Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5–10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome. Citrobacter rodentium infection models infection with human enterohemorrhagic (EHEC) and enteropathogenic Escherichia coli (EPEC) -L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester  for the first 6 days and thereafter refreshed every two days.The  surface  was base surface , 7.5 x 1C. rodentium strain ICC169 was grown for 20 h at 37˚C on MacConkey agar (Oxoid).in vitro mucosal surfaces were subjected to VIP and cytokine treatment for 96 h. The 96 h treatment duration was chosen to mimic in vivo exposure during C. rodentium infection  by oral gavage. At day 10, 14 and 19 post infection, control and infected mice were anesthetized with isoflurane and killed by cervical dislocation. The most distal 2 cm of colon were collected: the most proximal cm was collected for isolation of mitochondria in ice cold imidazole buffer  and the most distal cm was put in fresh Carnoy’s methanol fixative .Male C57BL/6 mice were infected with 100 μl of the bacterial suspension [5 x 10in vivo experiments with VIP treatment, an intraperitoneal (i.p.) saline (0.9%) administered dose of 0.5 nmol VIP per mouse/day was chosen as this dose had previously been shown to be the best dose for alleviating C. rodentium induced colitis, and this dose had no effect on body weight or behavior of uninfected mice [VIP treatment: For all ted mice . For theFor colitis analysis, 5 μm sections of Carnoy’s fixed tissue were stained with haematoxylin/eosin and the entire section was systematically scored for: crypt abscesses (0–3), aberrant crypt architecture (0–3), tissue damage (0–3), increased crypt length (0–3), goblet cell depletion (0–3), lamina propria neutrophil counts (0–3) and inflammatory cell infiltration (0–3) in a blinded fashion. The colitis score represents the sum of the individual scores for crypt abscesses, aberrant crypt architecture, tissue damage, increased crypt length, lamina propria neutrophil counts and inflammatory cell infiltration.The goblet cell depletion scores were confirmed using PAS/Alcian blue stain, as previously described : after iin vitro mucosal surface) at 95˚C, slides were then cooled for 40 min in room temperature. 3% hydrogen peroxide in PBS was used to block endogenous peroxidase activity (15 min) and non-specific antibody binding blocked by incubation in Dako protein block serum-free reagent  for 30 min, room temperature, prior to incubation with primary antibodies. The following rabbit polyclonal primary antibodies for subunits of the mitochondrial respiratory enzyme complexes were used: complex I , complex-II , complex-III , complex-IV , Caspase-3 , 3-Nitrotyrosine  and VIP  at optimal dilutions of 1:2000, 1:200, 1:2000, 1:2000, 1:500, 1:500 and 1:200 respectively, in antibody diluent . Incubation with the complex-II (SDHA) and Caspase-3 primary antibodies was for 16 h at 4˚C; incubation with VIP complex I, III, IV and 3-Nitrotyrosine antibodies for 3 h at 23˚C. Sections were then washed four times in PBS containing 0.05% Tween 20 and incubated with horseradish peroxidase (HRP) labelled anti-rabbit polymer  for 15 min. Bound polymer was visualized using 0.05% 3,3’-diaminobenzidine hydrochloride (DAB) chromogen  for 10 min, and tissue was counterstained with haematoxylin. Scoring of staining intensity  was performed blinded, and scores of key samples were verified by a second blinded independent observer. Results plotted in the graphs represent the average score of 1 cm distal colon.Immunohistochemistry was performed as previously described . Antigen2). Both the serosal and mucosal chambers were filled with 2 ml KREB’s buffer and gassed with 95% O2 and 5% CO2 at 37 ºC, pH 7.4. The serosal solution also contained 5.1 mM Na-L-Glutamate, 5.7 mM Na-Pyruvate and 10 mM D-Glucose (Riedel-Haen AG) and the mucosal solution contained 5.13 mM Na-L-Glutamate (Merck), 5.7 mM Na-Pyruvate (Sigma-Aldrich) and 10 mM D-Mannitol (BDH Laboratory supplies).Experiments were performed as previously described . Briefly5´GCTGCCTCCCGTAGGAGT-3´) was added to tissue sections and incubated at 45°C in a humidified chamber containing 40% formamide over-night (~18h). Subsequently, the slides were washed with 0.9 M NaCl, 20 mM Tris-HCl pH 7.4 at 50°C for 20 minutes, followed by a brief submersion in room temperature PBS. For co-staining, sections were incubated with monoclonal rabbit-anti-E. coli O152 antiserum (Denka Seiken) diluted 1:100 at 4°C over-night followed by another over-night incubation with Alexa Fluor 488-conjugated goat anti-rabbit diluted 1:100 at 4°C, rinses and reagents were used as mentioned above for immunofluorescence. DAPI was used to visualize DNA as described above.The slides were deparaffinized and dehydrated in 95% ethanol. Hybridization solution (40% (v/v) formamide, 0.1% SDS, 0.9 M NaCl, 20 mM Tris-HCl pH 7.4) containing 10 ng/μl of Cy3.5 5´ labeled eubacteria-specific probe  diluted (1:1000) in blocking buffer. Slides were then incubated for 1 h at RT with Alexa Fluor 488-conjugated anti-rabbit immunoglobulin . DAPI was used to visualize DNA as described above. Pictures were captured with an Eclipse 90i fluorescence microscope (Nikon).g and 4˚C. The supernatant was saved, the pellet resuspended in homogenizing buffer A and centrifuged for 10 min at 1000 g and 4˚C. Supernatants from these steps were pooled and centrifuged for 10 min at 1000 g and 4˚C. The pellet was used for mitochondrial isolation and the supernatant for the nitrite assay. The pellet was resuspended in homogenization buffer A without BSA and EGTA and centrifuged for 10 min at 1000 g and 4˚C. For measurement of mitochondrial respiratory complex activities, mitochondria were resuspended in 50 mM phosphate buffer, pH 7.4; for measurement of membrane potential, mitochondrial ATP and phosphorylation capacity, the mitochondrial pellet was resuspended in isotonic buffer . All aliquots were kept frozen at -20°C and used within a week.Murine colon: Mitochondria were isolated as previously described . BrieflyIn vitro mucosal surface: Mitochondria were isolated as previously described [g and 4˚C. The supernatant was saved, the pellet resuspended and centrifuged again for 10 min at 1000 g and 4˚C. The supernatants from these steps were pooled and centrifuged for 10 min at 1000 g and 4˚C. The supernatant obtained was stored at -20˚C for the nitrite assay and the pellet resuspended in homogenization B buffer containing digitonin (0.02%), centrifuged at 10000 g and resuspended in a buffer appropriate for further experimentation .escribed . BrieflyNADH-ferricyanide reductase (complex-I) activity was measured in accordance with the method of Hatefi  with ferThe activity of succinate cytochrome c reductase (complex II–III) was measured by analyzing the succinate supported reduction of ferricytochrome c to ferrocytochrome c at 550 nm in an assay mixture containing 100 mM phosphate buffer, 1 mM KCN, 2 mM succinate, 0.3 mM EDTA and 1.2 mg/ml cytochrome c (Sigma-Aldrich) in 0.4 ml [μM) in 10 mM phosphate buffer, pH 7.4 [The activity of cytochrome c oxidase (complex IV) was measured by quantifying the rate of decrease of absorbance at 550 nm at room temperature following the oxidation of reduced cytochrome c , 5 mM pyruvate, 10 mM succinate and 10 mM glucose, 5 units of hexokinase (Sigma-Aldrich) added, in total volume of 250 μl, and incubated for 30 min at 37˚C. The reaction was terminated by 5% ice cold trichloroacetic acid (TCA) and the inorganic phosphate was determined spectrophotometrically. Inorganic phosphate content was also assayed in a 0 min sample, where hexokinase addition was immediately followed by treatment with 5% cold TCA. Hexokinase and glucose in the reaction mixture acted as a trap for ATP to maintain the level of ADP in the system and to prevent release of free inorganic phosphate from ATP by phosphatases.Phosphate utilization was measured as previously described . BrieflyATP content was determined in aliquots of mitochondrial suspension using a commercial ATP assay kit .Murine colon: mitochondrial suspensions were incubated for 30 min at 37˚C in isotonic buffer A containing 10 mM succinate, 10 mM pyruvate, 1 mM ADP and 5 μM JC-1 . The dyed mitochondria were then collected by centrifugation, washed with buffer A and resuspended in the same buffer and fluorescence intensity read .In vitro mucosal surface: Mitochondrial transmembrane potential in intact cells was measured using a kit  in accordance with the manufacturer’s instructions.The method of Lowry  was usedAll analyses were performed using GraphPad Prism (version 3·0). Differences between groups were assessed using one-way analysis of variance (ANOVA) with Student Newman-Keuls Multiple Comparison test. Values were expressed as mean ± S.E.M and differences were considered significant if p < 0.05.C. rodentium density in the feces of infected C57BL/6 wild type (WT) mice occurred around day 10, tended to decrease at day 14 and by day 19 and 22 post infection the number of C. rodentium in feces had decreased by log 5–6 to 100–1000 CFU/g feces  compared to total bacteria in feces mirrored the fecal counts, with few O152 positive bacteria present before infection, large amounts present by day 10 and decreasing levels by day 14, and the proportion of 0152 negative eubacteria followed the opposite pattern . Similarly, the mitochondrial parameters measured below were similar in in vitro mucosal surfaces with and without VIP treatment in the absence of other stimuli  reversed the combined inhibitory impact of IFNγ and TNFα on complex-I activity both in uninfected  but not for complex II and III (in situ protein levels of complex-I (MTND6) and complex-IV (CCO-VIc) in response to TNFα and IFNγ under uninfected  and under un-infected conditions . We selected a range of therapeutic treatment regimens that all started at day 5, or after day 5, post infection  at day 10 and 14 post infection (p<0.05), but no VIP treatment regimen ameliorated the colitis (C. rodentium in contact with the colonic epithelium (C. rodentium in the spleen (2/12 VIP vs 3/7 control), although the difference was not statistically significant  that is generated when NO reacts with superoxide (O2.