRNA) or by inhibiting cytokine signaling using small molecules. In addition, gene modulation delivering anti-inflammatory cytokines or cytokine antagonists showed effectiveness in regulating autoimmunity. In this review, we summarize the potential target genes for gene or immunomodulation in autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel diseases (IBD) and multiple sclerosis (MS). This article will give a new perspective on understanding immunopathogenesis of autoimmune diseases not only in animals but also in human. Emerging approaches to investigate cytokine regulation through gene modulation may be a potential approach for the tailored immunomodulation of some autoimmune diseases near in the future.

In Vitro and in Vivo Anti-Hyperglycemic Activities of Taxifolin and Its Derivatives Isolated from Pigmented Rice (Oryzae sativa L. cv. Superhongmi).

Superhongmi is a new rice variety, which was developed for the enrichment of bioactive compounds through cross-breeding three varieties of rice breeds in Korea. The high-performance liquid chromatography coupled with a photodiode array detector quadrupole and tandem time-of-flight mass spectrometry (HPLC/PDA/QTOF-MS) analysis has revealed that superhongmi bran extract contained four taxifolin derivatives as well as cyanidin 3-glucoside. The high-performance countercurrent chromatography (CCC) and reversed-phase HPLC led to the isolation of aforementioned five compounds, and spectroscopic analysis identified cyanidin 3-glucoside (1), along with (2R,3R)-taxifolin 3-O-ß-d-glucopyranoside (2), (2R,3R)-4'-O-methyltaxifolin 3-O-ß-d-glucopyranoside (a novel compound) (3), (2R,3R)-taxifolin (4), and (2R,3R)-4'-O-methyltaxifolin (5). Compound 2 had the highest rat small intestinal sucrase inhibitory activity (0.54 mM) relevant for potentially managing postprandial hyperglycemia, followed by compound 1 (0.97 mM) and compound 4 (1.74 mM, IC50). The anti-hyperglycemic effect of compound 4 (taxifolin), a main peak in HPLC analysis was investigated using a Sprague-Dawley (SD) rat model. Compared to a control, taxifolin treatment (p < 0.001) reduced significantly after sucrose loading the observed postprandial blood glucose and the maximum blood glucose (Cmax) by 15% (203.60 ± 15.86 to 172.30 ± 12.74). These results indicate that taxifolin derivatives that inhibit the activity of carbohydrate-hydrolyzing enzymes resulting in reduced dietary carbohydrate absorption can potentially be used as a strategy to manage diabetes.

Effect of Steamed Onion (ONIRO) Consumption on Body Fat and Metabolic Profiles in Overweight Subjects: A 12-Week Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Objective: The aim of the study is to investigate the effect of Jeju steamed onion (ONIRO) on body fat and metabolic profiles in overweight subjects.Methods: This randomized, double-blind, placebo-controlled clinical intervention was conducted and completed at one clinical research site. The subjects (n = 70) were randomly divided into placebo or test group and were instructed to take before each meal either the placebo or ONIRO capsule for 12 weeks. Anthropometric as well as serum and metabolic parameters, including triglycerides, cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, leptin, adiponectin, C-peptide, and aspartate aminotransferase (AST) were measured at baseline and after 12 weeks. Body composition was also measured by dual-energy x-ray absorptiometry (DEXA) and computed tomography (CT). This trial is registered under the trial registration code clinicaltrials.gov: NCT03645382 (https://register.clinicaltrials.gov).Results: Compared to the placebo, ONIRO supplementation for significantly reduced the percentage of body fat and fat mass as measured by DEXA (p = 0.028 and 0.022, respectively) with no significant effects on lean body mass. CT analyses at the L1 level showed a significant decrease in the areas of whole fat, visceral fat, and subcutaneous fat (p = 0.009, p = 0.039, p = 0.020, respectively), while CT scan of L4 resulted in a significant reduction of whole fat area and subcutaneous area (p = 0.006 and p = 0.012, respectively). The levels of triglycerides (TG) and C-peptide were significantly lower after 12 weeks of ONIRO treatment.Conclusions: These findings suggest that ONIRO supplementation reduces total body fat, notably abdominal visceral fat, with positive changes of the clinically relevant metabolic parameters serum TG and C-peptide.

