AdipoRon alleviates fatty acid–induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30–40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 μM for 12 h) and times (25 μM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 μM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.     

Insulin-like growth factor-1 induces IRE1-XBP1–dependent endoplasmic reticulum biogenesis in bovine mammary epithelial cells

Insulin-like growth factor-1 (IGF-1) plays a key role in proliferation and galactopoiesis in mammary epithelial cells (MEC), but its definitive functions on endoplasmic reticulum (ER) during protein synthesis remain unknown. The present study aimed to elucidate the effects of IGF-1 on ER biogenesis in MEC in vitro and examined the expression of ER biogenesis-associated genes in the mammary gland during early lactation. We treated mammary alveolar cells–large T antigen cells (immortalized bovine MEC line established via stable transfection with simian virus-40 large T-antigen) with IGF-1 and examined ER biogenesis using the fluorescence intensity of an ER tracker and quantitative real-time PCR. We found IGF-1 significantly increased ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as CHKA (choline kinase α), PCYT1A (choline-phosphate cytidylyltransferase A), and SURF4 (surfeit locus protein 4). We focused on unfolded protein response to explore molecular mechanisms by which IGF-1 induces ER biogenesis. We found IGF-1 significantly increased mRNA levels of the XBP1 splicing form (XBP1s). Based on western blot analysis, IGF-1 induced the expression of (inositol-requiring kinase 1 α) protein, upstream of XBP1s, and phosphorylated-IRE1α. The inhibition of IRE1 endoribonuclease activity with 4-methylumbelliferone 8-carbaldehyde (4μ8C) significantly suppressed the increase in ER tracker fluorescence and ER biogenesis-related gene expression induced by IGF-1. Also, IGF-1–induced XBP1s and ER biogenesis-associated gene expression was inhibited by rapamycin, an inhibitor of mTORC1 (mammalian target of rapamycin complex 1), indicating that IRE1-XBP1 activation by IGF-1 is mediated by mTORC1. Moreover, to clarify the expression of XBP1s and ER biogenesis-associated genes expression under normal physiological conditions, mammary gland tissue from biopsies of dairy cows during late gestation and lactation were analyzed. In vivo data highlighted the significant increases in the mRNA levels of XBP1s and ER biogenesis-related genes in mammary gland tissue immediately after calving through 6 wk of lactation. The mRNA levels of IGF1R (IGF-1 receptor) in mammary glands increased during 6 wk of lactation. Therefore, the present study indicated for the first time that IGF-1 induces ER biogenesis by activating the IRE1-XBP1 axis under the regulation of mTORC1 in bovine MEC line.     

Glutamine pretreatment protects bovine mammary epithelial cells from inflammation and oxidative stress induced by γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP)

Glutamine (GLN) has many types of biological activity in rats, including anti-inflammatory, antioxidative stress, and anti-apoptosis effects. However, little is known about the effects of GLN on bovine mammary epithelial cells (BMEC). γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP) is a cell wall peptidoglycan component of gram-negative bacteria that can be recognized by the intracellular receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and can cause bovine mastitis. The goal of the present study was to investigate whether GLN protects BMEC from iE-DAP–induced inflammation, oxidative stress, and apoptosis. We cultured BMEC in a GLN-free medium for 24 h and then separated them into 4 groups: cells treated with 1× PBS for 26 or 32 h (control); cells stimulated by 10 μg/mL iE-DAP for 2 or 8 h (2- or 8-h iE-DAP); cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of 1× PBS treatment (8 or 4 mM GLN); and cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of iE-DAP treatment (DG). In the 2-h iE-DAP group, when levels of inflammation peaked, iE-DAP treatment increased both the mRNA and protein expression of NOD1, inhibitor of nuclear factor-κB (NFKBIA, IκB), and nuclear factor-κB subunit p65 (RELA, NF-κB p65), as well as the mRNA expression of IL6 and IL8 and levels of IL-6 and tumor necrosis factor-α in cell culture supernatants. In contrast, 8 mM GLN pretreatment inhibited the mRNA and protein expression of inflammatory-related factors by suppressing the NOD1/NF-κB pathway. In the 8-h iE-DAP group, iE-DAP treatment decreased the mRNA and protein expression of extracellular regulated kinase (Erk, ERK) and nuclear factor erythroid 2–associated factor2 (NFE2L2, Nrf2), as well as the mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT), coenzyme II oxidoreductase 1 (NQO1), and heme oxygenase 1 (HMOX1, HO1). In addition, iE-DAP treatment increased the expression of malondialdehyde in BMEC when oxidative stress levels peaked. Interestingly, 4 mM GLN pretreatment induced the mRNA and protein expression of antioxidative stress–related factors and inhibited the expression of reactive oxygen species in BMEC by promoting the ERK/Nrf2 pathway. Moreover, GLN reduced apoptosis caused by inflammation and oxidative stress in BMEC. This is the first report showing that GLN protects against iE-DAP-induced inflammation and oxidative stress via the NOD1/NF-κB and ERK/Nrf2 pathways in BMEC.     

