Animal protein hydrolysate reduces visceral fat and inhibits insulin resistance and hepatic steatosis in aged mice.

BACKGROUND/OBJECTIVES: An increasing life expectancy in society has burdened healthcare systems substantially because of the rising prevalence of age-related metabolic diseases. This study compared the effects of animal protein hydrolysate (APH) and casein on metabolic diseases using aged mice. MATERIALS/METHODS: Eight-week-old and 50-week-old C57BL/6J mice were used as the non-aged (YC group) and aged controls (NC group), respectively. The aged mice were divided randomly into 3 groups (NC, low-APH [LP], and high-APH [HP] and fed each experimental diet for 12 weeks. In the LP and HP groups, casein in the AIN-93G diet was substituted with 16 kcal% and 24 kcal% APH, respectively. The mice were sacrificed when they were 63-week-old, and plasma and hepatic lipid, white adipose tissue weight, hepatic glucose, lipid, and antioxidant enzyme activities, immunohistochemistry staining, and mRNA expression related to the glucose metabolism on liver and muscle were analyzed. RESULTS: Supplementation of APH in aging mice resulted in a significant decrease in visceral fat (epididymal, perirenal, retroperitoneal, and mesenteric fat) compared to the negative control (NC) group. The intraperitoneal glucose tolerance test and area under the curve analysis revealed insulin resistance in the NC group, which was alleviated by APH supplementation. APH supplementation reduced hepatic gluconeogenesis and increased glucose utilization in the liver and muscle. Furthermore, APH supplementation improved hepatic steatosis by reducing the hepatic fatty acid and phosphatidate phosphatase activity while increasing the hepatic carnitine palmitoyltransferase activity. Furthermore, in the APH supplementation groups, the red blood cell (RBC) thiobarbituric acid reactive substances and hepatic H2O2 levels decreased, and the RBC glutathione, hepatic catalase, and glutathione peroxidase activities increased. CONCLUSIONS: APH supplementation reduced visceral fat accumulation and alleviated obesity-related metabolic diseases, including insulin resistance and hepatic steatosis, in aged mice. Therefore, high-quality animal protein APH that reduces the molecular weight and enhances the protein digestibility-corrected amino acid score has potential as a dietary supplement for healthy aging.

Role of cytosolic and endoplasmic reticulum Ca2+ in pancreatic beta-cells: pros and cons.

Pancreatic beta cells utilize Ca2+ to secrete insulin in response to glucose. The glucose-dependent increase in cytosolic Ca2+ concentration ([Ca2+]C) activates a series of insulin secretory machinery in pancreatic beta cells. Therefore, the amount of insulin secreted in response to glucose is determined in a [Ca2+]C-dependent manner, at least within a moderate range. However, the demand for insulin secretion may surpass the capability of beta cells. Abnormal elevation of [Ca2+]C levels beyond the beta-cell endurance capacity can damage them by inducing endoplasmic reticulum (ER) stress and cell death programs such as apoptosis. Therefore, while Ca2+ is essential for the insulin secretory functions of beta cells, it could affect their survival at pathologically higher levels. Because an increase in beta-cell [Ca2+]C is inevitable under certain hazardous conditions, understanding the regulatory mechanism for [Ca2+]C is important. Therefore, this review discusses beta-cell function, survival, ER stress, and apoptosis associated with intracellular and ER Ca2+ homeostasis.

The animal protein hydrolysate attenuates sarcopenia via the muscle-gut axis in aged mice.

Age-related muscle loss and dysfunction, sarcopenia, is a common condition that results in poor quality of life in the elderly. Protein supplementation is a potential strategy for preventing sarcopenia and increasing muscle synthesis, but the effectiveness of protein type and level in improving sarcopenia is not well understood. In this study, we compared animal protein hydrolysate (APH), which has a high protein digestibility-corrected amino acid score (PDCAAS) and low molecular weight, with casein as a control group to investigate the effects and mechanisms of sarcopenia improvement, with a particular focus on the gut-muscle axis. APH supplementation improved age-related declines in muscle mass, grip strength, hind leg thickness, muscle protein level, muscle fiber size, and myokine levels, compared to the control group. In particular, levels of plasma cortisol, muscle lipids, and muscle collagen were markedly reduced by APH supplements in the aged mice. Furthermore, APH efficiently recovered the concentration of total SCFAs including acetic, propionic, and isovaleric acids decreased in aged mice. Finally, APH induced changes in gut microbiota and increased production of SCFAs, which were positively correlated with muscle protein level and negatively correlated with pro-inflammatory cytokines. In conclusion, APH can help to inhibit age-related sarcopenia by increasing muscle synthesis, inhibiting muscle breakdown, and potentially modulating the gut-muscle axis.