Stimulation of autophagy improves vascular function in the mesenteric arteries of type 2 diabetic mice.

NEW FINDINGS: What is the central question of this study? What is the role of autophagy in vascular dysfunction in type 2 diabetes? What is the main finding and its importance? Autophagy is decreased in the mesenteric arteries of type 2 diabetic mice, and stimulation of autophagy using rapamycin and trehalose improves vascular function, which is associated with normalization of myogenic response and endothelium-dependent relaxation. ABSTRACT: Vascular dysfunction is a major complication in type 2 diabetes (T2D). It has been suggested that dysregulation of autophagy is associated with various cardiovascular diseases. However, the relationship between autophagy and vascular dysfunction in T2D remains unclear. Thus, we examined whether reduced autophagy is involved in vascular dysfunction and whether stimulation of autophagy could improve vascular function in diabetes. Ten- to twelve-week-old male type 2 diabetic (db- /db- ) mice and their control (db- /db+ ) mice were treated with rapamycin or trehalose. Mesenteric arteries (MAs) were mounted for arteriography and their diameter was measured. Western blot analysis and immunofluorescence staining were assessed. Myogenic response (MR) was significantly increased, whereas endothelium-dependent relaxation (EDR) was significantly attenuated in the MAs of diabetic mice. These results were associated with increased expression of LC3II, p62 and beclin-1 in diabetic mice. Treatment with autophagy stimulators significantly reduced the potentiation of MR and improved EDR in the diabetic mice. Furthermore, autophagy stimulation normalized expression of LC3II, p62 and beclin-1 in the diabetic mice. In addition, phosphorylation level of endothelial nitric oxide synthase was decreased in diabetic mice and was restored by rapamycin and trehalose. T2D impairs vascular function by dysregulated autophagy. Therefore, autophagy could be a potential target for overcoming diabetic microvascular complications.

Chitosan oligosaccharide (GO2KA1) improves postprandial glycemic response in subjects with impaired glucose tolerance and impaired fasting glucose and in healthy subjects: a crossover, randomized controlled trial.

BACKGROUND: The antidiabetic and hypoglycemic effects of chitosan have been reported in previous studies. We have previously shown that chitosan oligosaccharide reduces postprandial blood glucose levels in vivo. We conducted a short-term crossover study to support the results of the previous study. METHODS: The study was a randomized, double-blind, controlled crossover trial completed at one clinical research site. Subjects with impaired glucose tolerance and impaired fasting glucose and healthy subjects were randomly assigned to consume one of two different experimental test capsules that differed in only the sample source (GO2KA1 vs placebo), and all subjects were instructed to consume the 75 g sucrose within 15 min. After a 7-day interval, the subjects consumed the other capsules that were not consumed on the first day. We assessed blood glucose levels using a 2-h oral sucrose tolerance test. The study was registered at clinicaltrials.gov (NCT03650023). RESULTS: The test group showed significantly lower blood glucose levels at 60 min (p = 0.010) and postprandial blood glucose areas under the curve (p = 0.012). The change in blood glucose levels at 60 min was significantly lower in the test group than in the placebo group (p = 0.017). CONCLUSIONS: Based on the results of this study, the consumption of chitosan oligosaccharide (GO2KA1) supplements with a meal can effectively reduce postprandial blood glucose levels, which is relevant to the prevention of diabetes.

Postprandial anti-hyperglycemic effect of vitamin B6 (pyridoxine) administration in healthy individuals.