-), alleviates it [of cells , as suppiates it .in vivo infection with C. rodentium. VIP treatment for day 5–14 and 5–10 alleviated the increase (p<0.01 and p<0.001), whereas VIP treatment for day 10–14 had less effect , and infection further increased it  increased during s effect , mirroris effect . Anothernfection . In vivop<0.001, . VIP  was chosen for our murine study as VIP in higher doses (1 and 5 nmol/ mouse) failed to provide beneficial impact on eceptors . AlthougC. rodentium induced colitis at 10 days post infection, although bacterial attachment was not affected [A previous study demonstrated that prophylactic VIP treatment decreased the affected . Togetheaffected ,38 furthaffected , this maC. rodentium in contact with epithelial cells tended to be lower in VIP treated mice compared to non-treated infected mice. That the improved health of the epithelial cells does not have overall beneficial effects on the infection may be due to that other factors fighting infection decrease simultaneously. Firstly, the infection induced mitochondrial damage and apoptosis is likely due to oxidative damage, since the mitochondria targeting antioxidant MitoQ, which scavenges peroxynitrite protects against these effects [C. rodentium, TNFα and IFNγ, both in vivo and in vitro. Although NO cause mitochondrial damage, several studies have shown a role for NO in mucosal defense against pathogens [C. rodentium infection. Furthermore, cellular components of the immune system  are also important for pathogen clearance [C. rodentium infection is unknown. However, the anti-inflammatory action of VIP is exerted via several mechanisms, including inhibition of production of proinflammatory cytokines (i.e. TNFα and IFNγ) and increased production of anti-inflammatory cytokines [Even though VIP had beneficial effects on mitochondria and epithelial cells in the 5–10 day treatment regimen, there was no clear benefit on colitis or infection clearance overall. The only potential beneficial parameters detected were related to pathogen localization: fewer VIP treated mice tended to have systemic infection (2/12) compared to the non-treated infected mice (3/7), and the amount of  effects . VIP redathogens ,60, and athogens –43, indilearance , 44–47. ytokines –18. Furtytokines  indicatiin vivo-like in vitro mucosal surfaces, we found that C. rodentium and TNFα, both individually and more severely in combination with IFNγ, decreased the levels and activity of the complex I and IV enzymes, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation. That VIP is protective in vitro, suggests that the decreased levels of VIP occurring in the colonic epithelial cells during infection may lead to that the infection induced expression of TNFα and IFNγ inflict even greater damage than what might have occurred otherwise. Although our results indicate that VIP i.p. injection is not a promising treatment for infection, we can thus not exclude that a more local treatment, for example restoring VIP in the epithelial cells only, may be more functional.Using C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The loss of VIP is mainly cytokine driven, and VIP supplementation alleviates mitochondrial dysfunction and epithelial apoptosis caused by C. rodentium infection, TNFα and IFNγ both in vivo and in vitro. The protective action of VIP occurs by inhibition of damaging NO-generation caused by the infection, both indirectly via cytokines and directly via pathogens. However, VIP treatment failed to alleviate colitis although it tended to affect pathogen localization in vivo.In summary, we found that that mucosal VIP decrease during murine


Animals were distributed in two experimental groups (n = 10) and fed a standard laboratory diet. Rats in the pterostilbene group were given a dose of 30 mg/kg body weight/d for six weeks. After sacrifice, serum glucose, transaminase, and insulin concentrations were quantified and the liver triacylglycerol content and fatty acid profile was analyzed. Different pathways of triacylglycerol metabolism in liver were studied, including fatty acid synthesis and oxidation, triglyceride assembly, fatty acid uptake, and glucose uptake. With pterostilbene administration, a reduction in insulin concentrations (consequently in the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR)) and hepatic triacylglycerol content were observed. No effects were observed in pterostilbene-treated rats in the activity of de novo lipogenesis enzymes. An improvement in the fatty acid profile was observed in pterostilbene-treated rats. In conclusion, pterostilbene is a useful molecule to reduce liver steatosis. Its delipidating effect is due, at least in part, to reduced fatty acid availability and triacylglycerol synthesis, as well as to an increased very low-density lipoprotein assembly and fatty acid oxidation.Excessive fat accumulation within the liver is known as “simple hepatic steatosis”, which is the most benign form of non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to determine whether pterostilbene improves this hepatic alteration in Zucker ( Excessive fat accumulation within liver is known as “simple hepatic steatosis”, which is the most benign form of non-alcoholic fatty liver disease (NAFLD). It is a major cause of chronic liver disease in western societies, and the burden is expected to grow with the increasing incidence of obesity and metabolic syndrome, which are closely associated with it ,2. In obfa/fa Zucker rat, a model of genetic obesity that exhibits a spontaneous mutation in the leptin receptor. This alteration decreases receptor affinity and changes the transduction signal, leading to the development of severe obesity. Many metabolic syndrome features, such as hyperphagia, hyperglycemia, hyperinsulinemia, hypercholesterolemia, adipocyte hypertrophy, and hyperplasia, and muscle atrophy are also observed in this model [Several animal models have been developed to study fatty liver disease. One of the most commonly used is the is model . Moreoveis model .The current treatment of liver steatosis is based on dietary energy restriction and physical activity ,8. In reAlthough positive effects on liver steatosis have been shown after resveratrol administration, this molecule shows an important problem related to its low bioavailability. Due to its strong metabolism in the intestine and liver, concentrations of resveratrol in plasma and tissues are low after oral administration ,15. As aIn this scenario, the aim of the present study was to determine whether pterostilbene improves steatosis, by using, as an animal model, the genetically obese Zucker rat. The analysis of several potential mechanisms of action underlying this effect was also undertaken.fa/fa) rats at 5 weeks old, purchased from Charles Rivers Laboratories , in accordance with the institution’s guide for the care and use of laboratory animals . The rats were individually housed in polypropylene cages and kept in an isolated room with a constantly regulated temperature (22 ± 2 °C) under a 12:12 h artificial light/dark cycle (light on at 21:00). After a 6-day adaptation period, the rats were randomly distributed in two experimental groups of ten animals each and fed a standard laboratory diet . Rats in the pterostilbene group were orally given this phenolic compound at a dose of 30 mg/kg body weight/d through an orogastric catheter for 6 weeks. Pterostilbene was diluted in 1 mL of ethanolic solution (20%). Rats from the control group (Control) received only the vehicle. All animals had free access to food and water. Food intake and body weight were measured daily. Pterostilbene (99.9% purity) was synthesized according to published procedures [The experiment was conducted with 20 Zucker . Liver and adipose tissue from epididymal, perirenal, mesenteric, and subcutaneous anatomical locations were dissected, weighed, and immediately frozen in liquid nitrogen. All samples were stored at −80 °C until analysis.At the end of the experimental period, and after an 8 to 12 h fasting period, rats were sacrificed by cardiac exsanguination under isoflurane anaesthesia. Serum was obtained from blood samples after centrifugation . A serum non-esterified free fatty acids (NEFAs)  insulin concentration was measured by using the commercial ELISA kit .The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated from basal insulin and glucose values using Matthews’ formula :HOMA-IR Total lipids were extracted from the liver, following the method described by Folch . The lipMoreover, a histological study was performed. Just after killing, a piece of liver was placed in 10% buffered formalin and subsequently embedded in paraffin. Liver sections were stained with haematoxylin and eosin using standard techniques. Two sections per sample (with two images per section) were viewed without knowing the treatment group to which each animal belonged. Biopsies were classified into four grades depending on fat accumulation using the Brunt et al. classification, assigning grade 0 when no fat was found in the liver, grade 1 when fat vacuoles were seen in less than 33% of hepatocytes, grade 2 when 33–66% of hepatocytes were affected by fat vacuoles, and grade 3 when fat vacuoles were found in more than 66% of hepatocytes . Two exp2, 0.5 mM dithiothreitol, and 10 mM N-acetyl-cysteine. Enzyme isolation was performed by centrifugation at 100,000× g, 4 °C for 40 min. The supernatants obtained were stored at −80 °C for further analysis. FAS activity was monitored spectrophotometrically by measuring the NADPH consumption, as previously described [To determine the fatty acid synthase (FAS) activity, 500 mg of liver were homogenized in 5 mL of a buffer (pH 7.6) containing 150 mM KCl, 1 mM MgClescribed ,22, and g, 4 °C for 10 min, the supernatant fraction was again centrifuged at 12,000× g, 4 °C for 15 min. Pellets were resuspended in 500 μL of a buffer (pH 7.4) composed of 70 mM sucrose, 220 mM mannitol, 2 mM HEPES, and 1 mM EDTA. CPT-1a activity was assayed by using the Bieber method [The activity of carnitine palmitoyltransferase-1a (CPT-1a) was assessed in the mitochondrial/peroxisomal fraction. Liver samples (500 mg) were homogenized in 1.5 mL of a buffer (pH 7.4) containing 25 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl. After centrifugation at 700× r method , based or method . CPT-1a To determine the citrate synthase (CS) activity, 200 mg of liver were homogenized in 5 mL of 0.1 M Triethanolamine-HCl buffer (pH 8.0) and diluted tenfold in the same buffer. Homogenates were incubated for 5 min at 30 °C with 0.1 M Tris-HCl of a buffer containing 0.1 mM DTNB, 0.25% Triton X-100, 0.5 mM oxalacetate, and 0.31 mM acetyl-CoA, and readings were taken at 412 nm. CS activity was expressed as nanomoles CoA formed per minute, per mg of protein [To assess the assembly of very low-density lipoproteins by the liver, microsomal triglyceride transfer protein (MTP) activity was determined fluorimetrically by using a commercial kit . MTP activity was expressed as a percentage of triacylglycerol transfer/mg protein/hour .