D-Allulose Ameliorates Dysregulated Macrophage Function and Mitochondrial NADH Homeostasis, Mitigating Obesity-Induced Insulin Resistance.

D-allulose, a rare sugar, has been proposed to have potential benefits in addressing metabolic disorders such as obesity and type 2 diabetes (T2D). However, the precise mechanisms underlying these effects remain poorly understood. We aimed to elucidate the mechanisms by which D-allulose influences obesity-induced insulin resistance. We conducted gene set enrichment analysis on the liver and white adipose tissue of mice exposed to a high-fat diet (HFD) along with the white adipose tissue of individuals with obesity. Our study revealed that D-allulose effectively suppressed IFN-γ, restored chemokine signaling, and enhanced macrophage function in the livers of HFD-fed mice. This implies that D-allulose curtails liver inflammation, alleviating insulin resistance and subsequently impacting adipose tissue. Furthermore, D-allulose supplementation improved mitochondrial NADH homeostasis and translation in both the liver and white adipose tissue of HFD-fed mice. Notably, we observed decreased NADH homeostasis and mitochondrial translation in the omental tissue of insulin-resistant obese subjects compared to their insulin-sensitive counterparts. Taken together, these results suggest that supplementation with allulose improves obesity-induced insulin resistance by mitigating the disruptions in macrophage and mitochondrial function. Furthermore, our data reinforce the crucial role that mitochondrial energy expenditure plays in the development of insulin resistance triggered by obesity.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

Chronic inflammation in high-fat diet-fed mice: Unveiling the early pathogenic connection between liver and adipose tissue.

Obesity-induced chronic inflammation has been linked to several autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. The underlying mechanisms are not yet fully understood, but it is believed that chronic inflammation in adipose tissue can lead to the production of pro-inflammatory cytokines and chemokines, which can trigger immune responses and contribute to the development of autoimmune diseases. However, the underlying mechanisms that lead to the infiltration of immune cells into adipose tissue are not fully understood. In this study, we observed a time-dependent response to a high-fat diet in the liver and epididymal white adipose tissue using gene set enrichment analysis. Our findings revealed a correlation between early abnormal innate immune responses in the liver and late inflammatory response in the adipose tissue, that eventually leads to systemic inflammation. Specifically, our data suggest that the dysregulated NADH homeostasis in the mitochondrial matrix, interacting with the mitochondrial translation process, could serve as a sign marking the transition from liver inflammation to adipose tissue inflammation. Taken together, our study provides valuable insights into the molecular mechanisms underlying the development of chronic inflammation and associated autoimmune diseases in obesity.

Luteolin Protects Against Obese Sarcopenia in Mice with High-Fat Diet-Induced Obesity by Ameliorating Inflammation and Protein Degradation in Muscles.

SCOPE: Although sarcopenia is mainly caused by aging, sarcopenia due to obesity has become an emerging issue given the increase in obesity among people of various ages. There are studies on obesity or sarcopenia, our understanding of obesity-mediated sarcopenia is insufficient. Luteolin (LU) has exhibited antiobesity effects, but no studies have investigated the LU effects on antisarcopenia. This study therefore investigated the effects of LU on obese sarcopenia in mice with high-fat diet (HFD)-induced obesity. METHODS AND RESULTS: To evaluate its inhibitory efficacy against obese sarcopenia, 5-week-old mice are fed an HFD supplemented with LU for 20 weeks. LU exerts suppressive effects on obesity, inflammation, and protein degradation in the HFD-fed obese mice. It also inhibits lipid infiltration into the muscle and decreases p38 activity and the mRNA expression of inflammatory factors, including TNF-α, Tlr2, Tlr4, MCP1, and MMP2, in the muscle. The suppression of muscle inflammation by LU leads to the inhibition of myostatin, FoxO, atrogin, and MuRF expression. These effects of LU affect inhibition of protein degradation and improvement of muscle function. CONCLUSION: Here, it demonstrates that LU's antiobesity and antiinflammatory functionality affect inhibition of muscle protein degradation, and consequently, these interactions by LU exerts a protective effect against obese sarcopenia.