Postprandial blood glucose lowering effect of vitamin B6 (pyridoxine) was evaluated in healthy individuals with normal blood glucose levels. Blood glucose levels were measured every 30 min for 2 h after oral sugar administration with or without 50 mg of pyridoxine. Pyridoxine significantly lowered the postprandial blood glucose levels at 30 min (from 165.95 ± 17.19 to 138.36 ± 20.43, p < 0.01) and 60 min (from 131.40 ± 17.20 to 118.50 ± 15.95) after administration. In addition, the area under the concentration-time curve (AUCt) was reduced by about 8.3% (from 257.08 ± 22.38 to 235.71 ± 12.33, p < 0.05) and the maximum concentration of blood glucose (Cmax) was reduced by about 13.8% (from 165.95 ± 17.19 to 143.07 ± 11.34, p < 0.01) when compared with those of the control group. Our findings suggest that pyridoxine supplementation may be beneficial for controlling postprandial hyperglycemia.

Inhibitory Effects of Roseoside and Icariside E4 Isolated from a Natural Product Mixture (No-ap) on the Expression of Angiotensin II Receptor 1 and Oxidative Stress in Angiotensin II-Stimulated H9C2 Cells.

Hypertension is a major risk factor for the development of cardiovascular diseases. This study aimed to elucidate whether the natural product mixture No-ap (NA) containing Pine densiflora, Annona muricate, and Monordica charantia, or its single components have inhibitory effects on hypertension-related molecules in Angiotensin II (Ang II)-stimulated H9C2 cells. Individual functional components were isolated and purified from NA using various columns and solvents, and then their structures were analyzed using ESI⁻MS, ¹H-NMR, and 13H-NMR spectra. H9C2 cells were stimulated with 300 nM Ang II for 7 h. NA, telmisartan, ginsenoside, roseoside (Roseo), icariside E4 (IE4), or a combination of two components (Roseo and IE4) were administered to the cells 1 h before Ang II stimulation. The expression and activity of hypertension-related molecules or oxidative molecules were determined using RT-PCR, western blot, and ELISA. Ang II stimulation increased the expression of Ang II receptor 1 (AT1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), tumor growth factor-ß (TGF-ß) mRNA, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of hydrogen peroxide (H2O2) and superoxide anion (•O2-) and reduced anti-oxidant enzyme activity. NA significantly improved the expression or activities of all hypertension-related molecules altered in Ang II-stimulated cells. Roseo or IE4 pretreatment either decreased or increased the expression or activities of all hypertension-related molecules similar to NA, but to a lesser extent. The pretreatment with a combination of Roseo and IE4 (1:1) either decreased or increased the expression of all hypertension-related molecules, compared to each single component, revealing a synergistic action of the two compounds. Thus, the combination of single components could exert promising anti-hypertensive effects similar to NA, which should be examined in future animal and clinical studies.

Inhibition of Osteoarthritis-Related Molecules by Isomucronulatol 7-O-ß-d-glucoside and Ecliptasaponin A in IL-1ß-Stimulated Chondrosarcoma Cell Model.

Osteoarthritis (OA) is the common form of arthritis and is characterized by disability and cartilage degradation. Although natural product extracts have been reported to have anti-osteoarthritic effects, the potential bioactivity of Ryupunghwan (RPH), a traditional Korean medicinal botanical formula that contains Astragalus membranaceus, Turnera diffusa, Achyranthes bidentata, Angelica gigas, Eclipta prostrata, Eucommia ulmoides, and Ilex paraguariensis, is not known well. Therefore, the inhibitory effects of single compounds isolated from RPH on the OA-related molecules were investigated using IL-1ß-stimulated chondrosarcoma SW1353 (SW1353) cell model. Two bioactive compounds, isomucronulatol 7-O-ß-d-glucoside (IMG) and ecliptasaponin A (ES) were isolated and purified from RPH using column chromatography, and then the structures were analyzed using ESI-MS, ¹H-NMR, and 13C-NMR spectrum. The expression or amount of matrix metalloproteinase 13 (MMP13), COX1/2, TNF-α, IL-1ß or p65 was determined by RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA). RPH pretreatment reduced the expression and amounts of MMP13, and the expression of collagen II, COX1/2, TNF-α, IL-1ß or p65, which were increased in IL-1ß-stimulated SW1353 cells. IMG reduced the expression of all OA-related molecules, but the observed inhibitory effect was less than that of RPH extract. The other single compound ES showed the reduced expression of all OA-related molecules, and the effect was stronger than that in IMG (approximately 100 fold). Combination pretreatment of both single components remarkably reduced the expression of MMP13, compared to each single component. These synergic effects may provide potential molecular modes of action for the anti-osteoarthritic effects of RPH observed in clinical and animal studies.

