Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and metabolic dysfunction in rat male offspring

Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and... Background: Maternal overnutrition including pre-pregnancy, pregnancy and lactation promotes a lipotoxic insult leading to metabolic dysfunction in offspring. Diet-induced obesity models (DIO) show that changes in hypothalamic mitochondria fusion and fission dynamics modulate metabolic dysfunction. Using three selective diet formula including a High fat diet (HFD), Cafeteria (CAF) and High Sugar Diet (HSD), we hypothesized that maternal diets exposure program leads to selective changes in hypothalamic mitochondria fusion and fission dynamics in male offspring leading to metabolic dysfunction which is exacerbated by a second exposure after weaning. Methods: We exposed female Wistar rats to nutritional programming including Chow, HFD, CAF, or HSD for 9 weeks (pre-mating, mating, pregnancy and lactation) or to the same diets to offspring after weaning. We determined body weight, food intake and metabolic parameters in the offspring from 21 to 60 days old. Hypothalamus was dissected at 60 days old to determine mitochondria-ER interaction markers by mRNA expression and western blot and morphology by transmission electron microscopy (TEM). Mitochondrial-ER function was analyzed by confocal microscopy using hypothalamic cell line mHypoA-CLU192. Results: Maternal programming by HFD and CAF leads to failure in glucose, leptin and insulin sensitivity and fat accumulation. Additionally, HFD and CAF programming promote mitochondrial fusion by increasing the expression of MFN2 and decreasing DRP1, respectively. Further, TEM analysis confirms that CAF exposure after programing leads to an increase in mitochondria fusion and enhanced mitochondrial-ER interaction, which partially correlates with metabolic dysfunction and fat accumulation in the HFD and CAF groups. Finally, we identified that lipotoxic palmitic acid stimulus 2+ in hypothalamic cells increases Ca overload into mitochondria matrix leading to mitochondrial dysfunction. Conclusions: We concluded that maternal programming by HFD induces hypothalamic mitochondria fusion, metabolic dysfunction and fat accumulation in male offspring, which is exacerbated by HFD or CAF exposure after weaning, potentially due to mitochondria calcium overflux. Keywords: Maternal overnutrition, Diet induced obesity (DIO), Hypothalamus, Mitochondria, Mitochondria dynamics, Fusion, Fission * Correspondence: acm590@hotmail.com; alberto.camachomr@uanl.edu.mx Departmento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autonoma de Nuevo Leon, Monterrey, Mexico Unidad de Neurometabolismo, Centro de Investigación y Desarrollo en Ciencias de la Salud, Universidad Autónoma de Nuevo Leon, Monterrey, Mexico Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 2 of 16 Background weaning by selective diets might modulate mitochondria Maternal obesity in humans associates with an increased fusion and fission dynamics in male offspring leading to risk of obesity metabolic-related disorders in offspring [1, metabolic dysfunction. 2]. It is known that obesity and maternal overnutrition create changes in uterine milieu during pregnancy leading Methods to developmental alterations and defects in organ function Animals, diets and nutritional programming model in and metabolism in offspring [3, 4]. Also, maternal nutri- offspring by HFD, CAF and HSD exposure tional programming by hypercaloric diets exposure that All the experiments were first performed using 8– simulate Western diets, such as high fat diet (HFD), cafe- 10 weeks-old female Wistar rats, 200–250 g (n = 24). teria diet (CAF) or high sugar diet (HSD) in murine Animals were handled according to the NIH guide for models, change offspring metabolism leading to insulin the care and use of laboratory animals (NIH Publications resistance, type 2 diabetes mellitus (T2DM) [5–7], cardio- No. 80–23, revised in 1996), with approval of the local vascular diseases and hypertension [8], non-alcoholic liver Animal Care Committee (BI0002). All efforts were made diseases and steatohepatitis [9, 10]. Defective molecular to minimize the number of animals used and their suf- pathways related to maternal programming by nutrient fering. Rats were housed individually in Plexiglas style oversupply can lead to failure in mitochondria dynamics cages, maintained at 22–23 °C and 12-h light/dark cycle. including modifications in mitochondria fission and Water and food was available ad libitum. Animals were fusion, which are potentially linked to metabolic com- acclimated to the animal facility 7 days prior to diets ex- promise and disease susceptibility [11]. posure. Females rats were randomized into four dietary Diet-induced obesity murine models alter mitochondrial groups: Control diet, HFD diet, CAF diet and HSD function and dynamics in selective organs including mus- diet (Table 1). The formulation of diets was: Control that cles, adipose tissue, liver and brain [11]. In particular, ma- contained a caloric density of 3.35 kcal/g divided in 71% ternal obesity in rodents is associated with altered carbohydrates, 11% lipids and 18% proteins (Research mitochondria function, reactive oxygen species generation Diets, New Brunswick, NJ, Cat. D12450B). High-fat diet and an increased mtDNA and mitochondrial biogenesis in (HFD) contained a caloric density of 4.9 Kcal/g divided oocytes and zygotes [12]. Likewise, oocytes and blastocysts in 45% lipids, 20% proteins and, 35% carbohydrates. from obese mice show reduced mitochondrial membrane Cafeteria (CAF) diet was made of liquid chocolate, bis- potential, high levels of autophagy and reduced mtDNA cuits, bacon, fries potatoes, standard diet and pork pate and mitochondrial biogenesis [13, 14], potentially linked based on a 1:1:1:1:1:1:2 ratio, respectively; total calories to hepatic lipotoxic insult in fetuses of obese females [13]. 