Oxidative stress resulting from the removal of endogenous catalase induces obesity by promoting hyperplasia and hypertrophy of white adipocytes.

Obesity is regarded as an abnormal expansion and excessive accumulation of fat mass in white adipose tissue. The involvement of oxidative stress in the development of obesity is still unclear. Although mainly present in peroxisomes, catalase scavenges intracellular H2O2 at toxic levels. Therefore, we used catalase-knockout (CKO) mice to elucidate the involvement of excessive H2O2 in the development of obesity. CKO mice with C57BL/6J background gained more weight with higher body fat mass with age than age-matched wild-type (WT) mice fed with either chow or high-fat diets. This phenomenon was attenuated by concomitant treatment with the antioxidants, melatonin or N-acetyl cysteine. Moreover, CKO mouse embryonic fibroblasts (MEFs) appeared to differentiate to adipocytes more easily than WT MEFs, showing increased H2O2 concentrations. Using 3T3-L1-derived adipocytes transfected with catalase-small interfering RNA, we confirmed that a more prominent lipogenesis occurred in catalase-deficient cells than in WT cells. Catalase-deficient adipocytes presented increased nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression but decreased adenosine monophosphate-activated protein kinase (AMPK) expression. Treatment with a NOX4 inhibitor or AMPK activator rescued the propensity for obesity of CKO mice. These findings suggest that excessive H2O2 and related oxidative stress increase body fat mass via both adipogenesis and lipogenesis. Manipulating NOX4 and AMPK in white adipocytes may be a therapeutic tool against obesity augmented by oxidative stress.

Orexin A-induced inhibition of leptin expression and secretion in adipocytes reducing plasma leptin levels and hypothalamic leptin resistance.

Orexin A (OXA) is a neuropeptide associated with plasma insulin and leptin levels involved in body weight and appetite regulation. However, little is known about the effect of OXA on leptin secretion in adipocytes and its physiological roles. Leptin secretion and expression were analysed in 3T3-L1 adipocytes. Plasma leptin, adiponectin and insulin levels were measured by ELISA assay. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the hypothalamus were evaluated by western blotting. OXA dose-dependently suppressed leptin secretion from 3T3-L1 adipocytes by inhibiting its gene expression while facilitating adiponectin secretion. The leptin inhibition by OXA was mediated via orexin receptors (OXR1 and OXR2). In addition to the pathway via extracellular signal-regulated kinases, OXA triggered adenylyl cyclase-induced cAMP elevation, which results in protein kinase A-mediated activation of cAMP response element-binding proteins (CREB). Accordingly, CREB inhibition restored the OXA-induced downregulation of leptin gene expression and secretion. Exogenous OXA for 4 weeks decreased fasting plasma leptin levels and increased hypothalamic pSTAT3 levels in high-fat diet-fed mice, regardless of increase in body weight and food intake. These results suggest that high dose of OXA directly inhibits leptin mRNA expression and thus secretion in adipocytes, which may be a peripheral mechanism of OXA for its role in appetite drive during fasting. It may be also critical for lowering basal plasma leptin levels and thus maintaining postprandial hypothalamic leptin sensitivity.

Ablation of catalase promotes non-alcoholic fatty liver via oxidative stress and mitochondrial dysfunction in diet-induced obese mice.

