All lactose-oxidizing enzymes of Pseudomonas taetrolens, a highly efficient lactobionic acid-producing microorganism, are pyrroloquinoline quinone-dependent enzymes.

In previous and present studies, four enzymes (GCD1, GCD3, GCD4, and MQO1) have been found to act as lactose-oxidizing enzymes of Pseudomonas taetrolens. To investigate whether the four enzymes were the only lactose-oxidizing enzymes of P. taetrolens, we performed the inactivation of gcd1, gcd3, gcd4, and mqo1 genes in P. taetrolens. Compared to the wild-type strain, the lactobionic acid (LBA)-producing ability of P. taetrolens ∆gcd1 ∆gcd3 ∆gcd4 ∆mqo1 was only slightly decreased, implying that P. taetrolens possesses more lactose-oxidizing enzymes. Interestingly, the four lactose-oxidizing enzymes were all pyrroloquinoline quinone (PQQ)-dependent. To identify other unidentified lactose-oxidizing enzymes of P. taetrolens, we prevented the synthesis of PQQ in P. taetrolens by inactivating the genes related to PQQ synthesis such as pqqC, pqqD, and pqqE. Surprisingly, all three knocked-out strains were unable to convert lactose to LBA, indicating that all lactose-oxidizing enzymes in P. taetrolens were inactivated by eliminating PQQ synthesis. In addition, external PQQ supplementation restored the LBA production ability of P. taetrolens ∆pqqC, comparable to the wild-type strain. These results indicate that all lactose-oxidizing enzymes in P. taetrolens are PQQ-dependent.

Malting barley carbon dots-mediated oxidative stress promotes insulin resistance in mice via NF-κB pathway and MAPK cascade.

BACKGROUND: Food-borne carbon dots (CDs) are widely generated during food processing and are inevitably ingested by humans causing toxicity. However, the toxic effects of food-borne CDs on the blood glucose metabolism are unknown. RESULTS: In this study, we brewed beer via a representative strategy and extracted the melting-barley CDs (MBCDs) to explore the toxic effects on blood glucose in mice. We found the accumulation of fluorescent labeled MBCDs in various organs and oral administration of MBCDs can cause visceral toxicity, manifested as liver damage. Mice were orally administered MBCDs (5 and 25 mg/kg) for 16 weeks, and increased levels of fasting blood glucose were observed in both MBCDs-treated groups. Transcriptomic analyses revealed that MBCDs activate oxidative stress, inflammatory responses, the MAPK cascade, and PI3K/Akt signaling in mice livers. Mechanistically, MBCDs exposure-induced reactive oxygen species (ROS) overproduction activates the nuclear factor-κB (NF-κB) signaling pathway and MAPK cascade, thereby promoting phosphorylated insulin receptor substrate (IRS)-1 at Ser307 and inducing insulin resistance (IR). Meanwhile, the IR promoted gluconeogenesis, which enhanced MBCDs-induced hyperglycemia of mice. Importantly, inhibition of the ROS significantly attenuated the MBCDs-induced inflammatory response and MAPK cascade, thereby alleviating IR and hyperglycemia in mice. CONCLUSION: In summary, this study revealed that MBCDs promote ROS overproduction and thus induced IR, resulting in imbalance of glucose homeostasis in mice. More importantly, this study was further assessed to reveal an imperative emphasis on the reevaluation of dietary and environmental CDs exposure, and has important implications for T2DM prevention research.

Efficient isolation of new lactobionic acid-producing microorganisms from environmental samples by colloidal calcium carbonate agar plate-based screening.

Lactobionic acid (LBA) has recently emerged as an important substance in various industries, such as cosmetics, foods, and pharmaceuticals. In this study, we developed a simple, efficient, and high-throughput method for screening LBA-producing microorganisms. First, an agar plate was prepared to isolate LBA-producing microorganisms by utilizing the property of LBA to solubilize colloidal calcium carbonate (CaCO3), resulting in the formation of a clear halo around colonies on a nutrient broth agar plate containing CaCO3. Subsequently, LBA production from the isolated microorganisms was confirmed using high-performance liquid chromatography (HPLC). Approximately 560 colonies from soil samples in Ulsan, Korea were screened and a clear halo was observed around three colonies on the prepared LBA-screening agar plate. The culture supernatants of these three colonies were analyzed by HPLC and it was found that these strains could produce LBA from lactose. Phylogenetic analysis by comparing their 16S rRNA nucleotide sequences revealed that these strains were Pseudomonas spp. and Alcaligenes faecalis. This is the first report highlighting that A. faecalis can produce LBA. As per the aforementioned results, the LBA-screening method that we devised here is highly effective for isolating and identifying new LBA-producing microorganisms.