3.72 kcal/g in 39% carbohydrates, 49% lipids, 12% pro- In fact, mitochondrial function in offspring after maternal teins and 513.53 mg of Sodium. High sugar diet (HSD) programming seems to be sex-dependent, showing insulin composition was Standard diet and condensed milk on a resistant and oxidative stress in males in compare with fe- 1:1.5 ratio, respectively; total calories 3.39 kcal/g in 69% males [15, 16]. Finally, in humans, placentas of obese carbohydrates, 18.5% lipids, 12.5% proteins and 228 mg mothers show a decrease in mtDNA and mitochondrial of Sodium. It is important to point out that these diets dysfunction which correlate with metabolic dysfunction in appear to be found in human population. After offspring [17]. randomization, female rats were fed for 9 weeks, includ- Mitochondrial functions are modulated by fission and fu- ing 3 weeks of pre-mating, mating, birth, and lactation. sion dynamic processes which assist to maintaining mito- Rats were mated with 12–14 weeks old Wistar males, chondria homeostasis [18]. Mitofusin 1 and 2 (MFN 1 and 300–350 g, during two days. Also, it is important to 2) and OPA1 and DRP1 modulate fusion and fission pro- highlight that body weight was quantified in these fe- cesses, respectively, helping to reduce cellular stress by join- males every week during pre-mating and gestation (See ing mitochondria or creating new mitochondria to assists Fig. 1a). We registered body weight of all offspring at deficient mitochondria [18]. Maternal programming by birth (approximately 15 rats/litter) and at the age of HFD exposure in rodent dams, decrease expression of 3 weeks we euthanized female offspring. Male offspring OPA1 and DRP1 proteins in skeletal muscles of female off- was grouped into 10–12 subjects per group and were al- spring and disrupt mitochondrial function in male offspring located into two groups: Group 1) exposure to Chow [19, 20], which might be transmitted across three genera- control diet to analyze maternal programming, including tions [20]. Furthermore, we and others have reported that three groups: Maternal HFD and offspring Chow control obese mice showed changes inMFN2expressioninhypo- diet (HFD-C), Maternal CAF diet and offspring Chow thalamus and other tissues [21–24], and importantly mito- control diet (CAF-C) and Maternal HSD and offspring chondria elongate in a Mfn1/2 dependent manner in Chow control diet (HSD-C); Group 2) exposure to the Agouti-related peptide neurons [25]. Here, we hypothesized same diet of their mothers until the age of 8 weeks, that maternal nutritional programming before and after resulting in three groups: Maternal HFD and offspring Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 3 of 16 Fig. 1 Effect of nutritional programming on dams and male offspring weight. a) Animal model. We fed female Wistar rats for 9 weeks according to the schedule to promote maternal programming or we fed offspring with hypercaloric diet exposure after weaning. b) Maternal body weight was followed for 6 weeks (pre-mating, mating and gestation) (Control n = 7, HFD n = 7, CAF n = 6 and HSD n = 4). c) Birth weight of offspring at day 0. d) Body weight after weaning for Control versus HFD-C, CAF-C, HSD-C for maternal programming groups, where HFD, CAF and HSD are maternal diet and C is control chow diet of offspring. e) Body weight after weaning for Control versus offspring fed with HFD, CAF and HSD after weaning (HFD-HFD, CAF-CAF, HSD-HSD groups). Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 HFD (HFD-HFD), Maternal CAF diet and offspring CAF Glucose tolerance and insulin tolerance test (GTT, ITT) diet (CAF-CAF) and Maternal HSD and offspring HSD assessments (HSD-HSD). We compared these groups with a group To test if maternal programming by selective hypercalo- where females and their offspring were fed with Chow ric diets leads to alterations in the tolerance of glucose Control diet. A total of seven experimental groups were and insulin, the GTT and ITT tests were carried out in included: Control, HFD-C, CAF-C, HSD-C, and the offspring. Males were 8 h–12 h fasted and then were HFD-HFD, CAF-CAF and HSD-HSD. Body weight, food intraperitoneally injected with 40% glucose body weight and calorie intake were quantified in offspring from or 1 U of insulin/100 g body weight. Blood glucose levels 3rd-7th week following by metabolic assessments as de- were quantified at 0 min, 15 min, 30 min, 45 min, scribed below (see Fig. 1a for details). The daily calorie 60 min, 90 min, and 120 min, as described previously intake was calculated from the weight of food consumed [26]. These tests were performed at the age of 7 weeks multiplied by the calories/gram of the food. for GTT and 8 weeks for ITT in each male rat. Table 1 Diet Composition Diet Percentage of macronutrients Ingredients Control Carbohydrates 71% Cystine 0.14 g, Coline 0.19 g, Vitamins 1.89 g, Celulose 2.89 g, Minerals 4.73 g, Soybean oil Lipids 11% 5.16 g, Starch 32.59 g, Dextrin 16.60 g, Sucrose 16 g, Casein 19.87 g per 100 g Proteins 18% Caloric density 3.35 kcal/g HFD Carbohydrates 35% Cystine 0.17 g, Coline 0.23 g, Vitamins 2.99 g, Celulose 1.72 g, Minerals 5.57 g, Inulin 1.72, Lipids 45% Soybean oil 5.75 g, Dextrin 29.46 g, Sucrose 10.35 g, Casein 24.14 g, Lard 17.72 g per 100 g Proteins 20% Caloric density 4.9 kcal/g CAF Carbohydrates 39% Lipids 49% Proteins 12% Chow diet 14.29 g, liquid chocolate 14.29 g, biscuits 14.29 g, bacon 14.29 g, fries potatoes Caloric density 3.72 kcal/g 14.29 g, pork pate 28.58 g 1:1:1:1:1:1:2 ratio per 100 g HSD Carbohydrates 69% Chow diet 40 g, condensed milk 60 g 1:1.5 ratio per 100 g Lipids 18.5% Proteins 12.5% Caloric density 3.39 kcal/g Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 4 of 16 Tissue samples collection particular interest were the following transitions: 16:0/36:4 Male rats were sacrificed by decapitation at 9 weeks of (872.8/599.6), 18:0/34:1 (878.8/577.5), 18:2/36:2 (900.9/ age. Blood samples were collected in 500 μL tubes 603.6) and 18:0/36:2 (904.9/603.6). Six species were present (Beckton Dickinson) and plasma fraction was isolated by at high concentrations, requiring quantification from the centrifugation at 4 °C and frozen at − 80 °C. Hypothal- [M + 1] isotopologue due to saturation of the detector by amus was dissected and divided into the left hemisphere the mono-isotopic ion. These were 16:1/34:2 (847.8/576.6), (for