Protective effect of tetrahydrocurcumin and chlorogenic acid against streptozotocin–nicotinamide generated oxidative stress induced diabetes

The present study was undertaken to investigate the protective effect of tetrahydrocurcumin (THC) and chlorogenic acid (CGA) against streptozotocin (STZ)–nicotinamide (NA)-induced type 2 diabetes in adult Wistar rats. Diabetes was induced in experimental rats weighing 180–220 g, by a single intraperitoneal (i.p.) injection of STZ (45 mg/kg BW), 15 min after the (i.p.) administration of NA (110 mg/kg BW). THC (80 mg/kg BW) and CGA (5 mg/kg BW) were orally administered to diabetic rats for a period of 45 days. Fasting plasma glucose, glycosylated haemoglobin (HbA_1C), thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) were significantly increased, whereas insulin, total haemoglobin (Hb), non-enzymic antioxidants (reduced glutathione (GSH), vitamin C, vitamin E and ceruloplasmin) were decreased significantly in diabetic rats. Though the diabetic rats treated with THC and CGA individual exerts beneficial effects in all the biochemical parameters in (STZ)-induced diabetic rats. The combined treatment with THC and CGA normalized all the above-mentioned biochemical parameters in STZ-induced diabetic rats. Normal pancreatic histological architecture in THC and CGA treated diabetic rats revealed that these phytochemical exert higher degree of protection when administered in combination than single treatment of individual compounds.     

Identification of hydroxycinnamic acid–tartaric acid esters in wine by HPLC–tandem mass spectrometry

In this work hydroxycinnamic acid esters of tartaric acid (mono-p-coumaroyl tartaric acid, monocaffeoyl tartaric acid, monoferuloyl tartaric acid esters) were separated, identified and quantified in several red wines by high performance liquid chromatography coupled with tandem mass spectrometry. The method features direct analysis of wines with no preparation and analysis of grape juice with minimum sample manipulation. For the identification was used both a triple quadrupole and a quadrupole–Tof, while for the quantification a triple quadrupole.     

Correction to: Amelioration of radiation‑induced liver damage by p-coumaric acid in mice

Food Science and Biotechnology -     

Perilla oil and α-linolenic acid ameliorated thrombosis in rats induced by collagen and epinephrine

Food Science and Biotechnology - Perilla frutescens is an annual herbaceous plant widely cultivated for oil production in China, Japan, and Korea. In this study, we investigated the effect of...     