Efficient production of cellobionic acid using whole-cell biocatalyst of genetically modified Pseudomonas taetrolens.

Pseudomonas taetrolens has previously been shown to convert cellobiose to cellobionic acid (CBA), which can potentially be used in cosmetics, food, and pharmaceutical industries. The cellobiose-oxidizing activity of the P. taetrolens strain, which expressed the homologous quinoprotein glucose dehydrogenase (GDH), was increased by approximately 50.8% compared to the original strain. Whole-cell biocatalyst (WCB) of the genetically modified P. taetrolens strain [pDSK-GDH] was prepared simply by fermentation and washing processes. Reaction conditions for the proper use of WCB, such as reaction temperature, cell density to be added, and cell harvest time for preparing WCB, were investigated. The highest CBA productivity (18.2 g/L/h) was achieved when WCB prepared in the late-exponential phase of cell culture was used at 35 °C with cell density of 10 at OD600nm. Under these conditions, 200 g/L of cellobiose was all converted to CBA in 11 h, and the WCB of P. taetrolens [pDSK-GDH] maintained the maximum catalytic activity during at least six cycles without a significant decline in the productivity. Our results suggest that the manufacture of WCB based on genetically engineered P. taetrolens and its optimized use could be further developed as an economically viable option for the large-scale production of CBA.

Whole-cell biocatalysis using genetically modified Pseudomonas taetrolens for efficient production of maltobionic acid from pure maltose and high-maltose corn syrup.

Maltobionic acid (MBA) can be applied to various fields such as food, cosmetics, and pharmaceutical industries. In this study, whole-cell biocatalysis for MBA production was performed using recombinant Pseudomonas taetrolens homologously expressing quinoprotein glucose dehydrogenase (GDH). Various reaction parameters such as temperature, cell density, and cell harvest time, were optimized for improving MBA production. Under the optimized reaction conditions using pure maltose as a substrate, the MBA production titer, yield, and productivity of whole-cell biocatalyst (WCB) were 200 g/L, 95.6%, and 18.18 g/L/h, respectively, which were the highest compared to those reported previously. Productivity, a key factor for industrial MBA production, obtained from whole-cell biocatalysis in this study, was enhanced by approximately 1.9-fold compared to that obtained in our previous work (9.52 g/L/h) using the fermentation method. Additionally, the WCB could be reused up to six times without a significant reduction in MBA productivity, indicating that the WCB is very robust. Although MBA productivity (8.33 g/L/h) obtained from high-maltose corn syrup (HMCS) as a substrate was 45.8% of that using pure maltose, HMCS can be a better substrate for commercial MBA production because its price is only 1.1% of that of pure maltose. The results of this study using a WCB to convert maltose into MBA may support the development of a potential industrial process for more economically effective MBA production in the future.

Secretory production of an engineered cutinase in Bacillus subtilis for efficient biocatalytic depolymerization of polyethylene terephthalate.

Polyethylene terephthalate (PET) waste has caused serious environmental pollution. Recently, PET depolymerization by enzymes with PET-depolymerizing activity has received attention as a solution to recycle PET. An engineered variant of leaf-branch compost cutinase (293 amino acid), ICCG (Phe243Ile/Asp238Cys/Ser283Cys/Tyr127Gly), showed excellent depolymerizing activity toward PET at 72 °C, which was the highest depolymerizing activity and thermo-stability ever reported in previous works. However, this enzyme was only produced by heterologous expression in the cytoplasm of Escherichia coli, which requires complex separation and purification steps. To simplify the purification steps of ICCG, we developed a secretory production system using Bacillus subtilis and its 174 types of N-terminal signal peptides. The recombinant strain expressing ICCG with the signal peptide of serine protease secreted the highest amount (9.4 U/mL) of ICCG. We improved the production of ICCG up to 22.6 U/mL (85 µg/mL) by performing batch fermentation of the selected strain in 2 L working volume using a 5-L fermenter, and prepared the crude ICCG solution by concentrating the culture supernatant. The recombinant ICCG successfully depolymerized a PET film with 37% crystallinity at 37 °C and 70 °C. In this study, we developed a secretory production system of the engineered cutinase with PET-depolymerizing activity to obtain high amounts of the enzyme by a relatively simple purification method. This system will contribute to the recycling of PET waste via a more efficient and environmentally friendly method based on enzymes with PET-depolymerizing activity.