RNA extraction and gene expression analysis) and 18:1/32:1 (849.8/550.5), 18:1/32:0 (851.8/552.6), 18:2/34:1 right hemisphere (frozen immediately at 80 °C for west- (875.8/578.6), 16:0/36:2 (877.8/604.6) and 18:1/36:2 (903.9/ ern blot analysis). Also, liver and retroperitoneal white 604.6). The concentration of each species was calculated adipose tissue was measured and collected. using MultiQuant 3.0.1 software (AB SCIEX, Framingham, MA, USA) by relating the peak area of each species to that Plasma biochemistry determination of the internal standard. Glucose was determined by glucose strips and Accu-check ® (Roche, Cat. 05987270), insulin (Millipore Western blot analysis Inc., Cat. EZRMI-13 k) and leptin (Millipore Inc., Cat. Frozen hypothalamus was homogenized in 500 μllysis buf- EZML-82 K) were determined by Elisa kits according to fer as described previously with minor modifications [27]. manufacturers’ instructions. Samples were subjected to SDS–PAGE, nitrocellulose membranes blocked for 2 h at RT in TBS-T buffer (10 mM Mass spectrometric determinations of triglycerides Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5) containing 5% Triglycerides were extracted from 10 μL of plasma using a BSA (Santa Cruz Biotecnology, Inc., cat. sc2323) and incu- single phase extraction method as described in Fuller et al. bated overnight with primary antibodies at 4 °C in TBS-T (2015), with the exception that 100 pmol of glyceryl tri- Buffer 1% BSA: anti-MFN2 (Abcam, Product code heptadecanoate (TG (17:0/17:0/17:0); Sigma-Aldrich, Cat. ab56889, 1:1000), anti-Drp1 (Cell Signaling, Cat. 8570, T2151) was added to each sample as the internal standard. Dilution 1:2000) and B-actin (Cell Signaling, Cat. 8457, Extracted triglycerides were first separated on a RRHD Dilution 1:3000). Anti-mouse and anti-rabbit horseradish Eclipse Plus C18 column (2.1 × 150 mm; 1.8 μm) main- Peroxidase-conjugated were used as secondary antibody in tained at 40 °C. The samples were maintained at 16 °C TBS-Buffer 5% BSA (Cell Signaling, Cat. 7076S & Cat. with 3 μL injected into the mobile phase which was at a 7074P29, Dilution 1:3000). Proteins were detected by flow rate of 0.4 mL/min. Mobile phase A consisted of 60% chemiluminescence in the Chemidoc XRS+ System H O, 40% CH CN containing 10 mM NH COOH and (BioRad) using Clarity Western ECL Blotting Substrates 2 3 4 solvent B was 90% (CH ) CHOH, 10% CH CN containing (BioRad, Cat. No. 1705061). Images were quantified densi- 3 2 3 10 mM NH COOH. Mobile phase conditions were 90% tometrically with ImageJ Software 1.50i (Wayne Rasband, solvent A and 10% solvent B at injection, which was National Institutes of Health, Bethesda, MD, USA). linearly ramped to 50% by 6 min and then to 100% solvent B at 24 min. This was maintained for 3 min before return- RNA isolation and real time (RT)-PCR ing to 90% solvent A at 28 min, where the column equili- RNA extraction from hypothalamic samples was performed brated for 4 min prior to the next injection. as described previously [28]. RT-PCR was performed by For the first 4 min column flow was diverted to waste be- High-Capacity cDNA Reverse Transcription Kit (Applied fore being directed into the electrospray source (ES biosystems, Cat. 4,368,814) using random primers and 5500 V) of an AB SCIEX QTRAP 6500 triple quadrupole following standardized protocols. tandem mass spectrometer with an ion source temperature of 250 °C. Nitrogen was used for curtain gas, 25 units; colli- Quantitative PCR sion gas set at medium; nebulizer gas 1, 20 units and auxil- Based on that, obesity modulates ER stress and mitochon- iary gas 2, 40 units. Declustering potential was 120 V; dria fusion and fission dynamics markers in animal models, entrance potential, 8 V, collision energy, 36 V and collision we identified changes in mRNA of these markers in the off- exit potential, 26 V. Individual species of triglycerides were spring exposed to maternal programming. We performed a measured by multiple reaction monitoring in positive ion quantitative PCR using cDNA (10 ng), Light Cycler SBYR mode using the ammonium adduct and corresponding green 480 Master Mix (Roche LifeScience, Product No. fragment arising from the neutral loss of one fatty acid. As 04707516001) and the following primers: MFN2 Forward such, only the sum composition (i.e. total number of car- (5’-CCATGTGTCGCTTATCCTTCT-3′), Reverse (5’-TGA bons and double bonds) of the remaining two remaining CTCCAGCCATGTCCAT-3′); Itpr Forward (5’-CACCTA fatty acyl chains could be determined. Sixty three transi- TGACCACACTGTCTC-3′), Reverse (5’-AAGAACFCCA tions were monitored with 57 species detected in plasma. TGAGAGTGAC-3′); Hspa5 Forward (5’-CCAGTCAGA Fatty acyl chain lengths ranged from 14:0 to 22:6 and of TCAAATGTACCCA-3′), Reverse (5’-ATCAGCCCACCG Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 5 of 16 TAACAATC-3′); Tfam Forward (5’-GTACACCTTCCACT (i.e. threshold), to acquire two separate binary images. The CAGCTTT-3′)Reverse (5’AGCTAAACACCCAGATGCA proportion of the cytoplasm (stained with ER-Tracker A-3′); Drp1 Forward (5’-AACCCTTCCCATCAATACA Green) occupied by the mitochondrial network (stained TCC-3′)Reverse (5’-TCCAGAGAGGTAGATCCAGAT with TMRM) was then calculated from the area of both G-3′) and GAPDH like endogenous gen Forward (5’-G images, calculating mitochondrial volume fraction occu- TAACCAGGCGTCCGATAC-3′), Reverse (5’-TCTCTGC pancy of the cytosol. Images were analysed using the soft- TCCTCCCTGTTC-3′) (Integrated DNA Techologies, Inc.) ware program Image J. in LightCycler ® 480 Instrument II (Roche LifeScience, 2+ Product No. 05015278001). Mitochondrial Ca levels We assessed if lipotoxicity induced by palmitic acid pro- Transmission electron microscopy motes failure in mitochondrial calcium homeostasis and To elucidate changes in mitochondrial and ER morphology mitochondrial dysfunction linked to calcium-overload. Cells in the hypothalamus linked to hypercaloric diets exposure, were grown on 22 mm coverslips and pre-treated with indi- a TEM study was performed. Tissue samples of hypothal- cated treatments and then were loaded with RM consisting amus of CAF-CAF and Control groups (n = 