Citric acid improves growth performance and phosphorus digestibility in Beluga (Huso huso) fed diets where soybean meal partly replaced fish meal

A 2 × 3 factorial experiment was conducted to evaluate the effect of partial substitution of dietary fish meal with soybean meal and citric acid (CA) supplementation on growth, food utilization, muscle composition and nutrient digestibility of Beluga, Huso huso. Three isonitrogenic and isoenergetic diets, as SBM₁ (soybean meal protein (SBP):fishmeal protein (FP) = 1:3), SBM₂ (SBP:FP = 2:3) and SBM₃ (SBP:FP = 1:1) containing two levels of CA (0 and 30 g kg⁻¹) were fed to triplicate groups of fish for 8 weeks. The results revealed that adding CA increased (P<0.05) the weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) and decreased (P<0.05) food conversion rate (FCR) whereas partial substitution of fishmeal with soybean meal decreased (P<0.05) growth performance. Partial replacement of fishmeal with SBM (P=0.021) as well as CA supplementation (P=0.019) and their interaction (P<0.001) affected hepatosomatic index (HSI). No differences (P>0.05) were observed for viscerosomatic index (VSI) among treatments (P>0.05). No differences (P>0.05) were detected in moisture, protein of muscle sample among treatments, but lipid content was reduced (P<0.05) while ash content increased (P=0.035) by CA. Soybean meal decreased (P<0.05) nutrient digestibility, whereas CA improved apparent protein and phosphorus (P) digestibility (P=0.011 and P<0.001 respectively). No interaction (P<0.05) between levels of SBM and CA was found on these parameters. Results of the present study indicate that Beluga has a limited ability to utilize SBM as a protein source in practical diets whereas CA can improve growth and nutrient utilization in Beluga.     

Effect of feed restriction or feeding high-fibre diet during the rearing period on body composition, serum parameters and productive performance of rabbit does

This study evaluated the effect of different feeding regimes from 11 weeks of age to first parturition on feed intake, leptin, non-esterified fatty acids (NEFA) and total protein serum levels, as well as productive performance in young rabbit does. In addition, body composition was estimated by bioimpedance analysis. Thirty-six 11-week-old does were randomly distributed in three groups. The AL-C group was fed ad libitum a control diet containing 350 g neutral detergent fibre (aNDFom)/kg, 11.6 MJ digestible energy (DE)/kg and 173 g crude protein (CP)/kg, and the does were inseminated at 16 weeks of age. The R-C group was fed 150 g/d of the same diet until 16 weeks of age, one week before artificial insemination (AI) at 17 weeks of age, and then fed ad libitum. The AL-F group was fed a diet containing 475 g aNDFom/kg, 9.4 MJ DE/kg and 174 g CP/kg ad libitum, and was inseminated at 17 weeks of age. During rearing (11-16 weeks), does in the R-C group had the lowest DE (1.54 MJ/d; P<0.003) and digestible protein (DP, 17.9 g/d; P<0.001) intake, as well as the lowest protein (172 g/kg; P<0.05) and energy (5.9 MJ/kg) body contents, leptin concentration at 16 weeks of age (2.48 ng/ml; P<0.001) and fertility (P<0.02) at first AI. Daily feed intake during pregnancy and lactation, as well as prolificacy and litter weight, were similar among groups. The highest percentage of body fat was observed for all the does when were inseminated for the first time (135 g/kg; P<0.001), consistent with the highest leptin (4.48 ng/ml; P<0.001) and total protein serum levels (6.87 g/dl; P<0.001) at this time. Serum NEFA level around parturition was higher (P<0.05) in groups AL-C and R-C (1.11 and 0.85 mmol/l) than in group AL-F (0.71 mmol/l), suggesting a lower lipid mobilization that tended to improve fertility rate for AL-F does on day 11 post-parturition (P<0.09). In conclusion, feed restriction during the rearing period delays reproductive development in young rabbits. In nulliparous does, ad libitum feeding during rearing with a high-fibre diet allows a similar productive performance to that of feeding with a less fibrous diet. Nevertheless, the use of high fibre diets during rearing does not affect feed intake throughout the first pregnancy and lactation.     