Enhancement of sophorolipids production in Candida batistae, an unexplored sophorolipids producer, by fed-batch fermentation.

Sophorolipids (SLs) from Candida batistae has a unique structure that contains ω-hydroxy fatty acids, which can be used as a building block in the polymer and fragrance industries. To improve the production of this industrially important SLs, we optimized the culture medium of C. batistae for the first time. Using an optimized culture medium composed of 50 g/L glucose, 50 g/L rapeseed oil, 5 g/L ammonium nitrate and 5 g/L yeast extract, SLs were produced at a concentration of 24.1 g/L in a flask culture. Sophorolipids production increased by about 19% (28.6 g/L) in a fed-batch fermentation using a 5 L fermentor. Sophorolipids production more increased by about 121% (53.2 g/L), compared with that in a flask culture, in a fed-batch fermentation using a 50 L fermentor, which was about 787% higher than that of the previously reported SLs production (6 g/L). These results indicate that a significant increase in C. batistae-derived SLs production can be achieved by optimization of the culture medium composition and fed-batch fermentation. Finally, we successfully separated and purified the SLs from the culture medium. The improved production of SLs from C. batistae in this study will help facilitate the successful development of applications for the SLs.

The toxic effects of titanium dioxide nanoparticles on plasma glucose metabolism are more severe in developing mice than in adult mice.

Titanium dioxide nanoparticles (TiO2 NPs) are authorized food additives, and children have the highest exposure. Therefore, children are likely more susceptible to the adverse effects of TiO2 NPs than adults. Previous study showed that oral administration of 50 mg/kg body weight (bw) TiO2 NPs increase plasma glucose in mice. However, few studies have directly compared the adverse effects of exposure to TiO2 NPs on plasma glucose metabolism of different age groups. In this study, the developing (age 3 weeks) and adult mice (age 10 weeks) were orally administered with 50 mg/kg bw TiO2 NPs per day. The TiO2 NPs induced hyperglycemia earlier in the developing mice than in the adult mice. Then mechanisms were analyzed after mice were oral administration of TiO2 NPs for 8 weeks and 26 weeks, respectively. Results showed that the treatment with TiO2 NPs activated xenobiotic biodegradation in livers of both developing and adult mice at the early stage. However, only in the developing mice, TiO2 NPs induced endoplasmic reticulum (ER) stress in livers and increased reactive oxygen species in livers and sera in the early stage. The ER stress and ROS activated an inflammation response and mitogen-activated protein kinase pathways, thereby inducing insulin resistance in the livers of developing mice at the early stage. The response of the adult mice was delayed, and these changes were observed in the late stage of the study. The results of this study all suggest that children are more susceptible than adults to the toxicity of orally administered TiO2 NPs.

C/EBPß mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes.

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPß gene. Notably, C/EBPß also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPß and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPß alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPß binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPß. C/EBPß binds directly to the promoter of the musclin gene and upregulates its expression.

The saturated fatty acid palmitate induces insulin resistance through Smad3-mediated down-regulation of FNDC5 in myotubes.

Elevated plasma free fatty acid (FFA) levels are associated with insulin resistance and can cause lipotoxicity in skeletal muscles. In response to FFAs, skeletal muscle can secrete a variety of cytokines. Irisin, one such muscle-secreted cytokine, can improve glucose tolerance, glucose uptake, and lipid metabolism. It is produced by the transmembrane protein fibronectin type Ⅲ domain containing 5 (FNDC5) by specific proteases. The purpose of this study was to investigate the regulatory mechanisms of the FNDC5 response to palmitate and their relationships with insulin resistance in C2C12 myotubes. RNA sequencing analysis results from C2C12 myotubes treated with palmitate showed that palmitate could activate the TGF-ß signaling pathway. Palmitate directly affected the expression of Smad3, but not its phosphorylation level, in C2C12 myotubes. Furthermore, knockdown and knockout of Smad3 alleviated the inhibitory effect of palmitate on the expression of FNDC5. In contrast, overexpression of Smad3 aggravated the inhibition of FNDC5 expression. There is a Smad3 binding motif in the -660 bp to -649 bp region of the Fndc5 promoter. CRISPR/Cas9 knockout of this region also alleviated the inhibition of FNDC5 expression in response to palmitate. More importantly, inhibition of FNDC5 expression mediated by Smad3 led to a decrease in insulin sensitivity in C2C12 myotubes. Collectively, these findings suggest that palmitate could induce insulin resistance through Smad3-mediated down-regulation of the Fndc5 gene.