4) were fixed of: 156 mM NaCl;3 mM KCl; 2mMMgSO4;1.25mM with 2.5% glutaraldehyde in 0.1 M sodium cacodylate KH2PO4; 10 mM D-Glucose; 2 mM CaCl2; 10 mM Hepes; (pH 7.4) for 2 h at room temperature, post-fixed with 1% with pH 7.3–7.4, containing 20 μMRhod-2 (Molecular OsO4 in 0.1 M sodium cacodylate, and counterstained in Probes, Invitrogen, Cat. R1245), AM and 0.02% Pluronic 1% uranyl nitrate. Tissue samples were dehydrated through F-127 for 30 min at RT. The fluorescence intensity was de- a graduated acetone series and embedded in Epon 812 resin termined every 15 s. Images were obtained using a Zeiss for sectioning. Ultrastructural images of thin sections were Axiovert 100 M confocal microscope with a Plan-Neofluar observed under a transmission electron microscope × 63/1.25 oil immersion objective lens and equipped with a Carl-Zeiss EM 109, and collected with a bottom-mount helium-neon laser at RT. Fluorescence images labelled with film-based camera. Rhod-2 AM were collected using an excitation wavelength of 514 nm. Rhod-2, AM fluorescence was normalized and Measurements of mitochondrial mass, membrane plotted using the software Image J. potential (ΔΨm) and ER activity Obesity favours a lipotoxic environment with palmitic acid Statistical analysis being one of the lipids related to this response and known Statistical analysis was performed with GraphPad Prism to increase ER stress and mitochondrial dysfunction. To Software (Version 7.0a Graph Pad Software Inc., La Jolla, evaluate the effects of lipotoxicity on the mitochondria of CA). Data were expressed as Mean ± SD. The data pre- the hypothalamus, we used an in vitro model of hypothal- sented were analyzed using Two-way analysis of variance amic cells. Hypothalamic mHypoA-CLU192 cells were (Two-way ANOVA), analysis of variance (ANOVA) or grown on 22 mm coverslips in growth medium (1× Student’s t-test with post-hoc test of Dunnett’s multiple DMEM with 10% fetal bovine serum, FBS), 25 mM glu- comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p cose and 1% penicillin/streptomycin) and maintained at < 0.0001 were considered significant. 37 °C with 5% CO2 for 24 h. Cells were pre-treated with indicated treatments and then cells were loaded with re- Results cording medium (RM) consisting of; 156 mM NaCl; Selective nutritional programming alters pregnancy ratio, 3 mM KCl; 2 mM MgSO4; 1.25 mM KH2PO4; 10 mM body weight and food intake in offspring D-Glucose; 2 mM CaCl2; 10 mM Hepes; with pH 7.3–7.4, Our aim was to evaluate the effect of nutritional program- containing 25 nM TMRM (Tetramethylrhodamine, Mo- ming by maternal hypercaloric diets in mitochondrial dy- lecular Probes, Invitrogen Cat. T668) for ΔΨmand 1 μM namics in the hypothalamus of the offspring and evaluate if ER-Tracker Green (Molecular Probes, Invitrogen, Cat. this effect is exacerbated when offspring is exposed to these E34251) for 30 min at room temperature (RT) and were formula after weaning. We determined the effect of hyper- washed with saline solution containing 25 nM TMRM. caloric diets on body weight of female Wistar rats Images were acquired using a Zeiss Axiovert 100 M con- pre-mating, mating and during gestation (Fig. 1b). Female focal microscope with a Plan-Neofluar × 63/1.25 oil rats were fed with hypercaloric formula (HFD, CAF or immersion objective lens at RT. TMRM fluorescence was HSD) do not modify body weight before mating; however, excited at 543 nm and ER-Tracker Green excited at there was a significant decrease in female body weight dur- 488 nm wavelength laser. Images were analysed using the ing CAF exposure at 5–7 weeks and there were no changes software Image J. For each plane of the z-stack, the two during HFD and HSD exposure (Fig. 1b). While alterations channels ER-Tracker Green and TMRM were separated, in insulin and leptin levels have been found in mothers fed following the subtraction of the background fluorescence hypercaloric diets during pregnancy and lactation [29–31], Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 6 of 16 it is important to point out that in our murine model, ma- HSD-HSDexposurewedofinda significantdecreasein ternal hypercaloric diet intake does not develop obesity in body weight at week 5 which caught up to control values at mothers. Our main aim at this stage was to identify meta- week6(Fig. 1e). bolic changes in male offspringlinked tomaternal over nu- Next, we quantified the total food intake and body trition. To address this aim, pups (male and female) from weight every day in male offspring linked to diets expos- mothers were randomized at the age of 3 weeks in 7 experi- ure between 23 to 49 days of age. Subjects from the mental groups as previously described. Initially, we found HFD-C, CAF-C and HSD-C groups did not show change that nutritional programming by CAF diet decreased off- in food intake consumption or total kcal/day intake spring weight whereas HSD litters increases their body (Fig. 2a). By contrast, hypercaloric diet exposure after pro- weight at birth (Fig. 1c). Next, we assessed weight during gramming in the HFD-HFD group showed a decrease in 6–7 weeks of age of male offspring. The HFD-C group food intake and had less kcal/day intake when it was com- showed an increase in body weight at 6–7 weeks old when pared to Chow control group (Fig. 2b), this contrast with compared to control group, similar to their mothers (Fig. their weight where we did not found difference in com- 1d). Offspring exposed to CAF diet programming (CAF-C) pare with control group at age of 7 weeks (Fig. 2b, c, d showed a decreased in total body weight at age of 4– and Fig. 1e). Worthy of note, the CAF-CAF group had hy- 5 weeks old and caught up control body weight at 6 weeks perphagic behavior from day 36 to day 45 of age and de- (Fig. 1d). Also, hypercaloric challenged after weaning show- creased weight when compared with control group from ing in the HFD-HFD offspring group, decreased weight at 4week until 7week ofage (Fig. 2b, c, d and Fig. 1e). 