Expression of basic fibroblast growth factor,protein kinase C and members of the apoptotic pathway in skeletal muscle of streptozotocin-induced diabetic rats

This study investigated the potential mechanisms that may underlie diabetes induced amyoatrophy. Sprague-Dawley rats were either injected intraperiotneally with STZ (test group; N = 8) to induce diabetic-like symptoms (blood glucose level ≥16.65 mmol/L) or with buffer (control group; N = 8). Differences in muscle structure between the STZ-induced diabetic and control groups were evaluated by histochemistry. Protein and mRNA levels of basic FGF (bFGF), bax, bcl-2, and caspase 3 in skeletal muscle were compared between the 2 groups using immunohistochemistry and quantitative PCR, respectively. Serum level of insulin and protein kinase C (PKC) were measured by competitive RIA and ELISA, respectively. Unlike control animals, the skeletal muscle fibers from STZ-induced diabetic animals were broken and pyknotic, the sarcomeric structure disrupted, and mild hyperplasia of interstitial adipose tissues was detected. The serum level of PKC was higher (P = 0.003) and the protein and mRNA levels of bFGF in skeletal muscle were lower (P = 0.001) in STZ-induced diabetic versus control animals. Protein and mRNA levels of the apoptosis promoting genes caspase-3 and bax were higher in skeletal muscle from STZ-induced diabetic rats as compared to control animals (P < 0.001 and P = 0.037, respectively), while mRNA and protein levels of bcl-2, an inhibitor of apoptosis, was lower in STZ-induced diabetic rats versus control animals (P = 0.026). Increasing apoptosis in skeletal muscle from STZ-induced diabetic rats was further demonstrated by TNNEL assay. Our findings suggest that enhanced PKC levels, reduction of bFGF expression, and increased in apoptosis might be associated with the development of diabetes-induced myoatrophy.     

Evidence for a pro-apoptotic phenotype in skeletal muscle of hypertensive rats

In this report, we demonstrate that soleus muscle of spontaneously hypertensive rats (SHR) had significantly lower protein levels of apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) as well as significantly higher protein levels of second mitochondria-derived activator of caspase (Smac) and procaspase-8 compared to normotensive Wistar-Kyoto (WKY) rats. In addition, soleus muscle from hypertensive rats had significantly increased caspase-8 proteolytic enzyme activity as well as significantly elevated reactive oxygen species (ROS) generation and higher hydrogen peroxide (H₂O₂) content. There was no change in the protein levels of the antioxidant enzymes, catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD). Interestingly, ARC protein migrated at approximately 32 kDa in SHR but at 30 kDa in WKY rat muscle; possibly indicating a post-translational modification. These results demonstrate that soleus muscle of hypertensive rats display a pro-apoptotic phenotype and augmented ROS generation.     

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP₂ and EP₄, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 μg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP₂, and EP₄ mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP₄ within 24 h after treatment (P < 0.05), whereas EP₂ mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP₂, and EP₄ mRNA expression were greatest in the smooth muscle layers (P < 0.05) and least in the luminal epithelium (P < 0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP₄ mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E₂ and activation of the PGE₂ receptors EP₂ and EP₄.     

A dynamic approach reveals non-muscle myosin influences the overall smooth muscle cross-bridge cycling rate

Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B^+/− strain (8.73 ± 1.10 Hz vs 16.56 ± 1.26 Hz, P < 0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B^+/−) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle V_max during force maintenance.     

Nutritional value and intake of aquatic ferns (Azolla filiculoides Lam. and Salvinia molesta Mitchell.) in sows