Hydrogen peroxide (H2O2) produced endogenously can cause mitochondrial dysfunction and metabolic complications in various cell types by inducing oxidative stress. In the liver, oxidative and endoplasmic reticulum (ER) stress affects the development of non-alcoholic fatty liver disease (NAFLD). Although a link between both stresses and fatty liver diseases has been suggested, few studies have investigated the involvement of catalase in fatty liver pathogenesis. We examined whether catalase is associated with NAFLD, using catalase knockout (CKO) mice and the catalase-deficient human hepatoma cell line HepG2. Hepatic morphology analysis revealed that the fat accumulation was more prominent in high-fat diet (HFD) CKO mice compared to that in age-matched wild-type (WT) mice, and lipid peroxidation and H2O2 release were significantly elevated in CKO mice. Transmission electron micrographs indicated that the liver mitochondria from CKO mice tended to be more severely damaged than those in WT mice. Likewise, mitochondrial DNA copy number and cellular ATP concentrations were significantly lower in CKO mice. In fatty acid-treated HepG2 cells, knockdown of catalase accelerated cellular lipid accumulation and depressed mitochondrial biogenesis, which was recovered by co-treatment with N-acetyl cysteine or melatonin. This effect of antioxidant was also true in HFD-fed CKO mice, suppressing fatty liver development and improving hepatic mitochondrial function. Expression of ER stress marker proteins and hepatic fat deposition also increased in normal-diet, aged CKO mice compared to WT mice. These findings suggest that H2O2 production may be an important event triggering NAFLD and that catalase may be an attractive therapeutic target for preventing NAFLD.

Ursolic Acid Attenuates Hepatic Steatosis, Fibrosis, and Insulin Resistance by Modulating the Circadian Rhythm Pathway in Diet-Induced Obese Mice.

The aim of the current study was to elucidate the effects of long-term supplementation with dietary ursolic acid (UR) on obesity and associated comorbidities by analyzing transcriptional and metabolic responses, focusing on the role of UR in the modulation of the circadian rhythm pathway in particular. C57BL/6J mice were divided into three groups and fed a normal diet, high-fat diet, or high-fat + 0.05% (w/w) UR diet for 16 weeks. Oligonucleotide microarray profiling revealed that UR is an effective regulator of the liver transcriptome, and canonical pathways associated with the "circadian rhythm" and "extracellular matrix (ECM)⁻receptor interactions" were effectively regulated by UR in the liver. UR altered the expression of various clock and clock-controlled genes (CCGs), which may be linked to the improvement of hepatic steatosis and fibrosis via lipid metabolism control and detoxification enhancement. UR reduced excessive reactive oxygen species production, adipokine/cytokine dysregulation, and ECM accumulation in the liver, which also contributed to improve hepatic lipotoxicity and fibrosis. Moreover, UR improved pancreatic islet dysfunction, and suppressed hepatic gluconeogenesis, thereby reducing obesity-associated insulin resistance. Therapeutic approaches targeting hepatic circadian clock and CCGs using UR may ameliorate the deleterious effects of diet-induced obesity and associated complications such as hepatic fibrosis.

Catalase and nonalcoholic fatty liver disease.

Obesity and insulin resistance are considered the main causes of nonalcoholic fatty liver disease (NAFLD), and oxidative stress accelerates the progression of NAFLD. Free fatty acids, which are elevated in the liver by obesity or insulin resistance, lead to incomplete oxidation in the mitochondria, peroxisomes, and microsomes, leading to the production of reactive oxygen species (ROS). Among the ROS generated, H2O2 is mainly produced in peroxisomes and decomposed by catalase. However, when the H2O2 concentration increases because of decreased expression or activity of catalase, it migrates to cytosol and other organelles, causing cell injury and participating in the Fenton reaction, resulting in serious oxidative stress. To date, numerous studies have been shown to inhibit the pathogenesis of NAFLD, but treatment for this disease mainly depends on weight loss and exercise. Various molecules such as vitamin E, metformin, liraglutide, and resveratrol have been proposed as therapeutic agents, but further verification of the dose setting, clinical application, and side effects is needed. Reducing oxidative stress may be a fundamental method for improving not only the progression of NAFLD but also obesity and insulin resistance. However, the relationship between NAFLD progression and antioxidants, particularly catalase, which is most commonly expressed in the liver, remains unclear. Therefore, this review summarizes the role of catalase, focusing on its potential therapeutic effects in NAFLD progression.