Myricetin modulates streptozotocin–cadmium induced oxidative stress in long term experimental diabetic nephrotoxic rats

Oxidative stress plays a pivotal role in the development of diabetic nephropathy. The present study was aimed to evaluate the modulatory potential of myricetin on streptozotocin (STZ)–cadmium (Cd) induced oxidative stress in diabetic nephrotoxic rats. Diabetic nephrotoxicity was induced by single intraperitoneal injection of STZ at a dose of (40 mg/kg body weight (b/w)) and Cd as cadmium chloride (CdCl₂) (100 p.p.m.). Myricetin was administered to diabetic nephrotoxic rats by intraperitoneally at 1.0 mg/kg b/w for a period of 12 weeks to assess its effects on fasting plasma glucose, plasma insulin, total haemoglobin, glycosylated haemoglobin, lipid peroxidation products viz., thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), protein carbonyl content (PCO) and non-enzymatic antioxidants namely vitamins C and E and reduced glutathione (GSH) and also enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR). Improvement of antioxidant status in myricetin supplemented diabetic nephrotoxic rats revealed its cellular protective effect. Histopathology of liver and kidney confirmed the protective effects of myricetin in diabetic nephrotoxic rats. The outcome of this study concludes that myricetin could be therapeutic flavonol for regulating oxidative mechanism in STZ–Cd induced diabetic nephrotoxic rats.     

Mild heat stress–induced adaptive immune response in blood mononuclear cells and leukocytes from mesenteric lymph nodes of primiparous lactating Holstein cows

Heat stress negatively affects the metabolism and physiology of the bovine gut. However, it is not known whether heat stress induces an inflammatory response in mesenteric lymph nodes (MLN), the primary origin of gut immune cells, and thus contributes to inflammatory processes in the circulation. Therefore, our objective was to elucidate the effects of chronic heat stress on the systemic activation of acute-phase response in blood, proinflammatory cytokine production in peripheral blood mononuclear cells (PBMC), and the activation of the toll-like receptor signaling (TLR) 2/4 pathway in MLN leucocytes and their chemokines and chemokine receptor profiles in Holstein cows. Primiparous Holstein cows (n = 30; 169 ± 9 d in milk) were exposed to a temperature-humidity index (THI) of 60 [16°C, 63% relative humidity (RH)] for 6 d. Thereafter, cows were evenly assigned to 3 groups: heat-stressed (HS; 28°C, 50% RH, THI = 76), control (CON; 16°C, 69% RH, THI = 60), or pair-feeding (PF; 16°C, 69% RH, THI = 60) for 7 d. On d 6, PBMC were isolated and on d 7 MLN. Plasma haptoglobin, TNFα, and IFNγ concentrations increased more in HS than CON cows. Concomitantly, TNFA mRNA abundance was higher in PBMC and MLN leucocytes of HS than PF cows, whereas IFNG mRNA abundance tended to be higher in MLN leucocytes of HS than PF cows, but not for chemokines (CCL20, CCL25) or chemokine receptors (ITGB7, CCR6, CCR7, CCR9). Furthermore, the TLR2 protein expression tended to be more abundant in MLN leucocytes of HS than PF cows. These results suggest that heat stress induced an adaptive immune response in blood, PBMC, and MLN leukocytes involving the acute-phase protein haptoglobin, proinflammatory cytokine production, and TLR2 signaling in MLN leucocytes. However, chemokines regulating the leucocyte trafficking between MLN and gut seem not to be involved in the adaptive immune response to heat stress.     

Machine milking–induced changes in teat canal dimensions as assessed by ultrasonography

Changes in teat canal diameter as induced by machine milking are thought to be related to teat canal penetrability and susceptibility to new intramammary infections. The objective of this study was to describe the effects of machine milking on teat canal dimensions as assessed by ultrasonography and to evaluate postmilking changes of teat canal dimensions throughout an 8-h milking interval. Ultrasonographic images were taken of the left front and right hind teats of 80 Holstein cows milked 3 times per day. Imaging occurred before attachment of the milking unit after completion of premilking udder preparation (t?1), immediately after removal of the milking unit (t0), and then every hour after milking until 8 h had elapsed (t1 to t8). Teat canal length and teat canal diameter (at the proximal, middle, and distal regions) were measured. General linear mixed model analysis indicated differences in the relative change of teat canal length (compared with t?1) with least squares means (LSM) and 95% confidence interval (95% CI) of 11.1% (8.7–13.4%) after machine milking (t0). Teat canal length decreased throughout the 8-h milking interval but remained elevated relative to t?1. Teat canal diameter at the proximal and middle regions increased near the end of the milking interval, whereas that at the distal region did not change meaningfully during the 8 h of observation. Our findings suggest that the teat canal and its surrounding tissue is in a state of near-constant remodeling in cows milked 3 times daily. Future work is warranted to study the association between ultrasonographically assessed changes of teat canal dimensions after machine milking and teat canal penetrability to mastitis-causing pathogens.     