5–6 weeks and caught up control body weight at 7 weeks Lastly, we evaluated food efficiency by showing the kcal/ (Fig. 1e). Moreover, CAF-CAF diet exposure decreased weight ratio and observed that HFD maternal program- body weight from week 4 to week 7 of age and they did not ming does not affect food efficiency (Fig. 2e); however, fat recover to control values (Fig. 1e). Finally, during the hypercaloric surplus after programming in the HFD-HFD Fig. 2 Effect of nutritional programming and hypercaloric diet exposure after programming (offspring diet) on food consumption. a and d) Food was weighted daily for 28 days for the different groups both maternal programming and offspring diet. b and e) Kcal per day was calculated by group in this time frame. c and f) Food efficiency by week was calculated. Data are means ± SD. *p ≤ 0.05 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 7 of 16 group, displayed a significantly decrease in food efficiency increases substantially the plasma levels of the TG species: (Fig. 2f). These results suggest that maternal program- 16: 0/36:2 + 1, 16: 0/36:4, 18: 0/34:1, 18: 0/36:2, 18: 1/32:0 + ming by HFD and CAF exposure led to failure in their 1, 18: 1/36:2 + 1, 18: 2/34:1 + 1, 18: 2/36:2 and decrease the weight and energy homeostasis, and these alterations were 16: 0/36: 2 + 1, 16: 1/34: 2 + 1, 18: 1/32:0 + 1 and 18: 1/32:1 exacerbated in their offspring fed with HFD and CAF diets +1 (Fig. 4d). Also, maternal programming promotes in- after weaning. crease in 16: 0/36:4, 18: 0/36:2, 18: 2/36:2 and 16: 0/36:2 + 1, 18: 1/32:0 + 1, 18: 1/36:2 + 1 and 18: 2/34:1 + 1 for HFD Cafeteria diet exposure during pregnancy and lactation and HSD, respectively; and 18: 1/36:2 + 1, 18: 2/34:1 + 1 for disrupts glucose sensitivity in male offspring both programming groups (Fig. 4d). Male offspring tolerance to glucose and insulin was ana- lyzedat7and8weeks old by performing GTTand ITT, re- Nutritional programming by cafeteria diet modifies liver spectively, to determine whether hypercaloric diets disrupt and white adipose tissue weight in male offspring glucose sensitivity. Initially, we found no significant differ- Next, we sought to identify the effect of diets exposure ences in basal glucose levels in the seven experimental during pregnancy and lactation on liver and retroperi- groups during GTT test (Fig. 3a, d). Additionally, there toneal white adipose tissue weight. We identified that were no changes in glucose sensitivity evaluated during the maternal programming by HFD, CAF or HSD exposure GTT in maternal programming groups (HFD-C, HSD-C) did not change liver weight in offspring males (Fig. 5a). and in the offspring hypercaloric exposure after program- On the other hand, hypercaloric surplus after weaning in ming (HFD-HFD, HSD-HSD and CAF-CAF) (Fig. 3b, c, e, the HFD-HFD group showed a decrease in liver weight f). Besides, nutritional programing by CAF-C exposure de- when compared to control group (Fig. 5b). Also, we creased plasma glucose concentration and showed a signifi- found that maternal metabolic programming by fat in cant decrease in AUC (area under the curve) during the the HFD-C group led to an increase of retroperitoneal GTT (Fig. 3b, c). Next, during the ITT the HFD-C group white adipose tissue weight when it was compared to displayed an increase in basal glucose plasma levels when control group (Fig. 5c). Finally, hypercaloric exposure by compared to Chow control group (Fig. 3g). Also, ITT fat or sugar intake after maternal programming, in showed reduced insulin sensitivity in HFD-C and CAF-C HFD-HFD, HSD-HSD and CAF-CAF groups, increased groups during versus Control (Fig. 3h, i) and significant in- retroperitoneal white adipose tissue weight with respect crease in AUC for HFD-C group (Fig. 3i). There were no to the Chow control group (Fig. 5d). changes in insulin sensitivity in HSD-C, CAF-CAF and HSD-HSD groups (Fig. 3j,k,l). These results suggest that Hypercaloric diet exposure during pregnancy and maternal programing by HFD and CAF exposure might lactation promotes hypothalamic mitochondria fusion affect glucose homeostasis in male young offspring. and ER stress response in male offspring Afterwards, we determined leptin and insulin plasma We sought to identify the effect of nutritional programming levels associated to maternal diet and hypercaloric diets in by hypercaloric diet exposure on hypothalamic mitochon- offspring after weaning. We found that maternal program- dria dynamics evidenced by changes in expression of pro- ming by HFD and CAF or HSD (HFD-C and CAF-C or teins involved in mitochondria fusion (MFN 2, Opa 1) and/ HSD-C) increase plasma insulin and leptin concentration, or fission (DRP 1). We found that HFD-C, HFD-HFD and respectively (Table 2). Also, the plasma insulin and leptin HSD-HSD diets exposure led to a significant decrease in concentration were found increased in the groups the hypothalamic DRP1 protein expression in offspring HFD-HFD and CAF-CAF when they were compared to (Fig. 6a and c). By contrast, HFD-C, HFD-HFD, CAF-CAF Chow control group (Table 2). and HSD-HSD diets showed an increase in the protein ex- Finally, total triglycerides (TG) and selective species were pression of MFN2 in hypothalamus (Fig. 6b and c). These identified by lipidomic approach in the offspring. We ana- data suggest that hypercaloric diet exposure during preg- lyzed 57 selective TG species and we found that CAF diet nancy potentially promoted positive hypothalamic mito- exposure was the most selective formula to show robust chondrial fusion. Next, based on the results of metabolism TG changes in plasma. On this context, maternal program- and protein expression found in the groups of HFD-C, ming by HFD, CAF or HSD does not change total TG HFD-HFD and CAF-CAF, we quantified their mRNA ex- levels in offspring (Fig. 4a); however, CAF exposure during pression of MFN2, Opa 1 and DRP 1 genes. We identified programming and after weaning in the CAF-CAF group that HFD-C, HFD-HFD or CAF-CAF groups showed de- promotes increase in total TG species (Fig. 4b). Also, ma- crease expression of MFN 2 mRNA (Fig. 6d). We did not ternal programming by CAF promotes increases in the se- find significant differences in the mRNA expression of lective TG species: 16: 0/36:4, 18: 2/34:1 + 1, and a decrease DRP1 and Opa 1 genes (Fig. 6e and f). We also analyzed in 16: 0/36: 2 + 1, 16: 1/34: 2 + 1, 18: 1/32:0 + 1 and 18: 1/ the expression of Ip3r1, which is related to calcium homeo- 32:1 + 1 (Fig. 4c). Of note, CAF diet exposure after weaning stasis between ER and mitochondria and it has been Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 8 of 16 Fig. 3 Effect of nutritional programming and offspring diet on glucose homeostasis. a, d, g and j) Basal glucose was measured for all groups. b, e, h and k) GTT and ITT Tests were performed at 15, 30, 45, 60, 90, and 120 min. c and f) Area Under Curve for GTT. i and l) Area Under Curve for ITT. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 9 of 16 Table 2 Concentrations of insulin and leptin Insulin Experiment Group Concentration ng/μLSD P value Maternal programming Control 0.0782 0.0033 N/A HFD-C 0.163 0.055 0.0009*** CAF-C 1.234 0.4971 0.6189 HSD-C 0.074 0.0142 0.9617 Maternal programming plus diet offspring Control 0.0782 0.0033 N/A HFD-HFD 0.2478 0.0808 0.0001**** CAF-CAF 0.2644 0.0542 0.0001**** HSD-HSD 0.08941 0.0071 0.951 Leptin Experiment Group Concentration ng/μLSD P value Maternal programming Control 0.0671 0.0191 N/A HFD-C 0.063 0.0099 0.7709 CAF-C 17.026 3.983 0.0001**** HSD-C 0.0526 0.0031 0.0355* Maternal programming plus diet offspring Control 0.0671 0.0191 N/A HFD-HFD 0.1036 0.0189 0.0007*** CAF-CAF 0.09779 0.0202 0.0002*** HSD-HSD 0.05895 0.00496 0.5173 Fig. 4 Lipidomic profile in plasma samples. Lipids were extracted from plasma samples following standard protocols and were analyzed as described in Methods. The concentration of each species was calculated using MultiQuant 3.0.1 software (AB SCIEX, Framingham, MA, USA) by relating the peak area of each species to that of the internal standard. Total plasma TG levels in maternal nutritional programming (a) and maternal and hypercaloric diet exposure after weaning (b). Selective plasma TG species in maternal nutritional programming (c) and maternal and hypercaloric diet exposure after weaning (d). Concentrations are expressed as the mean ± SEM with Chow and HFD, CAF or HSD. n =10–12. *p <0.05, **p <0.01 and ***p <0.001 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 10 of 16 Fig. 5 Effect of nutritional programming and offspring diet on liver and adipose tissue weight. Liver (a, b) and adipose tissue (c, d) were weighted after dissection from all groups. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 Fig. 6 Effect of nutritional programming and offspring diet on mitochondrial dynamics in hypothalamus. Relative densitometry for protein levels of DRP1 (a, c)or MFN 2(b, c) n =8 per group. (c) Pictures are representative of Western Blot for DRP 1, MFN2 and Actin as loading control. mRNA expression levels of MFN2 (d), DRP 1 (e), Opa 1 (f) and Ip3r1 (g) n = 7 per group. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 11 of 16 reported upregulated in obesity or lipotoxicity [21]. Off- physiological role of ER-mitochondria. It has been re- spring fed with HFD or CAF after maternal programming ported that positive energy balance during obesity pro- (HFD-HFD and CAF-CAF) increased the mRNA expres- motes enhanced ER-mitochondria contacts, modulated sion of Ip3r1 gene (Fig. 6g). mitochondria physiology by calcium fluxes [21]. We ana- Additionally, we identified morphological alterations in lyzed in an in vitro system whether the lipotoxicity insult ER and mitochondria in CAF-CAF, and Control experi- induced by the saturated fatty palmitic acid modulates cal- mental groups by using TEM. We chose CAF-CAF be- cium fluxes from ER to mitochondria triggering mito- cause is one of the groups with the greatest difference in chondrial dysfunction. Hypothalamic cells were loaded metabolism and mitochondrial dynamics. We observed with TMRM, treated with the indicated conditions and that nutritional programming and offspring intake of were quantified as described using confocal microscopy to hypercaloric diet after weaning in the CAF-CAF group determine changes in ΔΨm(Fig. 8a). Palmitic acid stimu- promotes evident hypothalamic ER disorganization, with lation during 1 and 3 h to hypothalamic cells induced an unstacked cisternae rims, which were extremely distended increase of ΔΨm, however after 6 h of treatment we found and surrounding mitochondria and bigger mitochondria a decrease of ΔΨm, this effect was exacerbated at 12 h (Fig. 7b and d). These results indicated an increase in the (Fig. 8b), showing that palmitic acid induces mitochon- ER-mitochondria contacts and confirm our previous find- drial dysfunction. ings at molecular level showing that CAF-CAF intake lead To assess mitochondrial mass and ER function, cells to mitochondrial fusion and ER dysfunction. were co-loaded with TMRM to label mitochondria and with ER-Tracker Green, which aid to label the cytosol, Lipotoxicity of saturated fatty palmitic acid induced allowing measurement of the volume occupancy of the decrease of ER signal, mitochondrial mass, membrane mitochondrial network within the dimensions of the potential (ΔΨm) and mitochondrial calcium overload cytosol and ER (Fig. 8b). At 1, 3 and 6 h after palmitic Based on our previous results showing that hypercaloric acid treatment we identified a significant decrease in diet exposure promoted an increase in mitochondrial fu- mitochondrial mass (Fig. 8b), which was more evident at sion and in endoplasmic reticulum Ip3r1 mRNA expres- 12 h of treatment with palmitic acid. Similarly, we ob- sion in hypothalamus, and, that CAF diet exposure after served a decrease in the ER-Tracker Green fluorescence weaning (CAF-CAF group) increases mitochondrial fusion intensity when hypothalamic cells were treated for 1 and and ER contacts in offspring, we sought to determine the 3 h and an increase at 6 h. However, at 12 h we observed Fig. 7 Effect of nutritional programming of CAF diet plus offspring CAF diet on hypothalamus ultrastructure. a) Control, b) CAF-CAF groups. Note that CAF-CAF diet promote appearance of bigger mitochondria and mitochondria-ER interactions in CAF-CAF (d, 3000×) in compare with Control (c, 3000×). Unstacked and extremely distended endoplasmic reticulum cisternae rims extended (* asterisk) around mitochondria (▼ arrow head) was observed in CAF-CAF group. N, nucleus; G, Golgi apparatus Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 12 of 16 Fig. 8 Lipotoxicity of palmitic acid induced decrease in mitochondrial mass, membrane potential (ΔΨm) and in ER signal. (a) Representative confocal microscopy images of ΔΨm, which was measured by the retention of TMRM (red), and ER-Tracker Green to define the cytosol. (b) Quantification of mitochondrial membrane potential. Mitochondrial mass, which was calculated from the images stained with TMRM and ER-Tracker Green. ER-Tracker Green fluorescence intensity. Data are mean ± SEM, and values are from three independent experiments. *p < 0.01 compared to control group. Scale bar = 10 μm again a decrease similar to the one observed at 1 and formula which has low fat or carbohydrates percentage. 