® reagent , according to the manufacturer’s instructions. RNA concentration and quality were determined using a Nanodrop spectrophotometer . Each RNA sample was subsequently submitted to two DNase treatments  to ensure the complete removal of possible contaminating genomic DNA. Next, 1.5 μg of purified total RNA was used for the synthesis of first-strand cDNA, using iScript reverse transcriptase , following the protocol provided by the company.The total RNA from 100 mg of liver samples was isolated using TRIzol−ΔΔCt method [SYBR-Green real-time reverse transcription-polymerase chain reaction technology  was used to perform the relative quantification of the fatty acid translocase/scavenger receptor cluster of differentiation (CD36) and respiratory electron transport chain complex II (cox-2) mRNA expression. β-actin was selected as the invariant internal control for reverse transcription quantitative polymerase chain reaction (RT-qPCR) and subsequent normalization. Specific primers were synthesized commercially . Primer sequences are detailed in t method . Data fr2PO4, and 7.5 mM Na2HPO4; pH 7.4) containing 1 mM PMSF and 0.1 mM iodoacetamide. Proteins were purified from the lysate by centrifugation at 800× g, 4 °C for 5 min and were stored at −80 °C for further analysis.The total number of proteins from 100 mg of liver samples were extracted using ice-cold PBS  was added to each protein sample before carrying out another centrifugation at 14,000× g, 4 °C for 1 min. Pellets were resuspended in 100 μL of a specific buffer, composed of 10 mM HEPES, 0.1 mM EDTA, 1.5 mM MgCl2 6H2O, 420 mM NaCl, glycerol 10% v/v, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.05 mM iodoacetamide and were subsequently incubated at 4 °C under rotation during 30 min, shaking them every 15 min.Nuclear proteins were extracted for sterol regulatory element-binding protein 1 (SREBP-1c) and peroxisome proliferator-activated receptor α (PPARα) quantification, performing a centrifugation at 300× g, 4 °C for 10 min and were stored at −80 °C for further analysis. After subjecting all the samples to the Western blot analysis, total protein content was quantified by the Bradford method. The number of proteins for each desirable target were denatured by heat, fractionated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinyl difluoride membranes , which had been previously activated by a methanol treatment. Next, membranes were blocked with 5% bovine serum albumin (BSA) in 0.01% tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h, before being incubated overnight with the suitable primary antibody dilution at 4 °C. After washing the membranes three times with TBST, they were incubated with an appropriate dilution of horseradish peroxidase (HRP)-conjugated secondary antibody for another 2 h. Finally, after three more washes, specific immunoreactive bands were detected by a chemiluminescent ECL assay kit . The intensity of the bands was determined by densitometric analysis and quantified by a ChemiDoc MP imaging system . Primary antibodies for acetyl-CoA carboxylase (ACC) , phosphorylated ACC  from Cell Signaling Technology , and SREBP-1c , PPARα , diacylglycerol O-acyltransferase 2 (DGAT2) , mitochondrial transcription factor A (mtTFA) , and mitochondrial uncoupling protein 2 (UCP2) monomer and dimer  were purchased from Santa Cruz Biotechnology . A specific secondary HRP-labeled rabbit (1:1000 antibody dilution), mouse (1:1000 antibody dilution), or goat (1:1000 antibody dilution) antibodies were employed depending on the primary antibody origin. β-actin (1:1000 antibody dilution) was used as an invariant internal control for sample normalization.Supernatants were obtained after centrifugation at 14,000× v/v) [2) cartridges (SPE columns) . The correct separation procedure was confirmed by thin layer chromatography.The analysis of the fatty acid composition of hepatic triacylglycerols and phospholipids was performed following a previously described procedure . Frozen samples (0.3 g) of liver were weighed, kept at 0 °C in an ice bath, and homogenized in chloroform:methanol 2:1 (v/v) . After dSodium methoxide in methanol (0.5 M)  was added (500 µL) to lipid species extracts to prepare fatty acid methyl esters (FAMEs). FAMEs were solved in 500 µL of hexane containing 50 ppm of butylated hydroxytoluene as a stabilizer and were transferred to gas chromatography (GC) vials and analyzed with a Hewlett Packard HP6890 gas chromatograph , connected to an automatic injector (Hewlett Packard HP7673), and equipped with a flame ionization detector (FID). Samples were continuously maintained at 4 °C with a refrigerated recirculation bath . The analytical column was a fused silica capillary SP-2380 column , purchased from Supelco . The oven temperature was initially programmed at 150 °C (hold 4 min) and raised to 220 °C at a 4 °C/min rate (hold 25 min). Injection (1 µL) was run in split (40:1) mode. Helium was the carrier gas at constant flow (1 mL/min) and make-up gas for the FID. The injector was maintained at 200 °C and the detector at 250 °C. FAMEs were identified by comparison with retention times with standards , and they were expressed as a percentage of total fatty acids in each lipid species (triacylglycerols or phospholipids).t test was used for comparisons between both experimental groups. Significance was assessed at the p value < 0.05 level. Effect size was calculated according to Cohen’s d formula with the recommendation of 0.2, 0.5, and 0.8 for small, moderate, and large effects, respectively.Results were presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using IBM SPSS Statistics 24.0. All the parameters were normally distributed, according to the Shapiro–Wilk’s test. The Student’s p = 0.04). Interestingly, a lower liver weight was observed in rats treated with pterostilbene. Although the differences were statistically non-significant (p = 0.092), the effect size was rated as moderate (Cohen’s d = −0.78). Triacylglycerol content was also reduced by pterostilbene (−16.9%). When steatosis was evaluated by microscopy, fat infiltration that reached grade 2 in the control rats was reduced to grade 1 in the pterostilbene-treated group (Pterostilbene significantly reduced adipose tissue mass (ed group . Moreoveed group .p = 0.03) and insulin concentrations (p = 0.03). Consequently, HOMA-IR was also significantly reduced (p = 0.02)  . No sign = 0.02) .de novo lipogenesis, in the present study the activity of FAS was measured by spectrophotometry. In addition, the ratio phosphorylated ACC/total ACC, measured by Western blot, was used as an index of ACC activity because this enzyme is unactivated by phosphorylation. In both cases, no effects were observed in pterostilbene-treated rats   A, wherea= 0.003) B.p = 0.001)  .p = 0.01) (p = 0.01)  . Moreove = 0.01) .p = 0.04) (p = 0.01) (p = 0.02)  A. In con = 0.04) B. In add = 0.01) C. Finall = 0.02) D when co = 0.02) E,F.n-6) and linolenic acid (C18:3 n-3) were significantly increased in the liver from rats treated with pterostilbene. Accordingly, the sum of polyunsaturated fatty acids (PUFA) was also increased.The fatty acid profile of the hepatic triacylglycerols was slightly modified by the pterostilbene treatment . Linolein-3), although other polyunsaturated fatty acids  were also increased without reaching statistical significance (cis-11) content was statistically reduced after the pterostilbene treatment. This decrease, along with that of palmitoleic acid (C16:1 cis-9) led to a reduction in total monounsaturated fatty acids (MUFA), which was nearly statistically significant .As regards to the fatty acid profile of hepatic phospholipids, the sum of polyunsaturated fatty acids (PUFA) was also statistically increased in pterostilbene-treated animals. This was mainly due to the increase in docosahexaenoic acid  showed a moderate steatosis because this analysis showed grade 2, according to Brunt scale, in terms of fat infiltration. Pterostilbene partially reduced this alteration because rats from the PT group showed grade 1, meaning that the number of hepatocytes affected was reduced. This delipidating effect was also observed when the hepatic triacylglycerol content was quantified by using a spectrophotometric method; in fact, rats treated with the phenolic compound showed a reduction of 17%. This effect was not due to a reduction in energy intake because no differences were found between both experimental groups. These results show that the presence of two methoxy groups in the pterostilbene molecule allows this phenolic compound to maintain the delipidating effect on fatty liver showed by its parent compound resveratrol. Unexpectedly, no reductions were observed in transaminases in the PT group. Concerning this issue, it is important to emphasize that, although transaminases are commonly used as biomarkers of fatty liver, they are not reliable markers because they can be normal, even in advanced NAFLD ,30. In aOne of the main factors involved in the ethipatogenesis of liver steatosis is insulin resistance. Indeed, it is one of the multiple hits predisposing to the development of NAFLD . This meThe amount of triacylglycerols accumulated in hepatocytes is regulated by various metabolic processes, fatty acid uptake, fatty acid synthesis, and esterification on the one hand (“input”) and fatty acid oxidation and triacylglycerol export on the other hand (“output”). Steatosis occurs when “input” exceeds “output” ,35. In oAs far as fatty acid uptake is concerned, the CD36 gene expression was measured. The CD36 transporter is involved in the uptake of both diet and lipolysis-derived free fatty acids. It should be emphasized that approximately two-thirds of hepatic triacylglycerols are derived from this pathway [de novo lipogenesis. Normally, this metabolic pathway accounts for 5% of hepatic fat content, but when obesity is developed, it can represent up to 25% [de novo lipogenesis was not affected by pterostilbene treatment. In a previous study from our group, we also observed this lack of effect when pterostilbene was administered to Wistar rats, together with a high-fat, high-sucrose diet [de novo lipogenesis [Another source of fatty acids for triacylglycerol synthesis is p to 25% . In factp to 25% . In the ose diet . This faogenesis ,39.DGAT2, one of two enzymes which catalyze the final reaction in the synthesis of triacylglycerols, in which diacylglycerol is covalently bound to long-chain fatty acyl-CoAs, is overexpressed in NAFLD ,41. A siLiver fatty acid oxidation is reduced in obese Zucker rats . When weFinally, the activity of MTP was increased in liver from rats treated with pterostilbene. This enzyme transfers triacylglycerols to nascent apolipoprotein B, producing very low density lipoproteins (VLDL) and removing lipid from hepatocytes. Consequently, it can be stated that this effect contributed to the steatosis reduction induced by this phenolic compound. The increase in MTP activity was not accompanied by enhanced values of serum triacylglycerols, suggesting that other processes taking place in tissues different from liver, and also involved in serum VLDL balance, could be modified by pterostilbene. This would be the case of the uptake of fatty acids from triacylglycerols circulating as VLDL, mediated by lipoprotein-lipase in skeletal muscle and white adipose tissue. A limitation of this work is that the study of tissues other than liver were not included.The role of UCP2 in NAFLD development has been intensively studied, but reported studies are controversial. Some studies have shown that hepatocellular UCP2 expression is increased in NAFLD, indicating its potential role in disease development ,43,44,45Finally, as regards to the lipid profile, hepatic steatosis is characterized by an altered profile with an increase in monounsaturated and a decrease in polyunsaturated fatty acids, mainly those of long -chain docosahexaenoic acid . These mIn conclusion, pterostilbene arises as a useful molecule to reduce liver steatosis. Its delipidating effect is due, at least in part, to reduced fatty acid availability, triacylglycerol synthesis, as well as to an increased very low-density lipoprotein assembly and increased fatty acid oxidation.