Involvement of inhibitor kappa B kinase 2 (IKK2) in the regulation of vascular tone.

Inhibitor kappa B kinase 2 (IKK2) plays an essential role in the activation of nuclear factor kappa B (NF-kB). Recently, it has been suggested that IKK2 acts as a myosin light chain kinase (MLCK) and contributes to vasoconstriction in mouse aorta. However, the underlying mechanisms are still unknown. Therefore, we investigated whether IKK2 acts as a MLCK or regulates the activity of myosin light chain phosphatase (MLCP). Pressure myograph was used to measure vascular tone in rat mesenteric arteries. Immunofluorescence staining was performed to identify phosphorylation levels of MLC (ser19), MYPT1 (thr853 and thr696) and CPI-17 (thr38). SC-514 (IKK2 inhibitor, 50 µM) induced relaxation in the mesenteric arteries pre-contracted with 70 mM high K+ solution or U-46619 (thromboxane analog, 5 µM). The relaxation induced by SC-514 was increased in the arteries pre-contracted with U-46619 compared to arteries pre-contracted with 70 mM high K+ solution. U-46619-induced contraction was decreased by treatment of SC-514 in the presence of MLCK inhibitor, ML-7 (10 µM). In the absence of intracellular Ca2+, U-46619 still induced contraction, which was decreased by treatment of SC-514. Furthermore, phosphorylation levels of MLC (ser19) and MYPT1 (thr853) were decreased by treatment of SC-514. IKK2 is involved in the vascular contraction through regulation of MLCP activity by phosphorylating MYPT1 at thr853 in rat mesenteric arteries. These findings suggest IKK2 could be a new pharmacological target for specific therapies of various vascular diseases.

Calorie Restriction Effect of Heat-Processed Onion Extract (ONI) Using In Vitro and In Vivo Animal Models.

Onion (Allium cepa L.) is widely consumed as food or medicinal plant due to its well-defined health benefits. The antioxidant and antihyperlipidemic effects of onion and its extracts have been reported well. However, very limited information on anti-hyperglycemic effect is available in processed onion extracts. In our previous study, we reported that Amadori rearrangement compounds (ARCs) produced by heat-processing in Korean ginseng can reduce carbohydrate absorption by inhibiting intestinal carbohydrate hydrolyzing enzymes in both in vitro and in vivo animal models. To prove the enhancement of anti-hyperglycemic effect and ARCs content by heat-processing in onion extract, a correlation between the anti-hyperglycemic activity and the total content of ARCs of heat-processed onion extract (ONI) was investigated. ONI has a high content of ARCs and had high rat small intestinal sucrase inhibitory activity (0.34 ± 0.03 mg/mL, IC50) relevant for the potential management of postprandial hyperglycemia. The effect of ONI on the postprandial blood glucose increase was investigated in Sprague Dawley (SD) rats fed on sucrose or starch meals. The maximum blood glucose levels (Cmax) of heat-processed onion extract were significantly decreased by about 8.7% (from 188.60 ± 5.37 to 172.27 ± 3.96, p < 0.001) and 14.2% (from 204.04 ± 8.73 to 175.13 ± 14.09, p < 0.01) in sucrose and starch loading tests, respectively. These results indicate that ARCs in onion extract produced by heat-processing have anti-diabetic effect by suppressing carbohydrate absorption via inhibition of intestinal sucrase, thereby reducing the postprandial increase of blood glucose. Therefore, enhancement of ARCs in onion by heat-processing might be a good strategy for the development of the new product on the management of hyperglycemia.