Aquatic ferns (AFs) such as Azolla filiculoides and Salvinia molesta are grown on swine lagoons in the tropics and used in diets for pigs. The present work is aimed at evaluating their potential as feed ingredients for sows. When presented with ad libitum AFs, gilts weighing 110 ± 14 kg (mean ± SD), were able to ingest 9.1–9.7 kg fresh AF per day (from 597 to 630 g dry matter (DM) per day) and from 1240 to 1428 g DM per day when presented in a dry, ground form. A digestibility study was conducted, using sows weighing 213 ± 9 kg (mean ± SD), which were fed diets containing maize, soybean meal and 0, 150 or 300 g AF kg⁻¹ diet. The presence of AFs had a negative impact on the faecal digestibility of the crude protein, NDF and energy content of the whole diet (P<0.001) and on the ileal protein digestibility, especially with 300 g AFs kg⁻¹ diet. The level of AFs in the diet had no effect on stomach weight (P>0.05) but increased the weight of the rest of the gastrointestinal tract (P<0.001). The rate of AF fibre fermentation in the pig large intestine was measured using an in vitro gas test. The rates were much lower than tropical tree foliage, which can also be used in pig diets in the tropics. This could partly explain the low apparent digestibility of AFs in pigs. In conclusion, the inclusion level of AFs in rations for sows should be limited to 150 g AFs kg⁻¹ diet due to the low digestibility and energy density, as well as the negative impact on the digestibility of the whole diet.     

Expression of the Cytoskeletal-Associated Protein Filamin in Adult Rat Organs

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.     

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.     

Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions

Aims

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle—the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.

Main methods

To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2^−/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).

Key findings

CR-AMPKα1/2^−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ^±±p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).

Significance

This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.     

Association of TGF-β1 − 509 C/T, 29 C/T and 788 C/T gene polymorphisms with chronic periodontitis: A case–control study

The aim of this study was to investigate the possible association between TGF-β1 − 509 C/T (rs1800469), 29 C/T (Prol10Leu, rs1800470) and 788 C/T (Thr263Ile, rs1800472) gene polymorphisms and chronic periodontitis (CP) in a sample of Iranian population. This case–control study was conducted on 100 CP patients and 100 healthy unrelated, age-, sex-, and ethnicity-matched. Genotyping was performed by tetra amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) technique. Our findings showed that there was a significant difference between the groups regarding TGF-β1 29 C/T (rs1800470) polymorphism (χ2 = 23.23, P < 0.0001). The CT and TT genotypes increased the risk of CP in comparison with the CC genotype (OR = 4.42, 95% CI = 2.16–9.06, P < 0.001 and OR = 5.84, 95% CI = 2.32–14.71, P < 0.001, respectively). The T allele increased the risk of CP (OR = 2.50, 95% CI = 1.66–3.74, P < 0.001) in comparison with C allele. No significant association was found among the groups regarding − 509 C/T and 788 C/T variants of TGF-β1 gene. This study shows that TGF-β1 29 C/T polymorphism, but not − 509 C/T and 788 C/T polymorphisms, may contribute to the development of CP in a sample of Iranian population. Further studies with larger sample sizes and different ethnicities are needed to validate our findings.     

Morphology of hypoxia following cryoablation in a prostate cancer murine model: Its relationship to necrosis, apoptosis and, microvessel density

The aim of this study is to investigate the tumor tissue changes in terms of hypoxia and demonstrate its relationship to vascularity and apoptosis following therapeutic cryoablation in a prostate tumor murine model. Total 67 male C57BL/J6 mice were assigned into sham-operation group and cryoablation group. Murine prostate tumors (RM-9) were inoculated subcutaneously in a right hind leg and treated with cryotherapy. Of 30 mice, tumor volumes were measured for 12 days following operation. Of 37 mice, tumor tissues were harvested in 24 h following operation, and histological/molecular changes were analyzed. Hematoxylin and eosin or immunohistochemical staining were utilized to quantify tumor necrosis, hypoxia (pimonidazole), vascularization (CD31), and apoptosis (cleaved caspase-3). The results showed that cryoablated tumors demonstrated significant delayed growth following treatment compared to controls. Pathological analysis revealed that the severity of hypoxia increased in the cryoablation arm compared to controls. Necrotic and apoptotic populations were also found to be increased in the cryoablation arm (P = 0.028 and 0.021). Hypoxia demonstrated a positive correlation with necrosis (r = 0.520, P = 0.001) and apoptosis (r = 0.474, P = 0.003), while showing negative correlation with microvessel density (MVD) (r = −0.361, P = 0.021). We concluded that in the peripheral areas from the cryoneedle impact site, strong hypoxic responses were found, which may play important role in tumor freezing injury. To our knowledge, this is the first report describing cryoablation-mediated changes of hypoxia at a molecular level in the prostate cancer murine model.     