We found, in OC, that increased mitochondrial number associated with increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and mitochondrial transcription factor A (TFAM) protein levels, as well as mtDNA content. The OC mitochondria presented an increased maximum length, as well as reduced cristae width and junction diameter, associated with increased optic atrophy 1 protein (OPA1) and prohibitin 2 (PHB2) protein levels. In addition, in OC tissues, augmented cAMP and sirtuin 3 (SIRT3) protein levels were observed. PCA of the 25 analyzed biochemical parameters classified OC patients in a distinct group from controls. We highlight a “mitochondrial signature” in OC that could result from cooperation of the cAMP pathway with the SIRT3, OPA1, and PHB2 proteins.Ovarian cancer (OC) is the most lethal gynecologic cancer characterized by an elevated apoptosis resistance that, potentially, leads to chemo-resistance in the recurrent disease. Mitochondrial oxidative phosphorylation was found altered in OC, and mitochondria were proposed as a target for therapy. Molecular evidence suggests that the deregulation of mitochondrial biogenesis, morphology, dynamics, and apoptosis is involved in carcinogenesis. However, these mitochondrial processes remain to be investigated in OC. Eighteen controls and 16 OC tissues (serous and mucinous) were collected. Enzymatic activities were performed spectrophotometrically, mitochondrial DNA (mtDNA) content was measured by real-time-PCR, protein levels were determined by Western blotting, and mitochondrial number and structure were measured by electron microscopy. Statistical analysis was performed using Student’s  Ovarian cancer (OC) is the most lethal gynecologic cancer in Western countries and includes different histological subtypes, of which the large majority are diagnosed as serous and mucinous . The absIn general, metabolic changes are observed in cancer cells. Previously, the dependence on glycolysis in cancer cells was ascribed to defects in mitochondrial bioenergetics; however, recent data showed that mitochondria are functional in many cancer cells and they contribute, together with glycolysis, to sustain cell proliferation . The mitMitochondrial biogenesis is the process via which cells increase their mitochondrial mass, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is one of the master regulators of this process . Recent In this work, in human serious and mucinous OC tissues, we analyzed several aspects of mitochondrial biogenesis, dynamics, structure, and function in order to acquire molecular insights. We found an increased number of mitochondria, increased maximum mitochondria length, and reduced width and junction diameter of cristae, suggesting alteration of mitochondrial biogenesis, dynamics, and structure, respectively. The alteration of mitochondrial dynamics and structure was associated to an increased level of OPA1 and PHB2 proteins. Increased mitochondrial biogenesis was confirmed by increased PGC1α and mitochondrial transcription factor (TFAM) protein levels, as well as mitochondrial DNA (mtDNA) content. Analysis of respiratory chain complexes revealed a strong decrease in complex I activity. In addition, in OC tissues, we found activation of the cAMP/PKA pathway and an increased level of SIRT3 protein. Interestingly, principal component analysis (PCA) of the 25 analyzed biochemical parameters allowed classifying patients in a distinct group from control (CT) subjects, endorsing a “mitochondrial signature” in OC.The study was conducted on 34 specimens: 18 controls (CT) and 16 OC tissues . Fixed specimens of CT and OC were processed for electron microscopy (EM) analysis. The images obtained by EM showed an increased mitochondria number in OC with respect to CT tissues A,B also. To analt-test) and a non-parametric (Mann-Whitney) test to exclude false positives due to the deviation of the distributions from normality. The results of t-tests with resampling and Mann-Whitney’s tests are in The descriptive statistical analysis showed that OC mitochondria were characterized by an increased maximum length and a decreased cristae width and cristae junction diameter . The resThe increase in mitochondrial length and the decrease in crista width and crista junction diameter in OC tissues prompted us to investigate molecular aspects. The antibody against OPA1, in the CT group, immuno-revealed L and S forms at canonical molecular weight A. A furtThe increase in mitochondrial number prompted us to investigate mitochondrial biogenesis and function. Thus, we analyzed the expression of proteins involved in mitochondrial biogenesis, PGC1α and TFAM, and the content of mtDNA. Furthermore, we investigated cAMP/PKA signaling and SIRT3, which can modulate mitochondrial biogenesis and dynamics ,15,23. QFinally, we performed an exploratory analysis on the whole dataset by PCA clustering . As can More recent studies revealed a key role of mitochondria in tumorigenesis , for botIn this work, we analyzed mitochondria in human ovarian cancer (serous and mucinous) tissues in order to further understand the molecular mechanisms responsible for altered mitochondrial functions. First of all, we performed EM analysis to obtain information on mitochondrial morphology and number. EM analysis revealed, in OC with respect to CT, a decrease in cristae width and cristae junction diameter and an increase in mitochondrial length and mitochondrial number. This suggests an alteration of mitochondrial dynamics, structure, and biogenesis. To evaluate the molecular mechanism at the bases of these alterations, since deregulation of OPA1 processing was reported in OC , OPA1 prThe enhanced mitochondria number in OC tissues can result in an increase in mitochondrial biogenesis. This finding is supported, at a molecular level, by the increase in PGC1α and TFAM protein levels, and the mitochondrial DNA content found in OC. PGC1α, a master regulator of mitochondrial biogenesis, promotes TFAM expression that, in turn, increases the transcription and translation of mitochondrial DNA ,23. IndeSummarizing, we show that serous and mucinous histological types of ovarian cancer tissues are characterized by increases in mitochondrial biogenesis and cristae remodeling. This could be the result of activation of cAMP/PKA and stabilization of SIRT3. In addition, the increased level of SIRT3 works together the increased level of PHB2 for the stabilization of OPA1, which, in addition to its role in dynamics, also controls mitochondrial morphology, keeping the cristae junction narrow, giving more resistance toward apoptosis ,34. HoweInterestingly, PCA of the 25 analyzed biochemical parameters allowed classifying OC patients as a distinct group from controls. We highlight, in the heterogeneity of ovarian cancer, a characteristic “mitochondrial signature”, characterized by increased mitochondrial biogenesis and altered mitochondrial structure that could result from the cooperation of cAMP pathway and the SIRT3, OPA1, and PHB2 proteins. The molecular parameters that define the OC group are interconnected among them and support a “mitochondrial signature”.The study was conducted on 34 subjects: 16 patients with ovarian cancer  and 18 control cases (CT). The control cases were represented by six samples of ovarian tissue and 12 samples of tubal fimbria tissues that are considered the histological origin of ovarian cancer cells . For allSamples were fixed in 2.5% glutaraldehyde and, after an overnight wash in the same buffer, the samples were post-fixed with 1% osmium tetroxide in PBS for 2 h at 4 °C. Fixed specimens were processed for embedding in Epoxy Resin-Araldite(M) CY212 . Semi-thin 2-µm-thick sections were stained with Toluidine blue. Ultra-thin sections were mounted on Formvar-coated nickel grids and stained routinely with uranyl acetate and lead citrate. Images of semi-thin sections were captured using a Nikon photomicroscope equipped with a Nikon Digital sight DS-U1 camera . Ultra-thin sections were observed using a transmission electron microscope Morgagni 268 . The mitochondria were chosen in the mitochondria aggregates in the different areas of cells. The count was performed at 18,000× by using the “touch count” function of the ITEM-SIS (SOFT IMAGING SYSTEM-Olympus) program of the electron transmission microscope. The mitochondria length and the mitochondria cristae parameters (width and junction diameter) were measured at 56,000× and 140,000×, respectively, by using the “arbitrary line” function of the electronic transmission microscope. The data regarding mitochondria number were obtained examining three fields for each sample. For length of mitochondria, 12 mitochondria for each sample were examined. For cristae width, five mitochondria for each sample were examined. For cristae junction diameter, three mitochondria for each sample were examined. All measurements were designed with the software function, indicating the two desired points and the software pressure.The sample tissues (100–400 mg) were homogenized in 0.25 M mannitol, 10 mM Tris, and 0.25 mM phenylmethylsulfonyl fluoride (PMSF) (Buffer A) and sonicated at 30% amperage for 20 s. The proteins were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% fatty-acid-free dry milk in 500 mM NaCl, 20 mM Tris, 0.05% Tween-20  for 3 h at 4 °C and probed with antibodies against OPA1 and COXIV , PHB2 , RIIα-PKA, C-PKA, TFAM , SIRT3 , PGC1α , and β-actin . After being washed in TTBS, the membrane was incubated for 60 min with anti-rabbit or anti-mouse IgG peroxidase-conjugated antibodies. Immunodetection was then performed, after further TTBS washes, with enhanced chemiluminescence (ECL) . Image acquisition was performed by the ChemiDoc imaging system , and densitometric analysis was performed by the Image Lab software .ND1 gene and the reference β-actin gene. The PCR mixture contained the specific primers (800 nM), specific TaqMan probes (200 nM), 5–10 ng of DNA, and 1× Taqman Fast Advanced MMIX (Life Technologies). Amplification conditions were 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of 95 °C for 1s and 60 °C for 20 s. The difference in threshold cycle values ΔCt, namely, CtND1/CtActin, was used as a measure of the relative abundance of the mitochondrial genome. In particular, the mtDNA/nDNA ratio is reported as 2−ΔCt.Total DNA was prepared from 20 mg of tissues using a Wizard Genomic DNA Purification Kit (Promega). MtDNA content was measured by real-time PCR using a QuantStudio 7 Flex real-time PCR (Applied Biosystems). The primers for the mtDNA were ND1-forward (For) (5’–CCCTAAAACCCGCCACATCT–3’) and ND1-reverse (Rev) (5’–TAGAAGAGCGATGGTGAGAGCTAA–3’) and were used together with the mtDNA probe (5’–FAM–CCATCACCCTCTACATCACCGGCC–TAMRA–3’). The primers for β-actin were Actin-For (5’–CCCAGCCATGTACGTTGCTA–3’) and Actin-Rev (5’–CGTCACCGGAGTCCATCAC–3’), and the probe was  were homogenized in Buffer A. The homogenate was then centrifuged at 600× 2, in the presence of 3 mM KCN, 1 μg/mL antimycin, and 200 μM decylubiquinone, using 70 μg of protein, by following the oxidation of 100 μM NADH at 340–425 nm (Δε = 6.81 mM−1∙cm−1). The activity was corrected for the residual activity measured in the presence of 1μg/mL rotenone.The NADH-UQ oxidoreductase activity (complex I) was performed in 40 mM potassium phosphate buffer, pH 7.4, 5 mM MgCl2 in the presence of 20 mM succinate, 2 mM KCN, 65 μM decylubiquinone, and 20 μM cytochrome c, using 50 μg of protein. The cytochrome c reduction was followed at 550–540 nm (Δε = 19.1 mM−1∙cm−1).Succinate-cytochrome c oxidoreductase (complex II + III) activity was performed in 25 mM potassium phosphate buffer, pH 7.4, 5 mM MgCl−1∙cm−1). Enzymatic activity was measured in 10 mM phosphate buffer, pH 7.4, using 50 μg of protein. This rate was inhibited over 95% by 2 mM KCN.Cytochrome c oxidase (complex IV) activity was measured by following the oxidation of 10 μM cytochrome c at 550–540 nm .ATP hydrolase activity was measured by an ATP-regenerating system. Firstly, 100 μg of protein was suspended in a buffer consisting of 375 mM sucrose, 75 mM KCl, 30 mM Tris-HCl pH 7.4, 3 mM MgClThe citrate synthase activity was measured using 50 μg of protein in a solution containing 0.1 M Tris pH 8, 0.2% Triton X-100 in the presence of 0.5 mM 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), 0.5 mM Acetyl-CoA, and 0.5 mM oxaloacetate; the reaction was followed at 419 nm.w/v), centrifuged at 1000× g for 10 min. The supernatants were used to determine cAMP concentration using a direct immunoassay kit (Assay Designs) as described by the manufacturer. Total protein concentration was determined by BioRad protein assay. The cAMP levels in the samples were normalized to the total protein concentration and expressed as pmol/mg protein.For analysis of cAMP level, the tissue samples (80 mg) were homogenized in 0.1 M HCl .All data presented in t-tests, followed by resampling through a bootstrap of t-values  and by non-parametric Mann-Whitney’s tests. Statistical computations were generated by the Real Statistics Resource Pack software (Release 4.3) [The most common descriptive statistics  were computed for the mitochondria number, mitochondrial maximum length, and cristae junction diameter in both the CT and OC samples. Shapiro–Wilk tests were performed for each dataset to assess significant deviations from normal distribution. Levene’s tests for homogeneity of variances were also performed between CT and OC for each variable. Significant differences were estimated by Student’s ase 4.3) . For priase 4.3) ,50. MissIn this work, we performed an analysis of mitochondria in human tissues of serous and mucinous ovarian cancers. We showed that OC tissues exhibit an increased mitochondrial biogenesis and altered cristae structure. The observed increase in mitochondrial biogenesis, in OC, is in agreement with recent works showing an increase in mitochondrial biogenesis in cancer cells ,32. Howe


Concentrations of 1,5-AG has been applied as supporting biomarker to diagnosis of the major forms of diabetes . The serum 1,5-AG reference interval is relevant to the appropriate clinical application of this biomarker. This article contains data regards to serum concentration of the biomarker primarily for healthy subjects, capture from the literature, in different populations. Correlation analysis between 1,5-AG and markers associated with diabetes and its complication were presented. The data was complementary to the study “Reference intervals for serum 1,5-anhydroglucitol in children, adolescents, adults, and pregnant women” . The data present in this article improve the comparisons for 1,5-AG in different conditions and methodologies. Specifications TableValue of the data•Data will facilitate the comparison of 1,5-AG in different studies.•The data showed the correlation among 1,5-AG and relevant parameters associated with diabetes.•The data provide comparison between 1,5-AG reference interval in different ethnicities, ages, gender and methodologies.•These data provide information to researchers and clinical laboratory professionals to improve the 1,5-AG diagnostic use.1In this article, we provide complementary data  of the studied groups , correlaTM; Tomen America, New York, NY, USA) in automated system Labmax 400 analyzer (Labtest Diagnostic).We studied healthy Euro-Brazilian subjects, classified as children (0–14 years old), adolescent (>14 and <18 years old) and adults (≥18 years old). Additionally, we analyzed pregnant women in four gestational periods, <23 weeks; 24–28 weeks, 29–32 weeks and >32 weeks of gestation. 1,5-AG was measure by enzymatic colorimetric method , nourishment  and lipid profile were compatible with healthy subjects .The correlation in healthy subjects between 1,5-AG and glycemia, HbA1c, age, BMI and creatinine were weak or none .22.1The population comprises 2303 unrelated Euro-Brazilian healthy subjects from Curitiba, State of Parana, South of Brazil n = 922) were collected from blood bank donors. Children (n = 580) and adolescent samples (n = 496) were obtained from Public Schools. Healthy pregnant women (n = 305) samples were obtained from the Curitiba Government Laboratory.Adult samples (The normoglycemic criteria applied for selected subjects in the study were fasting glycemia <5.5 mmol/L with an HbA1c range of 20.2–36.6 mmol/mol (4.0–5.7%) for children, adolescents, and adults. For pregnant women a fasting blood glucose <5.1 mmol/L was applied to exclude gestational diabetes.All subjects declared that were not using any medications or drugs.2.2Samples were serum obtained in non-fasting state for adults, children and adolescents, and fasting for those who were pregnant. Blood were collected in BD vacutainers SST II advance vacutainer with silica clot activator/gel (Becton Dickinson Co.). Bloods were separated in less than two hours from venipuncture and the serum stored in an ultrafreezer (−80 °C).2.3Concentrations of 1,5-AG were measured enzymatically with the Glycomark reagent  in an automated system . The reaction details and methodology performance were described in Nowatzkea et al. 2.4Clinical data acquisition and analytical procedures, for laboratory data, have been reported previously 2.5p-values) less than 5% (p < 0.05) were considered significant for all tests.Descriptive statistics, correlation analysis and reference intervals were calculated with MedCalc MedCalc version 17.6 . Probability values (


In frozen-thawed sperm, melatonin inhibited MPTP opening and lactate concentrations and improved sperm viability, motility, acetyl-CoA concentration and adenosine triphosphate (ATP) production. With regard to the underlying physiological mechanism, melatonin suppressed movement of citrate synthase, isocitrate dehydrogenase, oxoglutarate dehydrogenase complex, and F0F1-ATP synthase permeability from mitochondrial to cytosolic fractions induced by MPTP opening; furthermore, it increased mRNA expressions of respiratory chain complex components and activities of complexes I, II, III, and IV and thereby improved oxygen consumption capacity in frozen-thawed sperm. In conclusion, melatonin improved OXPHOS of frozen-thawed ram sperm, attributed to inhibition of cryopreservation-induced MPTP opening.Structural and functional damages to mitochondria of frozen-thawed sperm are a typical cryoinjury, with mitochondrial permeability transition pore (MPTP) formation being the hallmark change. Mitochondria are both a primary synthesis site and principle target for melatonin; this compound can directly inhibit MPTP formation and therefore confer protection at a mitochondrial level. The objective was to determine effects of melatonin on MPTP opening, viability, motility, and oxidative phosphorylation (OXPHOS) of frozen-thawed ram sperm. Ram semen was diluted in glucose-egg yolk buffer with 0 or 10 Cryopreserved semen is a common method for preserving and transporting genetic material in most domestic species . HoweverCryopreservation damages sperm mitochondria, inhibiting metabolism. Based on metabolomic and proteomic analyses, 63% of differentially expressed proteins in sperm with different cryo-tolerance and 23% of differentially expressed proteins between fresh and frozen sperm were associated with regulation of metabolism . FurtherThe MPTP is a multi-component protein aggregate in mitochondria, involving both inner and outer mitochondrial membranes. This complex has two functions: integration of oxidative phosphorylation (OXPHOS) for energy production and induction of cell death when converted into a non-specific channel . OpeningMelatonin (N-acetyl-5-methoxytryptamine) is synthesized from the essential dietary amino acid, tryptophan, in the pineal gland, and involved in the regulation of circadian and seasonal rhythms . It is aOur objective was to determine effects of melatonin on MPTP opening and sperm metabolism, including OXPHOS enzymes distribution, respiratory chain complex activity and oxygen consumption in frozen-thawed ram sperm.All procedures involving animals were approved by the Chinese Academy of Science Animal Care and Use Committee (Protocol No. 20120208). All chemicals were purchased from Sigma Aldrich , unless otherwise indicated.Semen was collected, diluted in freezing solution (± melatonin) and subjected to slow freezing. First, effect of melatonin on MPTP were determined based on detection of opening state and sperm viability and motility. Then, to determine involvement of metabolism in protective effect of melatonin regulating MPTP opening, concentrations of key tricarboxylic acid cycle (TCA) enzymes in mitochondria and cytoplasm, gene expressions of mitochondrial coding respiratory chain complexes subunits and activity of complexes were assessed. Furthermore, oxygen consumption, adenosine triphosphate (ATP), lactate and acetyl-CoA contents were also measured to determine if melatonin improved OXPHOS .ad libitum access to water and to a 60:40 forage:concentrate ration .The experiment was conducted during the breeding season  at the Changling Grassland Farming Ecological Experimental Station of Chinese Academy . Ten Small-tail Han sheep rams  were used. All rams had 9 sperm/mL and >80% motility were retained and pooled. A total of 20 pooled semen samples were used for subsequent analysis.Twice weekly for 10 wk, semen samples from each ram were collected using an artificial vagina (AV) containing water at 38–40°C. During each collection day, 10 rams were continuously