Silicon dioxide nanoparticles induce insulin resistance through endoplasmic reticulum stress and generation of reactive oxygen species.

BACKGROUND: Silicon dioxide nanoparticles (SiO2 NPs) are one of the most widely utilized NPs in various food sectors. However, the potential endocrine toxicity of SiO2 NPs has not been characterized. RESULTS: In the present study, mice were orally administered a series of doses of SiO2 NPs. All doses of SiO2 NPs were absorbed into the blood, liver, and pancreas of the mice. Administration of 100 mg/kg bw (body weight) of SiO2 NPs significantly increased blood glucose levels in mice. However, the same dose of SiO2 fine-particles (FPs) did not result in altered blood glucose. Whole-genome analysis showed that SiO2 NPs affected the expression of genes associated with reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stress. In addition, we showed that SiO2 NPs activated xenobiotic metabolism, resulting in ER stress. Endoplasmic reticulum stress resulted in increased ROS production, which activated the NF-κB pathway leading to expression of inflammatory cytokines. Increased inflammatory cytokine expression resulted in serine phosphorylation of IRS1, which induced insulin resistance (IR). Furthermore these inflammatory cytokines activated the MAPK pathway, which further promoted the serine phosphorylation of IRS1. Insulin resistance resulted in elevated blood glucose. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) inhibited SiO2 NP-induced ROS production. The ROS scavenger N-acetylcysteine (NAC) did not affect SiO2 NP-induced ER stress, but inhibited SiO2 NP-induced activation of the NF-κB and MAPK pathways, expression of inflammatory cytokines, SiO2 NP-induced serine phosphorylation of IRS1, and SiO2 NP-induced elevations of blood glucose. CONCLUSION: Silicon dioxide NPs induced IR through ER stress and generation of ROS, but SiO2 FPs did not. Therefore, lifelong exposure of humans to SiO2 NPs may result in detrimental effects on blood glucose. The results of this study strongly suggested that non-nanoformed SiO2 should be used as food additives.

Mechanisms of titanium dioxide nanoparticle-induced oxidative stress and modulation of plasma glucose in mice.

Titanium dioxide nanoparticles (TiO2 NPs) are reported to increase plasma glucose levels in mice at specific doses. The production and accumulation of reactive oxygen species (ROS) is potentially the most important factor underlying the biological toxicity of TiO2 NPs but the underlying mechanisms are unclear at present. Data from genome-wide analyses showed that TiO2 NPs induce endoplasmic reticulum (ER) stress and ROS generation, leading to the inference that TiO2 NP-induced ER stress contributes to enhancement of ROS in mice. Resveratrol (Res) effectively relieved TiO2 NP-induced ER stress and ROS generation by ameliorating expression of a common set of activated genes for both processes, signifying that ER stress and ROS are closely related. TiO2 NP-induced ER stress occurred earlier than ROS generation. Upon treatment with 4-phenylbutyric acid to relieve ER stress, plasma glucose levels tended toward normal and TiO2 NP increased ROS production was inhibited. These results suggest that TiO2 NP-induced ER stress promotes the generation of ROS, in turn, triggering increased plasma glucose levels in mice. In addition, Res that displays the ability to reduce ER stress presents a dietary polyphenol antioxidant that can effectively prevent the toxicological effects of TiO2 NPs on plasma glucose metabolism.

Hypoxia in 3T3-L1 adipocytes suppresses adiponectin expression via the PERK and IRE1 unfolded protein response.

Adiponectin, an adipocytokine produced by adipocytes, functions as an anti-inflammatory and anti-apoptotic substance, while also enhancing insulin sensitivity. Patients or model animals with obesity or diabetes typically present attenuated expression of adiponectin. Moreover, obesity and diabetes are often accompanied with hypoxia in adipose tissue, which may result in endoplasmic reticulum (ER) stress as well as low expression of adiponectin. The purpose of this study was to investigate the specific role of the unfolded protein response (UPR) involved in the low expression of adiponectin induced by hypoxia. Subjecting 3T3-L1 adipocytes to hypoxia significantly reduced adiponectin expression and activated the PERK and IRE1 signaling pathways in a time-dependent manner. The ATF6 signaling pathway showed no obvious changes with hypoxia treatment under a similar time course. Moreover, the down-regulated expression of adiponectin induced by hypoxia was relieved once the PERK and IRE1 signaling pathways were suppressed by the inhibitors GSK2656157 and 4µ8C, respectively. Overall, these data demonstrate that hypoxia can suppress adiponectin expression and activate the PERK and IRE1 signaling pathways in differentiated adipocytes, and this two pathways are involved in the suppression of adiponectin expression induced by hypoxia.