Physical changes induced by the Maillard reaction in a glucose–glycine solution

Some physical changes associated with the development of Maillard reaction in a glucose–glycine aqueous solution were detected by means of thermal and rheological analysis as well as by more conventional determinations. The increase in water content, water activity (A_w) and enthalpy of ice melting (ΔHm) during the reaction reflects both the formation of new water molecules and the consumption of low molecular weight species. The concomitant arrangement of more complex molecular structures was indicated by changes in the glass transition temperature of the maximally freeze-concentrated solution (Tg′) and of the freeze-dried solution (Tg _(dry)) as well as in the estimated unfrozen water (UFW). After fractionation of the samples by solid phase extraction, the non-polar fractions exhibited much higher Tg_(dry) than the non-fractionated samples. The Tgs_(dry) of samples at increasing heating time were treated with the Gordon–Taylor equation and results were discussed. The expected increase in viscosity due to polymerisation was compensated by the formation of water, but it could be detected when viscosities were measured on samples equilibrated at a same A_w     

Protein aggregation induced by phase separation in a pea proteins–sodium alginate–water ternary system

The physicochemical properties of a native, globular plant protein–linear anionic polysaccharide aqueous system at 20 °C were investigated in conditions where biopolymers carry a net negative charge (pH 7.2, 0.1 M NaCl). The pea proteins–sodium alginate mixtures showed a phase separation mainly by thermodynamic incompatibility, characterized at both the macroscopic and microscopic scale. Phase diagram was established and confocal laser scanning microscopy (CLSM) provided accurate data on the microstructure morphology of the system, regarding its phase behavior. In admixture, sodium alginate induced a protein aggregation, certainly by a local depletion of the polysaccharide. Protein aggregates were present in both single-phase and biphasic mixtures, while increasing the sodium alginate concentration provided larger and denser protein microdomains, leading to a non-equilibrium state. By phase separation, the pea protein aggregates entrapped a part of the sodium alginate phase, thus modifying the protein volume fraction. Along a tie-line, a phase inversion phenomenon was detected, from a sodium alginate to a pea proteins-continuous phase. Rheological properties of the mixed systems depended on the biopolymer composition and were modified with respect to individual components. The protein-enriched phase volume fraction modified the flow behavior to non-Newtonian for starting concentrated mixtures; a shear-thinning at high shear rates was evidenced, while mixtures with a particularly high sodium alginate concentration (>0.8 wt%) had an apparent yield stress for low shear rates. Mechanical spectra showed that both protein enrichment within microdomains in the presence of alginate and stronger protein–protein interaction with time impacted the viscoelastic properties (G′ and G″ moduli) of the whole mixture.     

Formation of heterocyclic amine–amino acid adducts by heating in a model system

Although mutagenic and carcinogenic heterocyclic amines (HCAs) are known to be formed in cooked meat and fish, human HCA exposure and carcinogenic risk have not been elucidated in sufficient detail. In this work, we investigated the formations of HCA–amino acid adducts in a model system by using a liquid chromatography–mass spectrometry to elucidate another source of human HCA exposure. The 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) adduct with glycine was formed easily by heating at 200 °C within 5 min, which is probably based on the dehydration condensation of the amino group of PhIP and carboxyl group of glycine. PhIP and other HCAs such as 2-amino-3-methyl-3H-imidazo[4,5-f]quinolone, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline and 3-amino-1,4-dimethyl-5H-pyrido[3,4-b]indole, also bound with various amino acids by heating. Among these amino acids, proline tends to form adducts with HCAs, but serine, cysteine and lysine hardly bound with HCAs. These results provided a basic understanding of the formation of HCA adducts with amino acids during cooking.