collected twice, with a 30-min interval between collections (first ejaculates were discarded and second ejaculates were pooled). A graduated collecting tube attached to the disposable sleeve inside the AV was used to evaluate semen volume. Sperm concentration was calculated using a sperm density meter . Motility was estimated under 400 x magnification using a phase contrast microscope . Ejaculates with volumes of 0.7–2.0 mL, concentrations >2.5 × 10−7 M (frozen+melatonin group) of melatonin.Basic semen extender (Tris-egg-yolk) contained an 80% (v:v) solution of 170 mM glucose, 104 mM sodium citrate, penicillin (10 IU/mL) and streptomycin (10 IU/mL) in distilled water and 20% (v:v) of egg yolk (fresh), whereas freezing solution contained 94% (v:v) of basic extender and 6% (v:v) glycerol. Freezing solution was supplemented with 0 (frozen group) or 109 sperm/mL with corresponding freezing solutions. Diluted semen was chilled to 4°C over a 2-h interval, then kept at 4°C for 80 min (equilibration) and finally loaded into 0.25 mL straws. Filled straws were placed 4 cm above liquid nitrogen for 7 min and then plunged into liquid nitrogen. Samples were stored for at least 2 wk and subsequently thawed (40°C water bath for 15 s) and analyzed.After each collection, pooled semen was incubated at 37°C in a water bath for 30 min, and then divided into multiple aliquots that were diluted to 1.0 × 10in situ indicator of sustained MPTP opening in mammalian cells. In the presence of CoCl2, Cal-AM fluorescence is quenched from the cytosol and nuclear compartments, but remains inside mitochondria. However, when the MPTP opens, Cal-AM fluorescence is lost from mitochondria. Briefly, fluorescence intensity (FI) of Cal-AM was measured to reflect MPTP opening. Motile sperm were loaded with Cal-AM  to a final concentration of 15 mM in the presence of 30 mM CoCl2 and 0.1% Pluronic F127 in a dark chamber in a humidified incubator . After 20 min incubation, sperm were washed twice with phosphate buffered solution (PBS) to remove excess stain and quenching reagent. Samples were assayed within 2 h using a flow cytometer . Data for 20,000 cells per sample were stored in the list mode using FACS Analyzer flow cytometry software (Becton Dickinson); thereafter, these data were passed through a Hewlett Packard computer  with Consort 30 and analyzed by SuperCyt Analyst 3 software .The MPTP opening assay was an established calcein cobalt loading procedure, incubating sperm with calceinacetoxymethyl ester , as described . Cal-AM TM ADP:ATP Ratio Assay Kit , according to manufacturer's instructions. Firefly luciferase is a 62 KD monomer protein that catalyzes oxidation of ATP-dependent luciferase. Through electron transfer, chemical energy is converted into light energy by oxyluciferin, which is positively correlated with amount of ATP. The ratio of ADP:ATP was obtained by quantifying ATP concentration and then converted into ATP in the presence of pyruvate kinase (PK) to quantify ADP concentration. Briefly, 10 mL of washed sperm (1 × 106) were mixed with 90 mL ATP reagent and vortexed for 30 s in a tube. After 1 min, the ATP relative light unit A (RLU A) was recorded and after 10 min, the reading was repeated to obtain ADP background RLU B (prior to measuring ADP). Immediately after reading RLU B, 5 ml ADP reagent was added to the tube and it was vortexed. After 1 min, ADP RLU C was read. We used this equation: ADP:ATP ratio = RLU C—RLU B/RLU A.The adenosine diphosphate (ADP):ATP ratio in sperm was measured with an EnzyLight6 /mL with PBS, centrifuged at 13,000 × g for 2 min, supernatant aspirated with a 1 mL needle tube and filtered with a 0.45 μm pore diameter fiber. Then, 100 μL of standard and sample were added to each well and the plate incubated for 2 h at 37°C. Liquid was removed and 100 μL of Biotin-antibody was added to each well and the plate incubated for 1 h at 37°C. Thereafter, each well was aspirated and washed . For this, each well was filled with 200 μL of Wash Buffer using a squirt bottle and after 2 min, all liquid was removed. After the last wash, residual Wash Buffer was removed by aspirating. Then, 100 μL of HRP-avidin were added to each well and the plate incubated for 1 h at 37°C. This aspiration and wash process was repeated five times. Thereafter, 90 μL of TMB Substrate were added to each well and plates were incubated for 30 min at 37°C (in the dark). After adding 50 μL of Stop Solution to each well, optical density of each well was determined within 5 min, using a microplate reader set to 450 nm. Absorbances were corrected by subtracting background reading. Concentrations of lactate and acetyl-CoA were calculated by interpolating values on a standard curve made for each plate. Each ELISA was performed in triplicate.Concentrations of lactate and acetyl-CoA concentrations were measured with ELISA kits , according to manufacturer's instructions. Briefly, sperm concentration was adjusted to 2 × 106/mL by addition of PBS. Then, 5 μL of 1.0 mM propidium iodide (PI) staining solution was added to diluted samples and they were incubated for 5 min in the dark. Thereafter, samples were centrifuged at 1,000 × g for 10 min, supernatant discarded and PBS added to adjust sperm concentration to 1 × 106/mL. Samples were assayed within 2 h using a flow cytometer . Red fluorescence emission for PI  were measured in the FL2 filter. Two different populations can be recognized in this assay . For each sample, five non-consecutive microscopic fields were randomly chosen on the slide and three slides per sample examined under 400 × magnification using a phase-contrast microscope .For each sample of sperm, the kinematics was determined using a computer-aided sperm analysis system  with the following settings: recording rate of 60 Hz, framers number of 30, minimum contrast of 60, minimum cell size of 10 pixels, low size gate of 0.60, high size gate of 8, low intensity gate of 0.25, high intensity gate of 1.50, non-motile head size of 5 pixels, non-motile head intensity of 55, progressive average path velocity (VAP) threshold of 50 mm/s, slow (static) cell VAP threshold of 10 mm/s, slow (static) cell straight line velocity (VSL) threshold of 0 mm/s, and straightness (STR) threshold of 80%. Cells exhibiting a VAP ≥ 50 mm/s and a STR ≥ 80% were considered progressive. Total motile sperm (TM), progressively motile sperm (PM), immotile (IM), curvilinear velocity (VCL), VAP, VSL, beat cross frequency (BCF), and average lateral (ALH) were assessed. In brief, sperm concentration was calculated using a sperm density meter (Minitube), diluted to 2.0 × 10Sperm cytosolic and mitochondrial fractions were prepared with a Mitochondria/Cytosol Fractionation Kit  according to manufacturer's instructions. Briefly, sperm were washed with 10 mL of ice-cold PBS and centrifuged at 600 × g for 5 min at 4°C. Supernatant was removed, sperm re-suspended in 1.0 mL of 1 × Cytosol Extraction Buffer Mix containing DTT and protease inhibitors and samples were incubated on ice for 10 min. Then, the sperm suspension was transferred to an ice-cold Dounce tissue grinder for 10 min, with sufficient strokes to effectively lyse cells. After microscopic examination to verify that homogenization was complete, homogenate was transferred to a 1.5 mL micro-centrifuge tube and centrifuged at 700 × g for 10 min at 4°C. Supernatant was collected into a fresh 1.5-mL tube and centrifuged at 10,000 × g for 30 min at 4°C. This supernatant was stored as the cytosolic fraction. The pellet was re-suspended in 0.1 mL Mitochondrial Extraction Buffer Mix containing DTT and protease inhibitors, vortexed for 10 s and saved as the mitochondrial fraction. To evaluate separation effect, a 5-μL drop of supernatant and mitochondrial suspension were each mixed with 0.2% Janus Green B dye solution of equal volume and dyed on slide for 3 min, rendering mitochondria blue-green when examined at 1000 X.9/mL and stored overnight at −20°C. After two freeze-thaw cycles to rupture cell membranes, sperm lysates were centrifuged at 5,000 × g for 5 min at 4°C. Supernatant was collected and assayed immediately according to manufacturer's instructions. Each ELISA was performed in triplicate. Absorbances were corrected by subtracting background readings. Concentrations of key OXPHOS enzymes were calculated by interpolating values on a standard curve made for each plate.Concentrations of Citrate synthase (CS), Isocitrate dehydrogenase (IDH), 2-Oxoglutarate dehydrogenase complex (OGDC) and F0F1-ATP synthase in cytosolic and mitochondrial fractions were measured with ELISA Kits . Sperm samples were centrifuged at 1,000 × g for 5 min at 4°C. Re-suspended cells were collected and diluted with PBS (PH 7.0) and re-centrifuged at 1,000 × g for 5 min at 4°C. Collected samples were diluted with PBS (PH 7.0) to 1 × 10+, with activity calculated by measuring (340 nm) rate of NADH oxidation. Reduced coenzyme Q, a catalytic product of complex II, can further reduce 2,6-dichloroindole phenol, which has a characteristic absorption peak at 605 nm. Activity of complex II can be calculated by measuring reduction rate of 2,6-dichloroindole phenol. Complex III has characteristic light absorption at 450 nm; therefore, an increasing rate of light absorption at 450 nm can reflect its activity. Complex IV catalyzes reductive cytochrome C to produce oxidized cytochrome C. Reduced cytochrome C has characteristic light absorption at 550 nm, so the rate of decrease in light absorption at 550 nm reflects activity of complex IV. Specific enzyme activity was expressed as: U/mg prot.Complex I (NADH- CoQ oxidoreductase), complex II (Succinate- CoQ oxidoreductase), complex III (CoQ- cytochrome c oxidoreductase) and complex IV (Cytochrome c oxidase) in mitochondrial fractions were measured with kits , according to manufacturer's instructions. The reaction was assessed by following the decrease in absorbance with a diode-array spectrophotometer (PerkinElmer). Complex I catalyzes dehydrogenation of NADH to NAD6 /mL and they were seeded on 20 wells of a XF24 plastic microplate coated with concanavalin A. However, four wells were left without cells (to determine background corrections). The plate was centrifuged for 2 min at 1,200 × g to ensure uniform adhesion of cells to the bottom of the well. The supernatant was removed and 500 μL of medium added to each well. All OCR and ECAR measurements were performed at 37°C under air. The OCR measurements were made from 42 to 90 min. After 54 min, 1 μM FCCP or mT medium (control group) were added to each well. Furthermore, 1 μM antimycin A and 1 μM rotenone were added to each well at 78 min after collection of samples. The OCR and ECAR values for each well were normalized by the number