Ginsenoside Re improves skin barrier function in HaCaT keratinocytes under normal growth conditions.

Ginsenoside Re (Re), a major ginsenoside of ginseng, enhanced the cornified cell envelope (CE) formation in HaCaT keratinocytes under normal conditions. In HaCaT keratinocytes, Re was also able to upregulate filaggrin protein and caspase-14 activity in a concentration-dependent manner. These findings reasonably imply that Re possesses a desirable property of improving skin barrier function.

Protective properties of ginsenoside Rb3 against UV-B radiation-induced oxidative stress in HaCaT keratinocytes.

This work aimed to evaluate the skin anti-photoaging properties of ginsenoside Rb3 (Rb3), one of the main protopanaxdiol-type ginsenosides from ginseng, in HaCaT keratinocytes. The skin anti-photoaging activity was assessed by analyzing the levels of reactive oxygen species (ROS), pro-matrix metalloproteinase-2 (proMMP-2), pro-matrix metalloproteinase-9 (proMMP-9), total glutathione (GSH), and superoxide dismutase (SOD) activity as well as cell viability in HaCaT keratinocytes under UV-B irradiation. When HaCaT keratinocytes were exposed to Rb3 prior to UV-B irradiation, Rb3 exhibited suppressive activities on UV-B-induced ROS, proMMP-2, and proMMP-9 enhancements. On the contrary, Rb3 displayed enhancing activities on UV-B-reduced total GSH and SOD activity levels. Rb3 could not interfere with cell viabilities in UV-B-irradiated HaCaT keratinocytes. Rb3 plays a protective role against UV-B-induced oxidative stress in human HaCaT keratinocytes, proposing its potential skin anti-photoaging properties.

Palmitate increases musclin gene expression through activation of PERK signaling pathway in C2C12 myotubes.

Musclin is a type of muscle-secreted cytokine and its increased gene expression induces insulin resistance in type 2 diabetes. However, the mechanism underlying increased musclin gene expression is currently unclear. Excessive saturated fatty acids (SFA) can activate the secretion of several muscle-secreted cytokines as well as endoplasmic reticulum (ER) stress pathway, thereby contributing to the development of type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the effect of palmitate, the most abundant SFA in the plasma, on the gene expression of musclin in C2C12 myotubes. Treatment of C2C12 myotubes with palmitate or tunicamycin significantly increased the expression of musclin as well as ER stress-related genes, but treatment with oleate did not. Pre-treatment of C2C12 myotubes with 4-phenyl butyrate suppressed the expression of ER stress-related genes, simultaneously, resulting in decreased expression of the musclin gene induced by palmitate or tunicamycin. These results indicate that ER stress is related to palmitate-induced musclin gene expression. Moreover, palmitate-induced musclin gene expression was significantly inhibited in C2C12 myotubes when PERK pathway signaling was suppressed by knockdown of the PERK gene or treatment with GSK2656157, a PERK autophosphorylation inhibitor. However, there was no difference in the palmitate-induced musclin gene expression when IRE1 and ATF6 signaling pathways were suppressed by knockdown of the IRE1 and ATF6 genes. These findings suggest that palmitate increases musclin gene expression via the activation of the PERK signaling pathway in C2C12 myotubes.

Antioxidative properties of ginsenoside Ro against UV-B-induced oxidative stress in human dermal fibroblasts.

Ginsenoside Ro (Ro), an oleanolic acid-type ginsenoside, exhibited suppressive activities on reactive oxygen species (ROS) and matrix metalloproteinase-2 (MMP-2) elevation in UV-B-irradiated fibroblasts. Ro could overcome the reduction of the total glutathione (GSH) contents in UV-B-irradiated fibroblasts. Ro could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Collectively, Ro possesses a potential skin anti-photoaging property against UV-B radiation in fibroblasts.

Titanium dioxide nanoparticles increase plasma glucose via reactive oxygen species-induced insulin resistance in mice.

