Embryo Lipid Accumulation Research Articles

Fetal hyperglycemia and the origin of adult obesity

Fetal Hyperglycemia (FH) plays a critical role in adult obesity. FH occurs upon fetal exposure to high maternal glucose during Gestational Diabetes, resulting in hyperinsulinemia and increased fetal fat deposition. At birth, these infants are larger than normal babies, hypoglycemic and hyperinsulinemic and have a higher risk of adult-onset metabolic disorders such as obesity, Type 2 Diabetes and non-alcoholic fatty liver disease (NAFLD). Although in utero glucose exposure provides evidence in the above-mentioned conditions the molecular mechanisms remain unclear. Using a Zebrafish model of FH, we aim to test the hypothesis that FH increases hepatic de novo lipogenesis causing obesity later in life. Methods: We developed a novel Zebrafish model of FH by exposing embryos to high glucose (4.5% w/v) at day 4 post fertilization (4 dpf) during the last day of embryogenesis. At 5 dpf the glucose media was replaced by regular media with or without the Acetyl-CoA Carboxylase (acca) inhibitor ND646 for additional 24 hrs followed by embryo collection. Adult fish were raised from glucose-exposed embryos on a normal diet. Fat mass was assessed by Echo-MRI and glucose levels by glucometer. The substrate of de novo lipogenesis, malonyl CoA level was quantified by ELISA and triglycerides by colorimetry. Insulin expression (visualized by whole mount in-situ hybridization), lipogenic (acca), beta oxidation [(Carnitine palmitoyltransferase, (cpt1/2)] and gluconeogenic [(Glucagon (gcga), Glucose-6-Phosphatase (G6Pase), Phosphoenolpyruvate carboxykinase (pck1)] markers were quantified by RT-qPCR. Oil-Red-O staining (ORO) was used to detect lipid accumulation and quantified using spectrophotometry, while Nile red staining was used to detect adipocyte accumulation. Results: Zebrafish embryos become transiently hyperglycemic and hyperinsulinemic after 24 hrs of glucose exposure at 5 dpf. Surprisingly, despite being normoglycemic glucose-exposed embryos at 6 dpf showed significantly higher levels of hepatic lipid accumulation (48%) and de novo lipogenesis (acca and malonyl CoA) as well as markers of gluconeogenesis (gcga, G6Pase, pck1) and beta oxidation (cpt1/2), compared to controls. Interestingly, adult fish (3 months) become obese (fat mass, 2.5±0.2 vs 3.7±0.2) and develop epicardial fat deposition, despite being normoglycemic. At the molecular level, adult fish have increased fasting hepatic de novo lipogenesis, as indicated by elevated acca expression and malonyl CoA levels which was associated with higher hepatic triglyceride levels (1.5-fold) compared to adult fish raised from unexposed embryos. ND 646 administration reduced lipid accumulation in embryos. The effect of ND 646 on adult fish is under evaluation. Conclusion: Together our results show that FH induces persistent increase in whole embryonic and adult hepatic de novo lipogenesis and its pharmacological inhibition could be promising therapeutic intervention to rescue FH-induced adulthood obesity. National Institute of Health grant (P20 GM104357) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

OR24-5 Zebrafish in Utero Glucose Exposure Induces Lipid Dysregulation That Is Abrogated by Acetyl CoA Carboxylase or Glycolysis Inhibition