Influences of diet during gestation on potential postpartum reproductive performance and milk production of beef heifers

The influences of nutritional protein and energy during early and mid pregnancy on milk production and postpartum reproductive parameters were determined in 70 beef heifers of two composite breeds (Bos indicus X Bos taurus). At artificial insemination (AI), heifers were divided into four dietary treatment groups identified by the level of protein, and to a lesser extent energy, fed during the first and second trimesters: high/high (HH), high/low (HL), low/high (LH), and low/low (LL). Milk production was lower in the heifers receiving high treatment in first trimester than that in heifers receiving the low treatment (P = 0.01). Milk production was negatively associated with dam body condition score (BCS; P = 0.01), nonesterified fatty acids (P = 0.001), and leptin (P = 0.02) and positively associated with urea (P < 0.001) concentrations during lactation. Increased dietary protein in the first trimester increased or decreased concentrations of colostral protein dependent upon genotype (P = 0.03). Colostral protein was positively associated with bovine pregnancy associated glycoprotein from late gestation (P = 0.007). Milk fat was negatively associated with BCS (P = 0.007) and influenced by genotype (P = 0.003). Dietary treatment did not affect the postpartum reproductive performance of beef heifers. Gestation length (P < 0.001) and the postpartum interval to first estrus (PPI; P = 0.02) were positively associated with calf size. Placental size was negatively associated with placental expulsion time (P < 0.01). Prepartum BCS of the heifers was negatively associated with PPI (P = 0.01). Overall, high levels of nutrition during early gestation are detrimental to milk production in beef heifers.     

Molecular characterization,transcriptional regulation and association analysis with carcass traits of porcine TCAP gene

TCAP (also known as titin-cap or telethonin) is one of the titin interacting Z-disk proteins involved in the regulation and development of normal sarcomeric structure. In this study, we cloned the cDNA and promoter sequences of porcine TCAP gene, which contained a 504 bp full-length coding region. Quantitative real-time PCR (qRT-PCR) analyses showed that porcine TCAP was highly expressed in the skeletal muscle, heart, and kidney. During postnatal muscle development, TCAP expression was down-regulated from 30 days to 120 days in Large White and Meishan pigs. One single nucleotide polymorphism c.334G>A in exon 2 of the TCAP gene was identified and detected by allele-specific primer-polymerase chain reaction (ASP-PCR). Association analysis revealed that the polymorphism had significant associations (P < 0.05 and P < 0.01) with some carcass traits. Analysis of the porcine TCAP promoter in different cell lines demonstrated that it is a muscle-specific promoter. In addition, we found that the porcine TCAP promoter can be activated by MyoD, MyoG and MEF2 in myotubes, which indicated that TCAP may play a role in the regulation of porcine skeletal muscle development. These findings provide useful information for the further investigation of the function of TCAP in porcine skeletal muscle.     

ANG-(1-7) reduces ANG II-induced insulin resistance by enhancing Akt phosphorylation via a Mas receptor-dependent mechanism in rat skeletal muscle

The nonapeptide angiotensin II (ANG II) induces vasoconstriction via the ANG II type I receptor, while its splice product ANG-(1-7) elicits an antihypertensive effect via the Mas receptor. Although a critical role of ANG II in the etiology of skeletal muscle insulin resistance is well documented, the role of the ANG-(1-7)/Mas receptor axis in this context is poorly understood. Therefore, we determined whether ANG-(1-7) is effective in ameliorating the negative effects of ANG II on insulin-stimulated insulin signaling and glucose transport activity in isolated soleus muscle from normotensive lean Zucker rats. ANG II alone (500 nM for 2 h) decreased insulin-stimulated glucose transport activity by 45% (P < 0.05). In the presence of 500-1000 nM ANG-(1-7), insulin-stimulated glucose transport activity in muscle exposed to ANG II improved by ∼30% (P < 0.05). Moreover, ANG-(1-7) treatment increased Akt Ser⁴⁷³ phosphorylation (47%, P < 0.05) without an effect on glycogen synthase kinase-3β Ser⁹ phosphorylation. The dependence of ANG-(1-7) action on the Mas receptor was assessed using A779 peptide, a selective Mas receptor antagonist. The positive effects of ANG-(1-7) on insulin-stimulated glucose transport activity and Akt Ser⁴⁷³ phosphorylation in soleus muscle were completely prevented in presence of 1000 nM A779. In conclusion, the present study demonstrates that ANG-(1-7), via a Mas receptor-dependent mechanism, can ameliorate the inhibitory effect of ANG II on glucose transport activity in mammalian skeletal muscle, associated with enhanced Akt phosphorylation. These results provide further evidence supporting the targeting of the renin-angiotensin system for interventions designed to reduce insulin resistance in skeletal muscle tissue.     