3 h, which indicates ER stress (Fig. 8b). These results Diet might be the major driving force behind changes in showed that lipotoxic insult promotes time-dependent parameters as microbiota [32] and also CAF diet is con- hypothalamic mitochondria and ER dysfunction. sidered a robust model of metabolic syndrome when it is Next, we sought to identify time-dependent changes in compared to HFD [33]. In this context, we identified that 2+ mitochondrial Ca levels during lipotoxicity of saturated selective maternal overnutrition and hypercaloric expos- fatty palmitic acid stimulation. After 3 h of treatment with ure in male offspring after weaning using the HFD or palmitic acid, cells were incubated with the mitochondrial CAF diets led to body weight alterations and increase in 2+ Ca indicator Rhod-2 AM. Palmitic acid-treated cells ex- glucose, insulin, leptin and TG levels. Also, we found that hibited a slight increase in Rhod-2 fluorescence, indicating HFD maternal programming is effective to promote hypo- 2+ elevated mitochondrial Ca levels (Fig. 9). thalamic mitochondria fusion as well as the consumption of the HFD, CAF and HSD diets by the offspring after ma- Discussion ternal programming. Obesity and maternal overnutrition during pregnancy lead Ourinitial resultsshowedthatdamssubjected to CAF for- to several metabolic changes that trigger chronic related mula weight significantly less before delivery when compare diseases including Type 2 diabetes mellitus (T2MD) in to control, HFD or HSD diets. In offspring, we found that offspring [7, 13, 14, 20]. We sought to identify whether CAF diet decreased birthweight and whereas HSD increased hypercaloric diets exposure including HFD, CAF and birthweight. It is known that maternal overnutrition and HSD, during pregnancy and lactation selectively modu- obesity in females might impact survival and normal devel- lates body metabolic parameters and hypothalamic mito- opment in offspring [13, 14] and also increase body weight chondrial dynamics in male offspring after weaning. We in rats and in non-human primates with diets like HSD [31, compared the hypercaloric formula to standard chow diet 34, 35]. In fact, there is evidence that maternal HFD Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 13 of 16 2+ Fig. 9 Lipotoxicity of palmitic acid induced changes in mitochondrial Ca levels. a) Hypothetical model of calcium over flux from ER to 2+ mitochondria and metabolic complications during lipotoxicity. b) Mitochondrial Ca after 3 h of treatment with palmitic acid and the normalised values of Rhod-2 AM fluorescence are shown in the histogram. Data are mean ± SEM, and values are from three independent experiments exposure in rats does not promote changes in offspring body food intake at adulthood to define time dependent effects weight at birth [36]. A recent meta-analysis study showed of hypercaloric exposure before and after weaning. that maternal HFD increased birth weight in male mice, and Our data showed that maternal programming by CAF-C there was a trend in male rats to show low birth weight [37]. decreases plasma glucose levels during the GTT analysis, We hypothesized that no correlation from our results com- whereas the HFD- C group showed an increase in plasma pared with previous studies might be potentially due to the glucose during ITT test. These results partially agree with murine strain, age and time of diet exposure, as reported previous reports showing maternal HFD effect on glucose previously [36, 38, 39]. increase at 6 weeks of age in offspring [31, 43] and with the Next, we identified the effect of nutritional program- fact that maternal HFD reduced insulin tolerance [42]. In ming after weaning in body weight of offspring. At this addition, CAF diet exposure increases plasma glucose levels stage, we modulated hypercaloric surplus in offspring by in male and female offspring at the age of 3 months [44]. exposure to Chow control diet or keep them with the No correlations between our results and the reported by hypercaloric mother’s diet. We found that maternal HFD Desai et al. 2014 and Melo et al. 2014, might be related to exposure (HFD-C group) increases body weight whereas the age of the subjects, 24 weeks versus 8 weeks from our HFD-HFD and CAF-CAF decrease body weight at 5th– studies [31, 43]. However, we do report that nutritional pro- 6th and 4th–7th weeks of age respectively, compared with gramming by HFD or CAF decrease insulin sensitivity and Control group. Also, CAF exposure after maternal pro- enhance glucose metabolism during the ITT and GTT, re- gramming (CAF-CAF group) had an increase in food in- spectively. Also, hypercaloric stimuli by HDF or CAF ex- take at 6th week. Supporting our findings, CAF formula posure after maternal programming lead to an increase in decreases rate of growth shortly after its exposure [40], plasma insulin, leptin and TG lipid species levels compared and on the other hand, maternal programming by HFD to control, which correlates with an increase in retroperi- exposure leads to body weight increase in mice offspring toneal white adipose tissue weight in these groups as well [31] tentatively associated to hyperphagia from 5 to as a decrease in liver weight in HFD-HFD and CAF-CAF. 6 weeks of age [41, 42]. In this context, we did not find