Abstract Background and Aim One in ten pregnancies suffer from Gestational Diabetes Mellitus (GDM). GDM exposes fetus to high uterine glucose that causes fetal hyperglycemia. At birth Fetal hyperglycemia results in hypoglycemia, macrosomia, and larger than normal infants and these infants are predisposed to a number of metabolic disorders such as obesity, Type 2 Diabetes and non-alcoholic fatty liver disease (NAFLD) in adulthood. Although in-utero glucose exposure provides evidence for fetal lipid dysregulation linked to metabolic disorders in adults, the molecular mechanisms are still unclear. Increase in circulating insulin and glucose are implicated in the enhanced activation of hepatic De novo lipogenesis (DNL) in dyslipidemia, hence, we aim to investigate the potential beneficial effect of the pharmacological inhibition of DNL or glycolysis in our well-characterized zebrafish model of fetal hyperglycemia that mimics the metabolic disorders in human GDM against lipid derangement mediated by excess glucose in embryos in a short term and avoid the long-term outcome of obesity. Methods We developed a zebrafish model of fetal hyperglycemia by exposing embryos to high glucose (4.5% w/v) during the last day of embryogenesis from 96 to 120 hours post-fertilization (hpf). Glucose level was detected in embryo homogenates by glucometer. Insulin expression was visualized by whole-mount in-situ Hybridization. Gene expression of glycolytic (Hexokinase) and lipogenic [Acetyl-CoA Carboxylase (acca)] markers by RT-qPCR and malonyl CoA levels (product of DNL) by ELISA were also assessed. Lipid accumulation was detected by Oil-Red-O staining (ORO) and quantified by spectrophotometry. Pharmacological intervention using metformin or ND646 (known inhibitors of acca) or 2-Deoxyglucose, 2DG (Known inhibitor of Hexokinase) with glucose for 24hours were utilized to attenuate fetal hyperglycemia-mediated lipid accumulation in embryos. Results Zebrafish embryos exposed to high glucose for 24 hours are transiently hyperglycemic while normoglycemia is restored 24 hours after removal from glucose media. At 120 hpf embryos exposed to high-glucose demonstrated increased insulin, hexokinase, and acca expression compared to untreated controls. We also observed a significant reduction in malonyl CoA levels in ND 646 and metformin cotreated embryos with glucose compared to controls. Interestingly, quantification of ORO staining revealed a significant increase in lipid deposition in glucose treated embryos compared to controls at 120 hpf (0.2±0.01 vrs 0.13±0.005), which was significantly attenuated in embryos co-treated with glucose and 2-DG- (0.15±0.003) or metformin- (0.11±0.007) compared to glucose-treated embryos. Similarly, a moderate reduction in ORO staining was observed in embryos that were co-treated with glucose and ND646 (0.17 ± 0.006). Conclusion and significance Together our results show that embryonic glucose exposure leads to increased lipid accumulation which can be rescued by blocking glycolysis or de novo lipogenesis. Intervening glycolysis and de novo lipogenesis could be a promising therapeutic approach for modulating fetal hyperglycemia-related lipid dysregulations. Presentation: Monday, June 13, 2022 12:00 p.m. - 12:15 p.m.

Is it possible to alter the embryo lipid accumulation with reduction of fetal bovine serum and use of l-carnitine for in vitro maturation of bubaline oocytes?

SummaryIn vitro embryo production (IVEP) is a procedure that can promote genetic improvement in a short time frame. However, the success rates obtained with this biotechnology in water buffaloes are still inconsistent, and can be associated with the high concentration of lipids in the cytoplasm of oocytes and embryos. The objective of this study was to evaluate the effects of reduced concentration of fetal bovine serum (FBS) and/or use of l-carnitine during in vitro maturation (IVM) on the preimplantation development and lipid accumulation in bubaline embryos. In a first experiment, the lowest concentration of FBS in the IVM medium (0%, 2.5%, 5% or 10%) was determined, and the lowest concentration that maintained good embryo development rates was 5%. In a second experiment, the addition of 5 mM of l-carnitine into the maturation medium was evaluated. The blastocysts produced were submitted to lipid evaluation involving staining followed by observation using optical (Oil Red O) and confocal (BODIPY 493/503) microscopy. No difference was observed between the 5% and 10% FBS groups, which were superior to the 0% and 2.5% groups. Furthermore, the performance of the groups treated with 5% and 10% FBS was better than the groups supplemented with l-carnitine. There was no difference regarding embryo lipid accumulation. The results indicated that it is possible to reduce the FBS concentration to 5% in in vitro maturation medium for production of bubaline embryos, and supplementation with 5 mM l-carnitine does not increase embryo production.

90 LIPID CONTENT OF IN VIVO- AND IN VITRO-PRODUCED JERSEY AND HOLSTEIN CATTLE EMBRYOS AND THE EFFECT OF FORSKOLIN ON EMBRYO LIPID REDUCTION