Naringenin, a citrus flavonoid, increases muscle cell glucose uptake via AMPK

Naringenin, a flavonoid found in high concentrations in grapefruit, has been reported to have antioxidant, antiatherogenic, and anticancer effects. Effects on lipid and glucose metabolism have also been reported. Naringenin is structurally similar to the polyphenol resveratrol, that has been reported to activate the SIRT1 protein deacetylase and to have antidiabetic properties. In the present study we examined the direct effects of naringenin on skeletal muscle glucose uptake and investigated the mechanism involved. Naringenin stimulated glucose uptake in L6 myotubes in a dose- and time-dependent manner. Maximum stimulation was seen with 75 μM naringenin for 2 h (192.8 ± 24%, p < 0.01), a response comparable to maximum insulin response (190.1 ± 13%, p < 0.001). Similar to insulin, naringenin did not increase glucose uptake in myoblasts indicating that GLUT4 glucose transporters may be involved in the naringenin-stimulated glucose uptake. In addition, naringenin did not have a significant effect on basal or insulin-stimulated Akt phosphorylation while significantly increased AMPK phosphorylation/activation. Furthermore, silencing of AMPK, using siRNA approach, abolished the naringenin-stimulated glucose uptake. The SIRT1 inhibitors nicotinamide and EX527 did not have an effect on naringenin-stimulated AMPK phosphorylation and glucose uptake. Our data show that naringenin increases glucose uptake by skeletal muscle cells in an AMPK-dependent manner.     

Cytotoxicity from sulfide exposure in a sulfide-tolerant marine invertebrate

It is unknown whether sulfide-tolerant marine invertebrates suffer cytotoxicity from sulfide exposure in vivo at environmentally-relevant concentrations. We tested this with the mudflat polychaete Glycera dibranchiata. Exposure of live animals to sulfide up to 2.4 mmol l^− 1 for 24 h did not affect animal survival, and animal condition (gross appearance and activity) was unaffected at sulfide concentrations up to 0.25 mmol l^− 1. However, animal condition was decreased at higher sulfide concentrations (P < 0.0001, n = 33). Coelomic fluid obtained from the animals showed decreased erythrocyte count (P = 0.0035, n = 14), indicating cell loss, and increased propidium iodide and Hoechst 33324 staining (P < 0.0001 and P = 0.0003, respectively, n = 14), indicating loss of plasma membrane integrity, even at sulfide concentrations that produced no change in animal condition. When G. dibranchiata were allowed a 72 h recovery period following 24 h sulfide exposure, there was no overall improvement in condition (P ≥ 0.12, n = 7-8), and worms that had been exposed to 1 mmol l^−  1 sulfide still had an erythrocyte count that was less than half that of control animals (P = 0.028, n = 7). The inability to completely recover the cell count was at least partially due to impaired production of new erythrocytes, since analysis of BrdU incorporation indicated that erythrocyte proliferation was reduced from 2% per day in control animals to 0.12% per day in animals exposed to 1 mmol l^− 1 sulfide (P = 0.010, n = 21). Together, these findings indicate that at least some sulfide-tolerant marine invertebrates experience significant cellular injury and impaired tissue proliferation when exposed to environmentally relevant sulfide concentrations, even when the appearance and behavior of the animal appear unaffected.