Epac2a-knockout mice are resistant to dexamethasone-induced skeletal muscle atrophy and short-term cold stress.

Exchange protein directly activated by cAMP (Epac) 2a-knockout (KO) mice exhibit accelerated diet-induced obesity and are resistant to leptin-mediated adipostatic signaling from the hypothalamus to adipose tissue, with sustained food intake. However, the impact of Epac2a deficiency on hypothalamic regulation of sympathetic nervous activity (SNA) has not been elucidated. This study was performed to elucidate the response of Epac2a-KO mice to dexamethasone-induced muscle atrophy and acute cold stress. Compared to age-matched wild-type mice, Epac2a-KO mice showed higher energy expenditures and expression of myogenin and uncoupling protein-1 in skeletal muscle (SM) and brown adipose tissue (BAT), respectively. Epac2a-KO mice exhibited greater endurance to dexamethasone and cold stress. In wild-type mice, exogenous leptin mimicked the responses observed in Epac2a-KO mice. This suggests that leptin-mediated hypothalamic signaling toward SNA appears to be intact in these mice. Hence, the potentiated responses of SM and BAT may be due to their high plasma leptin levels. [BMB Reports 2018; 51(1): 39-44].

Tomatidine inhibits tumor necrosis factor-α-induced apoptosis in C2C12 myoblasts via ameliorating endoplasmic reticulum stress.

In this study, we examined the effect of tomatidine on tumor necrosis factor (TNF)-α-induced apoptosis in C2C12 myoblasts. TNF-α treatment increased cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) protein levels in a dose- and time-dependent manner. Pretreatment of cells with 10 µM tomatidine prevented TNF-α-induced apoptosis, caspase 3 cleavage, and PARP cleavage. Cells were treated with 100 ng/mL TNF-α for 24 h, and flow cytometry was utilized to assess apoptosis using annexin-V and 7-aminoactinomycin D. TNF-α up-regulated activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression. This effect was suppressed by pretreatment with tomatidine. Pretreatment with 4-phenylbutyric acid (a chemical chaperone) also inhibited TNF-α-induced cleavage of caspase 3 and PARP and up-regulation of ATF4 and CHOP expression. In addition, tomatidine-mediated inhibition of phosphorylation of c-Jun amino terminal kinase (JNK) attenuated TNF-α-induced cleavage of PARP and caspase 3. However, tomatidine did not affect NF-κB activation in TNF-α-treated C2C12 myoblast cells. Taken together, the present study demonstrates that tomatidine attenuates TNF-α-induced apoptosis through down-regulation of CHOP expression and inhibition of JNK activation.

A Model-Based Joint Identification of Differentially Expressed Genes and Phenotype-Associated Genes.

Over the last decade, many analytical methods and tools have been developed for microarray data. The detection of differentially expressed genes (DEGs) among different treatment groups is often a primary purpose of microarray data analysis. In addition, association studies investigating the relationship between genes and a phenotype of interest such as survival time are also popular in microarray data analysis. Phenotype association analysis provides a list of phenotype-associated genes (PAGs). However, it is sometimes necessary to identify genes that are both DEGs and PAGs. We consider the joint identification of DEGs and PAGs in microarray data analyses. The first approach we used was a naïve approach that detects DEGs and PAGs separately and then identifies the genes in an intersection of the list of PAGs and DEGs. The second approach we considered was a hierarchical approach that detects DEGs first and then chooses PAGs from among the DEGs or vice versa. In this study, we propose a new model-based approach for the joint identification of DEGs and PAGs. Unlike the previous two-step approaches, the proposed method identifies genes simultaneously that are DEGs and PAGs. This method uses standard regression models but adopts different null hypothesis from ordinary regression models, which allows us to perform joint identification in one-step. The proposed model-based methods were evaluated using experimental data and simulation studies. The proposed methods were used to analyze a microarray experiment in which the main interest lies in detecting genes that are both DEGs and PAGs, where DEGs are identified between two diet groups and PAGs are associated with four phenotypes reflecting the expression of leptin, adiponectin, insulin-like growth factor 1, and insulin. Model-based approaches provided a larger number of genes, which are both DEGs and PAGs, than other methods. Simulation studies showed that they have more power than other methods. Through analysis of data from experimental microarrays and simulation studies, the proposed model-based approach was shown to provide a more powerful result than the naïve approach and the hierarchical approach. Since our approach is model-based, it is very flexible and can easily handle different types of covariates.