Jersey embryos have been suggested to have higher lipid content and lower tolerance to cryopreservation. In addition, in vitro-produced (IVP) bovine embryos have darker cytoplasm as a consequence of higher lipid accumulation than in vivo-derived embryos, associated with impaired embryo quality and reduced cryotolerance. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. We hypothesised that the lipid content of in vivo-produced and IVP Jersey embryos is higher than respective Holstein embryos and that forskolin would reduce lipid content of IVP embryos. The objectives of this experiment were (1) to analyse lipid content of in vivo and IVP Jersey and Holstein cattle embryos and (2) to evaluate the effect of forskolin added to IVP culture media. The factorial experimental design used two breeds (Holstein and Jersey) and three embryo production methods (in vivo, IVP, and IVP + forskolin). IVP embryos (n = 27 blastocysts) were collected from super-stimulated donors by routine procedures 7.5 days after AI. IVP embryos (n = 259 blastocysts) were produced by standard procedures; briefly, oocytes were aspirated from 2- to 8-mm follicles from slaughterhouse ovaries and matured for 24 h in SMM medium (BoviPro, MOFA Global, Verona, WI, USA). Matured oocytes were fertilized using semen from two different bulls for each breed, and embryos were cultured in BBH7 medium (BoviPro, MOFA Global) alone or with the addition of forskolin (10 µM) at Day 5 of culture at 38.5°C in 5% O2, 5% CO2, and 90% N2. The lipid content of embryos was quantified by staining Day 7 blastocysts with 1 μg mL–1 Nile red dye (580–596 nm), after which a digital photograph of the equatorial part of the embryo was taken at 40×, and fluorescence intensity (FI) was measured with Image Pro software. Data (Table 1) were analysed by ANOVA, and means were compared using Tukey’s HSD. Jersey and Holstein IVP embryos had higher lipid content than Holstein in vivo-produced embryos (P < 0.05), but were not different than Jersey in vivo-derived embryos (P > 0.1). Forskolin lowered the lipid content (P < 0.05) of both IVP Jersey and Holstein embryos and was not different (P > 0.1) than in vivo-produced embryos. Addition of forskolin to embryo culture media has the potential to lower embryo lipid accumulation and possibly improve embryo viability and cryotolerance of IVP embryos. Further studies including cryopreservation and transfer of IVP + forskolin embryos to recipients are necessary to corroborate the findings of the present study. Table 1.Fluorescence intensity of in vivo-produced and IVP Jersey and Holstein embryos

Reduction in cytoplasmic lipid content in bovine embryos cultured in vitro with linoleic acid in semi-defined medium is correlated with increases in cryotolerance.

We examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 μM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P < 0.05) in the embryonic development rate ( 25.8% versus LA: 18.5%), but the proposed system was effective in promoting the decrease (P = 0.0130) in the intracellular lipid content ( 27.3 ± 0.7 versus LA: 24.6 ± 0.7 arbitrary fluorescence units of embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming ( 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.

Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos.

Proper oocyte maturation is crucial for subsequent embryo development; however, oocyte mitochondrial and lipid-droplet behaviour are still poorly understood. Although excessive lipid accumulation during in vitro production (IVP) of bovine embryos has been linked with impaired cryotolerance, lipid oxidation is essential for adequate energy supply. Fetal bovine serum (FBS) and bovine serum albumin (BSA) are supplements used during IVP, containing high and low lipid content, respectively. This study aimed to understand how these supplements influence oocyte mitochondrial and lipid behaviour during in vitro maturation (IVM) in comparison to in vivo maturation, as well as their influence on development rates and embryo lipid accumulation during IVP. We demonstrate that only in vivo-matured oocytes maintained correlation between lipid content and active mitochondria. IVM media containing FBS increased total lipid content 18-fold and resulted in higher lipid accumulation in oocytes when compared with media with BSA. IVM using a lower FBS concentration combined with BSA resulted in satisfactory maturation and embryo development and also reduced lipid accumulation in blastocysts. In conclusion, IVM causes changes in mitochondrial and lipid dynamics, which may have negative effects on oocyte development rates and embryo lipid accumulation. Moreover, decreasing FBS concentrations during IVM may reduce embryo lipid accumulation without affecting production rates.

238 THE ROLE OF PHOSPHODIESTERASE (PDE) 5A AS MEDIATOR OF LIPOLYSIS IN BOVINE OOCYTES AND ITS EFFECT ON THE DEVELOPMENT AND LIPID CONTENT OF EMBRYOS PRODUCED IN VITRO

Intracellular levels of cyclic adenosine monophosphate modulators (cAMP) and cGMP, in adipocytes, are important for the regulation of the lipolysis rate. The phosphodiesterases (PDE) control cGMP and cAMP levels by degradation. Different PDE isoforms are expressed in bovine oocytes and cumulus cells. Previously, we found that using an inhibitor of PDE5A (sildenafil, SILD) increased cGMP levels in bovine oocytes during in vitro maturation (IVM). In the current study we investigated if inhibition of PDE5A during maturation reduces the lipid content in IVF embryos. For this, oocytes were cultured for 24 h in maturation medium with 10% FCS and 10–7 M SILD (treatment I), 10% FCS (treatment II) and 0.4% BSA (control; N ± 160 COC/groups submit to IVF). After COC were in vitro fertilized, cleavage (Day 4) and blastocyst rates (Day 7) were measured. Blastocysts were stained with Nile Red (1 μg mL–1) for lipid content quantification, by mean fluorescence intensity per μm2, measured in the ImageJ program (fluorescence intensity, f.i.). Four replicates were transformed to log10 and subjected to statistical analysis using the SAS system (SAS Institute Inc., Cary, NC, USA) by ANOVA followed by Tukey test with a significance level of 5%. No difference in cleavage (Day 4) and blastocyst (Day 7) rates were observed in all groups (82 and 41.9%, respectively), showing that presence of FCS, SILD, or both in IVM medium did not affect embryo development. Treatment I had higher lipid content (40.35 f.i.) than treatment II (31.12 f.i.), which in turn was also superior to control (22.31 f.i.). According to the results, the presence of FCS in IVM media generates embryos with higher lipid content, and association of FCS and SIL further increased lipid content. Although inhibition of PDE5 increases cGMP levels and leads to higher lipolysis, such an effect was not observed when SIL was used as the PDE5 inhibitor. Reasons for such findings are still unclear, but a possibility would be the activation of a negative feedback mechanism by the increased cGMP generated by SIL, because this nucleotide activates PKG, which in turn inhibits cGMP synthesis by guanylate cyclase. During development the lower cGMP levels could reduce lipolysis, resulting in increased lipid accumulation in embryos. Further studies are needed to address this possibility.