of sperm present and reported as: amol of O2 min−1 sperm−1 and nano-pH min−1 sperm−1. Basal mitochondrial OCR was calculated by subtracting OCR values obtained after addition of antimycin A and rotenone from OCR values at 54th min. Maximum mitochondrial OCR was calculated by subtracting OCR values obtained after addition of antimycin A and rotenone from OCR values recorded at 78th min in FCCP group. Spare OCR capacity was calculated by subtracting basal OCR values from maximum OCR values. Basal ECAR was calculated averaging ECAR values obtained from 42 to 63 min of the experiment.An XF24 extracellular flux analyzer  was used to measure real time OCR and ECAR. The carbonylcyanidep-trifluo-romethoxyphenylhydrazone  is a proton carrier, making many protons flow back and consuming much oxygen. However, this proton flow does not form ATP. Therefore, increased oxygen consumption after FCCP represents maximum oxygen consumption capacity of mitochondria (indirectly measuring maximum respiratory capacity), whereas the high value of its relative and basic value represents respiration potential. Finally, antimycin A and oligomycin were added; these respiratory chain inhibitors completely prevent mitochondrial oxygen consumption. Sperm concentration was adjusted to 2 × 102, 10 mM of each dNTP and 0.5 mg oligo (dT)15 primers to synthesize cDNA. After the reaction mixture reached 42°C, 20 units of reverse transcriptase was added to each tube and the sample incubated for 1 h at 42°C. Reverse transcription was stopped by denaturing the enzyme at 95°C. The final PCR mixture contained 2.5 μL cDNA, 1 × PCR buffer, 1.5 mM MgCl2, 200 μM dNTP mixture, 1 U of Taq DNA polymerase, 1 μM sense and antisense primers, and 5.0 μL sterile water. The qRT-PCR was conducted using the CFX96TM Real-Time PCR Detection System  under standard conditions. Transcripts of mitochondrial coding complex I (MT-C I): NADH dehydrogenase (ND)1, ND3, ND7, complex III (MT-C III): Cytochrome (Cyt) b, complex IV (MT-C IV): Cytochrome oxidase (COX) I, COX III, and complex V (MT-C V): ATPase (ATP) 6, ATP 8 were quantified in triplicate for each sample, with β-actin used as a reference. Expression levels were calculated using the comparative Ct (2−ddCt) method  and RNase-free DNase used to remove genomic DNA. Integrity and concentration of RNA were determined by measuring absorbance at 260 nm. Total RNA (1.0 μg) from each sample was re-suspended in a 20 μL final volume of reaction buffer, containing 25 mM Tris-HCl, 37.5 mM KCl, 10 mM dithiothreitol, 1.5 mM MgCl) method . PrimersP<0.05 considered significant and Duncan's multiple range test used to locate differences. The homogeneity of variances was checked through Levene test.Statistical analyses were performed using SPASS18.0 for Windows . Initially, a Shapiro-Wilk test was done to test the hypothesis that numerical data were normally distributed. All data were expressed as mean ± SD and were analyzed by analysis of variance (ANOVA), with P < 0.05, P < 0.05, P < 0.05, P < 0.05) viability ((P < 0.05) ADP:ATP ratio in frozen-thawed sperm after melatonin treatment  compared to those of the frozen group, but lower compared to the control group  compared to those of the frozen group, but higher compared to the control group  compared to those of the frozen group, but lower compared to the control group (P < 0.05). In addition, there were higher activities (P < 0.05) of complex I, II, III, and IV  leads to ATP synthesis through the OXPHOS pathway. Expression and activity of respiratory chain complexes determined efficiency of ATP synthesis. As mature sperm are unable to synthesize nuclear genome encoded new proteins, we only detected mitochondrial genome encoded complex subunits by real-time quantitative PCR. In the melatonin group, mRNA expression of nd ATP 8  were hig, and IV  in frozeP < 0.05; P < 0.05; Based on optimizing distribution of key TCA enzymes inside and outside mitochondria and increasing activity and expression of respiratory chain complexes, we analyzed sperm oxygen consumption to further assess respiratory efficiency of mitochondria in frozen-thawed sperm. In the melatonin group, basal OCR, maximum OCR and OCR/ECAR were 45.1, 35.5, and 123% higher  optimizing distribution of OXPHOS enzymes in sperm and (2) increasing activities of respiratory complexes. Consequently, melatonin not only reduced MPTP opening, but also improved mitochondrial metabolism in frozen-thawed ram sperm. Therefore, melatonin was an effective cryo-protectant; it improved OXPHOS to ATP production by optimizing distribution of TCA key enzymes in mitochondria and cytoplasm and by increasing activity of ETC in frozen-thawed sperm. Furthermore, it improved sperm viability and motility, attributed to inhibition of cryopreservation-induced MPTP opening. This study yielded new knowledge regarding cryopreservation-induced reductions in ram sperm quality, and may provide a basis for novel, evidence-based approaches to improve quality of frozen-thawed ram sperm.The datasets generated for this study are available on request to the corresponding author.All procedures involving animals were approved by the Chinese Academy of Science Animal Care and Use Committee (Protocol No. 20120208).YF and RZ conceived and designed experiments and wrote the paper. YF, CZ, HX, and XZ performed experiments. YF and CZ analyzed data. YF, HX, and XZ contributed reagents, materials, and analysis tools. All authors contributed to interpretation of data and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory.Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the β-oxidation pathway, which further increased the total triterpenoid yield . Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L  in shake flask cultures.In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.Here, we systematically engineered The online version of this article (10.1186/s12934-019-1127-8) contains supplementary material, which is available to authorized users. Betulinic acid is a pentacyclic lupane-type triterpenoid that widely exists in plants and possesses many pharmacological activities, such as antiviral  , 2, antiYarrowia lipolytica is shown in Fig. ERG9) and then converted to 2,3-oxidosqualene via a reaction catalyzed by squalene epoxidase (encoded by ERG1). The first step in the exogenous biosynthesis of betulinic acid from endogenous 2,3-oxidosqualene is catalyzed by lupeol synthase (LUS), which is followed by three sequential oxidations catalyzed by lupeol C-28 oxidases , leading to the generation of betulin, betulinic aldehyde, and betulinic acid. As increasing CYPs have been mined by bioinformatics analysis [Saccharomyces cerevisiae. The titer of betulinic acid in yeast could be enhanced to 0.16 mg/L/OD600 by applying a multiple-approach strategy, which included using a novel CYP, employing a better yeast strain and upregulating the expression of key genes in the betulinic acid biosynthesis pathway [AtLUP1 and CYP716Al1 with single-copy integration of ATR2 and overexpression of endogenous ERG1 and tHMG1 could improve betulinic acid production to ~ 28 mg/L in shake flask cultures in S. cerevisiae [tHMG1 and ERG9, betulinic acid biosynthesis was achieved in Y. lipolytica and the yield was improved to 26.53 mg/L by substitution of glycerol for glucose as a carbon source [The biosynthetic pathway of betulinic acid from glucose in analysis , 15, amianalysis –18, and analysis , heterolanalysis , 20–23. analysis  were benrevisiae . FurtherEscherichia coli and S. cerevisiae for the production of value-added compounds. By partitioning the biosynthetic pathway of the diterpene taxadiene into two modules—an upstream methylerythritol-phosphate (MEP) module and a heterologous downstream terpenoid-forming module—and balancing the two modules by using various promoters and gene copy numbers in E. coli, production of the taxadiene was increased 15,000-fold compared to that in the parent strain and a yield of 1.02 ± 0.08 g/L achieved in fed-batch bioreactor fermentations [S. cerevisiae was improved from 0.5 to 12.5 mg/L by engineering MVA and heterologous synthase modules [600 [Multimodular metabolic engineering is an efficient strategy for addressing perturbances such as imbalance in pathway flux, module incompatibility or accumulation of intermediates caused by manipulation of endogenous genes and the introduction of heterologous pathways, and this method enables systematic manipulation of strains and metabolic pathways , 25. Thintations . Similar modules . Recentlles [600 . These sYarrowia lipolytica, as a GRAS  and oleaginous microorganism, has become a potential platform for the production of highly hydrophobic compounds [Y. lipolytica has contributed to the convenience of genetic manipulation in this organism [Y. lipolytica has been used to produce various hydrophobic terpenoids, such as linalool  [Y. lipolytica was even higher than that in S. cerevisiae. For example, maximum production of β-carotene in Y. lipolytica was realized at 6.5 g/L [ompounds , 30 becaompounds , 32 and ompounds , 34. In organism –38. Basealcohol) , α-farnealcohol) , squalenalcohol) , β-carotalcohol) –45. In s 6.5 g/L , which dY. lipolytica for production of the hydrophobic betulinic acid and related triterpenoids by dividing the biosynthetic pathway into four functional modules, including the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. By introducing exogenous genes or overexpressing endogenous key genes associated with these modules, we obtained a 51.87 ± 2.77 mg/L yield of betulinic acid and a 204.89 ± 11.56 mg/L yield of triterpenoids, both of which were the highest yields reported in shake flask cultures.In this study, we used a multimodular metabolic engineering strategy to systematically engineer RcLUS gene from Ricinus communis under the control of the TEFin promoter was integrated into the Ku70 locus (YALI0C08701 g) of Y. lipolytica to produce lupeol  from Betula platyphylla [CrAO (CYP716AL1) from Catharanthus roseus [MtAO12 (CYP716A12) from Medicago truncatula [VvAO15 (CYP716A15) and VvAO17 (CYP716A17) from Vitis vinifera [Ku80 locus (YALI0E02068g), which generated strains YLJCC1, YLJCC7, YLJCC13, YLJCC19, YLJCC25. However, the desired product was not detected  [S. cerevisiae [Y. lipolytica. VvAO15 shared close evolutionary relationships and high homology with BPLO  Fig. b, c. We Finally, because no further improvement was observed upon fusion of CYP with CPR or upon addition of the ER-targeting peptide, the two best production strains from the above optimization, namely, YLJCC5 and YLJCC6 Fig. a, were sY. lipolytica, the quantities of betulinic acid produced by strains YLJCC5 and YLJCC6 remained low. To increase the supply of the 2,3-oxidosqualene precursor, we