Phlorizin Supplementation Attenuates Obesity, Inflammation, and Hyperglycemia in Diet-Induced Obese Mice Fed a High-Fat Diet.

Obesity, along with its related complications, is a serious health problem worldwide. Many studies reported the anti-diabetic effect of phlorizin, while little is known about its anti-obesity effect. We investigated the beneficial effects of phlorizin on obesity and its complications, including diabetes and inflammation in obese animal. Male C57BL/6J mice were divided into three groups and fed their respective experimental diets for 16 weeks: a normal diet (ND, 5% fat, w/w), high-fat diet (HFD, 20% fat, w/w), or HFD supplemented with phlorizin (PH, 0.02%, w/w). The findings revealed that the PH group had significantly decreased visceral and total white adipose tissue (WAT) weights, and adipocyte size compared to the HFD. Plasma and hepatic lipids profiles also improved in the PH group. The decreased levels of hepatic lipids in PH were associated with decreased activities of enzymes involved in hepatic lipogenesis, cholesterol synthesis and esterification. The PH also suppressed plasma pro-inflammatory adipokines levels such as leptin, adipsin, tumor necrosis factor-α, monocyte chemoattractant protein-1, interferon-γ, and interleukin-6, and prevented HFD-induced collagen accumulation in the liver and WAT. Furthermore, the PH supplementation also decreased plasma glucose, insulin, glucagon, and homeostasis model assessment of insulin resistance levels. In conclusion, phlorizin is beneficial for preventing diet-induced obesity, hepatic steatosis, inflammation, and fibrosis, as well as insulin resistance.

Evidence for glucagon-like peptide-1 receptor signaling to activate ATP-sensitive potassium channels in pancreatic beta cells.

Glucagon-like peptide-1 (GLP-1) is a gut peptide that promotes insulin release from pancreatic beta cells. GLP-1 has been shown to confer glucose-insensitive beta cells with glucose sensitivity by modulation of the activity of the ATP-sensitive potassium (KATP) channel. The channel closing effect of GLP-1, interacting with corresponding G-protein-coupled receptors, has been well established; however, to our knowledge, no study has shown whether GLP-1 directly induces activation of beta-cell KATP channels. Here, we aimed to evaluate whether the activation of beta-cell KATP channels by GLP-1 exists and affects intracellular Ca(2+) levels ([Ca(2+)]i). KATP channel activity was measured in isolated rat pancreatic beta cells by whole-cell perforated patch-clamp recordings with a diazoxide-containing pipette solution. Changes in [Ca(2+)]i and the subcellular localization of KATP channels were observed using the calcium-sensitive dye fura-4/AM and anti-Kir6.2 antibodies in INS-1 beta cells, respectively. To eliminate the well-known inhibitory effects of GLP-1 on KATP channel activity, channels were fully inhibited by pretreatment with methyl pyruvate and epigallocatechin-3-gallate. In the pretreated beta cells, GLP-1 and exendin-4 promptly activated the channels, reducing [Ca(2+)]i. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocked the effects of GLP-1 on channel activity. Moreover, phosphatidylinositol-3,4,5-trisphosphate mimicked the effects of GLP-1. These results suggested that beta-cell GLP-1 receptor signaling involved activation of KATP channels via a PI3K-dependent pathway. This alternative mechanism of GLP-1 function may act as a negative feedback pathway, modulating the glucose-dependent GLP-1 inhibition on KATP channel activity.

A nutrigenomic framework to identify time-resolving responses of hepatic genes in diet-induced obese mice.