Mitochondrial content and gene expression profiles in oocyte-derived embryos of cattle selected on the basis of brilliant cresyl blue staining

The aim of this study was to investigate the developmental rate, lipid and mitochondrial distribution and gene expression in oocyte-derived embryos selected on the basis of brilliant cresyl blue (BCB) staining. Lipid content and mitochondrial distribution in Day 8 blastocysts were evaluated by fluorescence intensity, while gene expression was analyzed by real-time PCR. The proportion of blastocysts (30.9%) was greater (P<0.05) in BCB+ than in BCB− oocytes (13%) but not different (P>0.05) from the control group (28.2%). Total cell number was also greater in BCB+ (155.1±36.2) than in BCB− (116.6±40.5) and control (127.5±35.7) blastocysts. Furthermore, the apoptotic cell number was less in BCB+ (3.7±4.4) than in BCB− blastocysts (8.7±8.7) but not different from the control group (5.9±3.9). BCB+ embryos contained more mitochondria compared to BCB− embryos (P<0.05). There was no significant difference in intercellular lipid accumulation in embryos from all groups. Interferon-τ (IFNτ), transforming growth factor β1 (TGFB1) and secreted seminal-vesicle Ly-6 protein 1 (SSLP1) gene expression was greater in BCB+ than in BCB− blastocysts. By contrast, Bcl2-associated X protein (BAX) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) gene expression was greater in BCB− than in BCB+ and control embryos. In conclusion, oocyte-derived embryos selected on the basis of BCB staining showed differences in developmental rate, quality, mitochondrial content and target gene expression compared to control embryos.

Cryosurvival of bovine blastocysts is enhanced by culture with trans-10cis-12 conjugated linoleic acid ( 10t, 12c CLA)

An excessive lipid content in embryo cells is a consequence of embryo culture in the presence of serum which is suggested to be responsible for their high susceptibility to cryopreservation. The objective of the present study was to examine the effects of supplementing serum-containing culture media with trans-10 cis-12 conjugated linoleic acid ( 10t, 12c CLA) on embryo lipid accumulation and its subsequent cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro (IVF = day 0). On day 1, presumptive zygotes ( n = 3390) were randomly placed in: (I) (MS), granulosa cell monolayer cultured with M199 and 10% serum; (II) (SCLA), granulosa cell monolayer cultured with M199, 10% serum and 100 μM 10t, 12c CLA and (III) (SOF), modified synthetic oviduct fluid, where embryo culture proceeded for 8 days. Cleavage rates or D7/D8 embryo quality did not vary among treatments. D7/D8 embryo production rate was significantly ( P < 0.001) lower in SOF (17.9 ± 1.6%) than in groups MS (29.8 ± 2.5%) and SCLA (27.8 ± 2.0%). After cytoplasmic lipid droplets observation under Nomarski microscopy, classified embryos were the leanest when cultured in SOF, intermediate in SCLA and the fattest in MS ( P < 0.02). Post-thawing intact blastocyst rates where significantly higher in the SCLA group (84.7 ± 4.1%) than in SOCS (50.3 ± 4.8%, P = 0.0007) or SOF (65.3 ± 6.9%, P = 0.03) groups. Post-thawing re-expanding rates were significantly lower when embryos were cultured in MS (34.7 ± 3.7%) than in SCLA (63.7 ± 5.3%, P = 0.0006) or SOF (49.0 ± 4.6%, P = 0.04). Moreover, re-expanding rates were lower ( P = 0.05) in SOF than in SCLA cultured embryos. These results clearly show that addition of CLA to serum-containing media reduced lipid accumulation during in vitro culture and significantly improved cryopreservation survival.