engineered the MVA module with three structural genes, namely, ERG1, ERG9 and HMG1. These genes were overexpressed individually or in combination. Expression cassettes of these structural genes were integrated into the rDNA site of the Y. lipolytica genome , which is responsible for the first oxygenation step in sterol biosynthesis and is recognized as the key enzyme in this pathway [ERG9), which converts FPP to squalene and represents the branching point of synthetic pathways of triterpenoids, sterols and sesquiterpenoids. The resulting strains, namely, YLJCC52 and YLJCC55, exhibited a further increase in betulinic acid and total triterpenoid production  is a commonly used approach to improve the production of terpenoids because this method can overcome the tight regulation of HMG1 in host cells [HMG1 was more effective than overexpression of tHMG1 for production of the monoterpene limonene in Y. lipolytica [HMG1 improved squalene synthesis 3.2-fold in Y. lipolytica [HMG1 rather than tHMG1 in Y. lipolytica. Our results suggested that overexpression of ERG1 and ERG9 only slightly enhanced betulinic acid production in Y. lipolytica. Further overexpression of HMG1 led to a marked improvement in the yield of the product.Previously, overexpression of naxadiol . It has n plants –57 and βrevisiae . Overexpst cells , 59–61. polytica . Recentlpolytica . Hence, EMC, EMT and Rtme encoding malic enzyme and Gapc encoding G3P dehydrogenase  increased slightly, although this increasing trend was not observed in YLJCC68 and YLJCC69 compared with YLJCC6. In contrast, when Gapc, which is responsible for NADH generation, was overexpressed, triterpenoid production decreased (YLJCC66). These results demonstrated that manipulation of the NADPH supply was more beneficial than manipulation of the NADH supply for betulinic acid production, which might be because NADPH is the direct redox supplier for endogenous MVA and the heterogenous CYP/CPR system. A previous study also demonstrated that improvement of the NADPH supply was an efficient method for improvement of betulinic acid production by introduction of mBDH1, which can increase cellular NADPH level with acetoin supplementation [One of the key factors required for triterpenoid biosynthesis is sufficient redox supply . Here, wentation .Fig. 4EnY. lipolytica, which has been characterized as a nonconventional yeast, is able to accumulate large amounts of intracellular lipids, which seems to be a primary competition pathway for terpene production. To increase the carbon flux into the MVA pathway, we tried to recover acetyl-CoA from lipids by enhancing β-oxidation of fatty acids via overexpression of four structural genes, namely, the triacylglycerol lipase gene (TGL3), long-chain fatty acid transporter gene (PXA1), multifunctional β-oxidation enzyme gene (MFE1), 3-ketoacyl-CoA thiolase gene (POT1), and one regulatory gene, namely, the regulatory gene PEX10, which is necessary for correct peroxisomal biogenesis and morphology for the β-oxidation process  to directly increase the synthesis of acetyl-CoA. In summary, the engineered strains YLJCC71-YLJCC84 were constructed, and the fermentation results are shown in Fig. Acetyl-CoA is the biosynthetic precursor of lipids, terpenes and sterols.  gene PXA, multifuylACL1) has been previously shown to increase the level of cytosolic acetyl-CoA 1.5-fold in Y. lipolytica [ACL1 (YLJCC71) failed to improve the production of triterpenes, while overexpression of ACL2 (YLJCC72) increased the titers of total triterpenoids and betulinic acid 1.23- and 1.30-fold, respectively  exhibited the most significant enhancement of production with a triterpene yield of 66.77 ± 2.79 mg/L. Similarly, this elevated production did not occur in the strains generated from YLJCC6, which might be caused by module incompatibility.Overexpression of the endogenous ATP citrate lyase gene  further increased squalene accumulation 1.72-fold, which might represent an increased carbon flux to the MVA pathway and terpenoid synthesis.To identify the impact of the engineering on cell growth and metabolism, we determined the dry cell weights (DCWs) and the levels of squalene and lipids in the parent strain 201249 and engineered strains marked with *  and acetyl-CoA generation module (overexpressing MFE1). Both betulinic acid and total triterpenoid production achieved the highest reported yields in microbes in shake flask cultures. This study can serve as a foundation for the construction of efficient triterpene production strains of Y. lipolytica.Herein, we systematically engineered E. coli was used for plasmid construction and propagation, and cell growth was performed at 37 °C in Luria–Bertani (LB) broth with 100 mg/L ampicillin or 50 mg/L kanamycin and shaking at 220 rpm. The incipient Y. lipolytica strain ATCC 201249 was kindly provided by Professor Ying-Jin Yuan . All the Y. lipolytica strains in this article 2SO4, 1.7 g/L yeast nitrogen base without amino acids, 2 g/L complete supplement mixture (CSM) lacking uracil (SC-Ura) or leucine (SC-Leu). YPD-hum medium containing 200 mg/L hygromycin was also used to screen the recombined strains, and 20 g/L agar was added for preparation of solid plates. Rich YPD medium, consisting of 50 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract, was used for fermentation.The proper auxotrophic medium used to grow transformants consisted of 20 g/L glucose H600 of 0.2 for 168 h. A 1/5 volumetric ratio of isopropyl myristate (IPM) was added to the shake flasks at 120 h to extract the products. All the titers presented in this research were based on the volume of the fermentation media. All assays were performed in at least triplicate (at least three independent assays for three different clones).The seed cultures were first prepared in 25-mL polypropylene tubes containing 5 mL of YPD medium. After 24 h, the seed cultures were inoculated into a 250-mL shake flask containing 50 mL of rich YPD medium with an initial ODBPLO from Betula platyphylla, CrAO from Catharanthus roseus, MtAO12 from Medicago truncatula, VvAO15 and VvAO17 from Vitis vinifera), five CPR-coding genes , four genes coding enzymes related to NADH or NADPH generation  and sfGFP were codon optimized and synthesized by GenScript  and listed in Additional file ERG1, ERG9 and HMG1), the long-chain fatty acid transporter coded gene PXA1, 3-ketoacyl-CoA thiolase coded gene POT1, multifunctional beta-oxidation enzyme coded gene MFE1, transcription factor coded gene PEX10, ATP citrate lyase subunit a coded gene ACL1, ATP citrate lyase subunit b coded gene ACL2, and triacyl -glycerol lipase coded gene TGL3 were amplified from genomic DNA of Y. lipolytica. All the genes were amplified with the proper sticky ends and incorporated into the corresponding integration plasmids by Golden Gate assembly or Gibson assembly.All the plasmids used in this study are listed in Table Fusion protein-encoding genes were obtained by overlap extension polymerase chain reaction (PCR). All the primers used to construct fusion protein-encoding genes were synthesized by Genewiz Inc. (China) and are listed in Additional file Y. lipolytica using the Zymogen Frozen EZ Yeast Transformation Kit II (Zymo Research Corporation). Two micrograms of linearized DNA were used in the transformation reaction, and then, the cells were harvested and plated on selective solid plates. The Ura3 selection maker was recycled by incubating the transformants in 4 mL of YPD for 4 days and diluting 10,000 times on SC solid medium containing 1.2 mg/mL 5-fluoroorotic acid for 2 days. The genotypes of all the strains were confirmed by colony PCR using KOD FX DNA polymerase . The restriction endonucleases and ligase used for plasmid construction were purchased from NEB. Plasmid extraction and DNA fragment purification were performed using the TIANprep Mini Plasmid Kit and TIANgal Midi Purification Kit, respectively . Gibson assembly was carried out using the ClonExpressII One Step Cloning Kit .All the integrative fragments were transformed into N-methyl-N-(trimethylsilyl) trifluoracetamide (MSTFA) at 80 °C for 1 h and filtering with a 0.22-μm film. Appropriate concentrations of lupeol, betulin  and betulinic acid  were dissolved in IPM to prepare standard curves via GC–MS analysis. The methods used for betulinic acid detection were based on previous studies with some modification [The IPM phase was collected by centrifugation of 10 mL of fermentation broth mixture  followed by filtration with a 0.22-μm film. Then, 50 μL of the resulting extract was trimethylsilylated by mixing with 150 μL of fication , 20. A RExtraction and analysis of squalene were performed according to Guo XJ et al.  with somExtraction and analysis of lipids were performed according to Wang et al. . Cells wFull-length amino acid sequences were obtained from GenBank and aligned by ClustalW. A phylogenetic tree was constructed using the neighbor-joining method with 1000 replicates using MEGA6 software. Amino acid sequence homology analysis was performed with BLASTP.Cell imaging was performed using UltraView Vox  after washing with ddH2O. Excitation/emission wavelengths were 488 nm/530 nm for GFP.Additional file 1: Figure S1. Structures of the endogenous compound 2,3-oxidosqualene and the heterogenous compounds lupeol, betulin, betulinic aldehyde and betulinic acid.Additional file 2: Figure S2. Production of lupeol in the parent strain 201249 and YLJCC0 (expressing RcLUS from R. communis).Additional file 3: Table S1. Triterpenoid production by strains with or without introduction of a plant CPR (LiCPR or MTR). No matter whether heterologous CPRs were expressed, no betulinic acid was observed, which indicated that the native P450 enzymes in Y. lipolytica did not catalyze betulinic acid production.Additional file 4: Figure S3. GC–MS profiles and mass spectra of chemical standards and fermentation extracts from YLJCC6. (a) GC–MS profiles of the (1) lupeol standard, (2) betulin standard, (3) betulinic acid standard, and (4) metabolite extracts of YLJCC6. (b) Mass spectra of lupeol and betulinic acid. Red represents the mass spectrum of the metabolite extracts of YLJCC6, and gray represents the mass spectrum of the standards.Additional file 5: Figure S4. (a) Phylogenetic tree of plant CYPs that were identified or deduced as lupeol c-28 oxidases constructed by MEGA6. (b) Homology analysis of CrAO, MtAO12, VvAO15 and VvAO17 with BPLO by BLASTP.Additional file 6: Figure S5. Confocal image of strains that expressed CYP or CPR fused with sfGFP. sfGFP was fused to the C-terminus of CYP or CPR.Additional file 7: Table S2. Oligonucleotides used in construct plasmids in this study. Table S3. The Codon-optimized gene sequences involved in this study.