There have been few reports about the possible toxic effects of titanium dioxide (TiO2 ) nanoparticles on the endocrine system. We explored the endocrine effects of oral administration to mice of anatase TiO2 nanoparticles (0, 64 and 320 mg kg(-1) body weight per day to control, low-dose and high-dose groups, respectively, 7 days per week for 14 weeks). TiO2 nanoparticles were characterized by scanning and transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their physiological distribution was investigated by inductively coupled plasma. Biochemical analyzes included plasma glucose, insulin, heart blood triglycerides (TG), free fatty acid (FFA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and reactive oxygen species (ROS)-related markers (total SOD, GSH and MDA). Phosphorylation of IRS1, Akt, JNK1, and p38 MAPK were analyzed by western blotting. Increased titanium levels were found in the liver, spleen, small intestine, kidney and pancreas. Biochemical analyzes showed that plasma glucose significantly increased whereas there was no difference in plasma insulin secretion. Increased ROS levels were found in serum and the liver, as evidenced by reduced total SOD activity and GSH level and increased MDA content. Western blotting showed that oral administration of TiO2 nanoparticles induced insulin resistance (IR) in mouse liver, shown by increased phosphorylation of IRS1 (Ser307) and reduced phosphorylation of Akt (Ser473). The pathway by which TiO2 nanoparticles increase ROS-induced IR were included in the inflammatory response and phosphokinase, as shown by increased serum levels of TNF-α and IL-6 and increased phosphorylation of JNK1 and p38 MAPK in liver. These results show that oral administration of TiO2 nanoparticles increases ROS, resulting in IR and increasing plasma glucose in mice.

4-Phenylbutyric acid increases GLUT4 gene expression through suppression of HDAC5 but not endoplasmic reticulum stress.

BACKGROUND: The chemical chaperone 4-phenylbutyric acid (4-PBA) has been shown to relieve endoplasmic reticulum (ER) stress. Therefore, it improves insulin sensitivity and promotes glucose metabolism in skeletal muscle. Glucose transporter type 4 (GLUT4), as a major glucose transporter protein, plays a central role in glucose metabolism. Until now, it has been unclear whether 4-PBA affects GLUT4 gene expression and thus, contributes to glucose metabolism. METHODS: C2C12 myotubes were treated with 4-PBA, tunicamycin or butyrate and subjected to Western blot and RT-PCR. RESULTS: 4-PBA-treated C2C12 myotubes increased GLUT4 expression and promoted glucose metabolism. Most interestingly, GLUT4 gene expression induced by 4-PBA was not associated with ER stress even in the presence of tunicamycin, an ER stress inducer. Moreover, we also found that 4-PBA inhibited histonedeacetylase 5 (HDAC5) expression in C2C12 myotubes, resulting in hyperacetylation of the histone H3 at the myocyte enhancer factor 2 (MEF2) binding site. This increased the binding of MEF2A to the site on the GLUT4 promoter, resulting in increased GLUT4 expression. CONCLUSIONS: Our data indicate that 4-PBA increases GLUT4 expression by acetylating the MEF2 site to increase the MEF2A binding through a mechanism that involves suppression of the HDAC5 pathway, but without involving ER stress.

Butylparaben induces glycolipid metabolic disorders in mice via disruption of gut microbiota and FXR signaling.

Butylparaben, a common preservative, is widely used in food, pharmaceuticals and personal care products. Epidemiological studies have revealed the close relationship between butylparaben and diabetes; however the mechanisms of action remain unclear. In this study, we administered butylparaben orally to mice and observed that exposure to butylparaben induced glucose intolerance and hyperlipidemia. RNA sequencing results demonstrated that the enrichment of differentially expressed genes was associated with lipid metabolism, bile acid metabolism, and inflammatory response. Western blot results further validated that butylparaben promoted hepatic lipogenesis, inflammation, gluconeogenesis, and insulin resistance through the inhibition of the farnesoid X receptor (FXR) pathway. The FXR agonists alleviated the butylparaben-induced metabolic disorders. Moreover, 16 S rRNA sequencing showed that butylparaben reduced the abundance of Bacteroidetes, S24-7, Lactobacillus, and Streptococcus, and elevated the Firmicutes/Bacteroidetes ratio. The gut microbiota dysbiosis caused by butylparaben led to decreased bile acids (BAs) production and increased inflammatory response, which further induced hepatic glycolipid metabolic disorders. Our results also demonstrated that probiotics attenuated butylparaben-induced disturbances of the gut microbiota and hepatic metabolism. Taken collectively, the findings reveal that butylparaben induced gut microbiota dysbiosis and decreased BAs production, which further inhibited FXR signaling, ultimately contributing to glycolipid metabolic disorders in the liver.