Obesity and its related complications have emerged as global health problems; however, the pathophysiological mechanism of obesity is still not fully understood. In this study, C57BL/6J mice were fed a normal (ND) or high-fat diet (HFD) for 0, 2, 4, 6, 8, 12, 20, and 24 weeks and the time course was systemically analyzed specifically for the hepatic transcriptome profile. Genes that were differentially expressed in the HFD-fed mice were clustered into 49 clusters and further classified into 8 different expression patterns: long-term up-regulated (pattern 1), long-term downregulated (pattern 2), early up-regulated (pattern 3), early down-regulated (pattern 4), late up-regulated (pattern 5), late down-regulated (pattern 6), early up-regulated and late down-regulated (pattern 7), and early down-regulated and late up-regulated (pattern 8) HFD-responsive genes. Within each pattern, genes related with inflammation, insulin resistance, and lipid metabolism were extracted, and then, a protein-protein interaction network was generated. The pattern specific sub-network was as follows: pattern 1, cellular assembly and organization, and immunological disease, pattern 2, lipid metabolism, pattern 3, gene expression and inflammatory response, pattern 4, cell signaling, pattern 5, lipid metabolism, molecular transport, and small molecule biochemistry, pattern 6, protein synthesis and cell-to cell signaling and interaction and pattern 7, cell-to cell signaling, cellular growth and proliferation, and cell death. For pattern 8, no significant sub-networks were identified. Taken together, this suggests that genes involved in regulating gene expression and inflammatory response are up-regulated whereas genes involved in lipid metabolism and protein synthesis are down-regulated during diet-induced obesity development.

Randomized double-blind placebo-controlled trial of powdered Brassica rapa ethanol extract on alteration of body composition and plasma lipid and adipocytokine profiles in overweight subjects.

We evaluated the effects of Brassica rapa ethanol extract (BREE) on body composition and plasma lipid profiles through a randomized, double-blind, and placebo-controlled trial in overweight subjects. Fifty-eight overweight participants (age 20-50 years, body mass index23.0-24.9) were randomly assigned to two groups and served BREE (2 g/day) or placebo (starch, 2 g/day) for 10 weeks. Body compositions, nutrients intake, plasma lipids, adipocytokines, and hepatotoxicity biomarkers were assessed in all subjects at baseline and after 10 weeks of supplementation. The plasma total cholesterol (total-C) concentration was significantly increased after 10 weeks compared to the baseline in both groups. However, BREE supplementation significantly increased the high-density lipoprotein cholesterol (HDL-C) concentration and significantly reduced the total-C/HDL-C ratio, free fatty acid, and adipsin levels after 10 weeks. No significant differences were observed in body compositions, fasting blood glucose, plasma adipocytokines except adipsin, and aspartate aminotransferase and alanine aminotransferase activities between before and after trial within groups as well as between the two groups. The supplementation of BREE partially improves plasma lipid metabolism in overweight subjects without adverse effects.

Time-dependent network analysis reveals molecular targets underlying the development of diet-induced obesity and non-alcoholic steatohepatitis.

Prolonged high-fat diet leads to the development of obesity and multiple comorbidities including non-alcoholic steatohepatitis (NASH), but the underlying molecular basis is not fully understood. We combine molecular networks and time course gene expression profiles to reveal the dynamic changes in molecular networks underlying diet-induced obesity and NASH. We also identify hub genes associated with the development of NASH. Core diet-induced obesity networks were constructed using Ingenuity pathway analysis (IPA) based on 332 high-fat diet responsive genes identified in liver by time course microarray analysis (8 time points over 24 weeks) of high-fat diet-fed mice compared to normal diet-fed mice. IPA identified five core diet-induced obesity networks with time-dependent gene expression changes in liver. These networks were associated with cell-to-cell signaling and interaction (Network 1), lipid metabolism (Network 2), hepatic system disease (Network 3 and 5), and inflammatory response (Network 4). When we merged these core diet-induced obesity networks, Tlr2, Cd14, and Ccnd1 emerged as hub genes associated with both liver steatosis and inflammation and were altered in a time-dependent manner. Further, protein-protein interaction network analysis revealed Tlr2, Cd14, and Ccnd1 were interrelated through the ErbB/insulin signaling pathway. Dynamic changes occur in molecular networks underlying diet-induced obesity. Tlr2, Cd14, and Ccnd1 appear to be hub genes integrating molecular interactions associated with the development of NASH. Therapeutics targeting hub genes and core diet-induced obesity networks may help ameliorate diet-induced obesity and NASH.