Aim: Paracrine interaction between macrophages and adipocytes in obese visceral fat tissues is thought to be a trigger of chronic inflammation. The immunomodulatory effect of the short chain fatty acid, butyric acid, has been demonstrated. We hypothesize that sodium butyrate (butyrate) attenuates inflammatory responses and lipolysis generated by the interaction of macrophages and adipocytes. Methods: Using contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the production of tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and the release of free glycerol, free fatty acids (FFAs) into the medium. We also examined the activity of nuclear factor-kappaB (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) in co-cultured macrophages, as well as lipase activity and expression in co-cultured adipocytes. Results: We found increased production of TNF-α, MCP-1, IL-6, and free glycerol, FFAs in the coculture medium, and butyrate significantly reduced them. Butyrate inhibited the phosphorylation of MAPKs, the activity of NF-κB in co-cultured macrophages, and suppressed lipase activity in co-cultured adipocytes. Lipase inhibitors significantly attenuated the production of TNF-α, MCP-1 and IL-6 in the co-culture medium as effectively as butyrate. Butyrate suppressed the protein production of adipose triglyceride lipase, hormone sensitive lipase, and fatty acid-binding protein 4 in co-cultured adipocytes. Pertussis toxin, which is known to block GPR41 completely, inhibited the antilipolysis effect of butyrate. Conclusion: Butyrate suppresses inflammatory responses generated by the interaction of adipocytes and macrophages through reduced lipolysis and inhibition of inflammatory signaling.

Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of Sphingomonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). The OPH was purified to homogeneity with a yield of 40 % and a 5?9-fold increase in specific activity. The enzyme was a homodimer consisting of 35 kDa subunits. Its activity was inhibited by PMSF, Hg 2+ and Zn 2+. The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and p-nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono-or diketones tested. K m and V max values for oxidized PVA and PNPA were 0?2 and 0?3 mM, and 0?1 and 3?4 mmol min "1 mg "1 , respectively. The gene for OPH was cloned and sequenced. Sequencing analysis revealed that the open reading frame consisted of 1095 bp, corresponding to a protein of 364 amino acids residues, encoding a signal peptide and a mature protein of 34 and 330 amino acids residues, respectively. The presence of a serine-hydrolase motif (a lipase box; Gly-X-Ser-X-Gly) strongly suggested that the enzyme belongs to the serine-hydrolase family. The protein exhibited homology with OPH of the Pseudomonas sp. strain VM15C (63 % identity) and the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti (29-32 % identity). The oph gene was expressed in Escherichia coli under the control of the lac promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day. Graphical Abstract

BackgroundInteractions between adipocytes and macrophages are associated with metabolic disorders. Production of pro-inflammatory mediators and the release of free fatty acids (FFAs) increase when these cells are co-cultured; butyrate significantly diminishes these effects by suppressing both the macrophage inflammatory and adipocyte lipolysis pathways. Butyrate is known to up-regulate the expression of prostaglandin E2 (PGE2). Therefore, we hypothesized that PGE2 is associated with the suppression of lipolysis by butyrate in co-culture.MethodsUsing contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the release of PGE2 into the medium and on lipolysis in adipocytes. To elucidate the underlying mechanism, we examined the effects of butyrate on cyclooxygenase-2 (COX2) and phospholipase A2 (PLA2) in co-cultured cells, and cyclic adenine monophosphate (cAMP) and protein kinase A type 1-α regulatory subunit (PRKAR1A) in co-cultured adipocytes. Silent interfering (si)RNA targeting of G-protein–coupled receptor (GPR)41 and 109A was employed to examine the effect on lipolysis in TNF-α–stimulated adipocytes.ResultsCo-culture increased PGE2 release into the medium, compared with cells cultured separately. Butyrate significantly increased PGE2 production. Co-culture elevated COX2 expression in macrophages and adipocytes, and butyrate further enhanced this effect. Co-culture enhanced cytosolic PLA2 activity in macrophages, which was further enhanced by butyrate. As for lipolysis, co-culture increased the release of FFAs and free glycerol into the medium, whereas butyrate (and to a lesser extent, PGE2) suppressed FFAs and free glycerol release. An inhibition study using a prostaglandin E receptor 3–selective antagonist suggested that approximately 40% of the suppressive effect of butyrate depends on the PGE2-mediated pathway, whereas 60% depends on a non-PGE2–mediated pathway. Co-culture increased cAMP and PRKAR1A levels in adipocytes, whereas butyrate restored the levels to those of the control. Similarly, in TNF-α–stimulated adipocytes, butyrate reduced FFAs and free glycerol release. siRNA inhibition of GPR41 and GPR109A suggested that the GPR109A-mediated pathway predominates, but the GPR41-mediated pathway also regulates the effect of butyrate on lipolysis in TNF-α–stimulated 3T3-L1 cells.ConclusionsButyrate attenuates lipolysis in adipocytes co-cultured with macrophages via non-PGE2–mediated and PGE2-mediated pathways.

Polyvinyl alcohol (PVA)-utilizing Sphingopyxis sp. 113P3 (re-identified from Sphingomonas sp. 113P3) removed almost 0.5% PVA from culture supernatants in 4 days. Faster degradation of 0.5% PVA was performed by the periplasmic fraction. The average molecular size of PVA in the culture supernatant or cell-bound PVA was gradually shifted higher, suggesting that lower molecular size molecules are degraded faster. Depolymerized products were found in neither the culture supernatant nor the cell-bound fraction; however they were recovered from the periplasmic fraction. As extracellular or cell-associated PVA oxidase activity was almost undetectable in strain 113P3, degradation of PVA must be performed by periplasmic PVA dehydrogenase after uptake into the periplasm. Following the consumption of PVA, a dent appeared on the cell surface on day 2 and increased in size and depth for 4 days and was maintained for 8 days. Ultrastructural change on the cell surface was only observed in PVA medium, but not in nutrient broth (NB), suggesting that the change is induced by PVA. Fluorescein-4-isothiocyanate-labeled PVA was bound more to cells grown in PVA than to cells grown in NB. No binding was found with PVA-grown cells treated with formaldehyde. Thus, a dent on the cell surface seems to be related to the uptake of PVA.