These results agree with previous data reported where in- difference between HFD-C and Control group in food in- creased leptin and insulin concentrations were found in take; however, the HFD-HFD group where dams and off- young rats and obese rats on HFD [45–47]. Our data also spring were fed HFD displayed a substantial decrease in show that compare to HFD and HSD which also show glu- food intake, disruption in food efficiency and body weight cose, leptin and insulin deregulation, the consumption of versus Control group. These results suggest that develop- CAF diet after weaning in offspring seems to be the most mental nutritional programming might set up a threshold significant nutritional formula to promote changes in total to increase the susceptibility to metabolic dysfunction. In TG plasma concentrations and in selective TG species. The any case, it would be potentially important to determine change in the profiles of triglycerides, ceramides and dhCer Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 14 of 16 have been linked to insulin resistance and obesity [48]and effect, as has been reported in other animal models [20]. alterations in the expression of ceramide synthase and dihy- Overall, our data propose that hypercaloric nutritional pro- droceramide desaturase increasing lipotoxicity [49, 50]. graming leads to exacerbation of mitochondria fusion in Additionally, our data correlate with negative effects on hypothalamus, which correlates, with metabolic comprom- normal organ weight during hypercaloric nutritional pro- ise in offspring. gramming [31]. Overall, we speculate that initial metabolic, Finally, we tested the hypothesis whether increase in hormonal and organ weight alterations in offspring at mitochondria fusion and ER-mitochondria contacts are 8 weeks, found in our experimental models, might com- potential negative modulators of mitochondria metabolism. promise basal metabolic settings and potentially increase It has reported that the onset of mitochondrial dysfunction 2+ the susceptibility to glucose imbalance in the adulthood. is secondary to ER stress and Ca release [59, 60]. We ini- Metabolic programming during maternal overnutrition tially identified that HFD-C decreases expression of Hspa5 and obesity leads to ER stress activation [13, 14, 31, 51, 52], and HFD-C, HFD-HFD and CAF-CAF manipulation in- and mitochondrial dysfunction in oocytes and embryos [14, creases Ip3r1 expression, two ER calcium markers. We 20], and impairs hypothalamic glucose metabolism in male found that palmitic acid stimulation to hypothalamic cells offspring [53]. It is known that mitochondrial dynamics is leads to ER stress, which correlates with a decrease in both regulated by cellular bioenergetic demands [54]. For in- mitochondrial membrane potential and mitochondrial mass stance, during high energy demand mitochondrial fusion at 6 h. Of note, palmitic acid stimulation increases mito- 2+ results in extended mitochondrial networks which provides chondrial Ca levels at earlier time-course such as 3 h after advantage to cell homeostasis; however, disruption of mito- treatment. This supports the hypothesis that lipotoxic 2+ chondrial fusion has been shown to result in energy failure stimulation with palmitic acid promotes initial Ca release and mitochondrial dysfunction [54]. Here we added new from ER leaking to mitochondria matrix and tentatively evidence by showing that HFD-C, HFD-HFD, CAF-CAF promote mitochondrial dysfunction. These results also and HSD-HSD exposure decrease the mitochondrial fission agree with the increase of the expression of Ip3r1 in mater- 2+ marker DRP1 and increase the fusion MFN 2 protein nal hypercaloric diets, given its role on Ca flux from ER marker expression in the hypothalamus of offspring. Of im- to mitochondria [21, 59, 61]. portance, mitochondrial dysfunction shows a lower expres- sion of the mitochondrial fusion marker protein OPA1, Conclusions which correlates with increased expression of the mito- Maternal programming by HFD, CAF or HSD increase chondrial fission marker protein DRP1 [54]. In our case, we insulin and leptin plasma levels, respectively, however, HFD found a negative feedback caused by maternal program- maternal programming exposure is potentially effective in ming and offspring diet that increase MFN 2 and decrease promoting hypothalamic mitochondrial fusion and ER stress in DRP 1 protein levels, which potentially suggest positive in the offspring. Positive mitochondria fusion is replicated in mitochondria fusion. We have not found correlation be- male offspring programmed by HFD, CAF and HSD and ex- tween mRNA expression and total protein levels of MFN 2 posure to HFD, CAF and HSD after weaning, which correl- and DRP1, our observations by TEM suggest an enhance in ate with failure in glucose, leptin and insulin sensitivity and mitochondria-ER contacts in the offspring of CAF-C group fat accumulation for the HFD and CAF nutrient exposure. and positive hypothalamic mitochondria fusion, bigger We suggest that lipotoxic insults related to lipid overload mitochondria and enhance mitochondria-ER contacts in might lead to mitochondrial dysfunction linked to calcium the CAF-CAF group. Hypothalamic DRP1 regulates ROS overload by the ER-mitochondria crosstalk. signaling in glucose sensing [55] and also modulates leptin Abbreviations sensitivity in POMC neurons [56]. DRP1 ablation leads to C: Control; CAF: Cafeteria diet; DIO: Diet induced obesity; DRP1: Dynamin- higher ROS production and dysfunctional mitochondria related protein 1; ER: Endoplasmic reticulum; GTT: Glucose tolerance test; HFD: High Fat Diet; HSD: High Sugar Diet; Ip3R1: Inositol 1,4,5-Trisphosphate [57]. On the other hand, others and we have reported up Receptor Type 1; ITT: Insulin tolerance test; MFN2: Mitofusin 2; regulation of hypothalamic MFN2 protein levels of obese mRNA: Messenger RNA; OPA1: Optic Atrophy type 1; mice [21, 22], and defective MFN2-expression in POMC or POMC: Proopiomelanocortin; ROS: Reactive Oxigen Species; T2DM: Type 2 diabetes mellitus; TEM: Transmission electron microscopy; Agrp neurons promotes or prevents obesity in a rodent TMRM: Tetramethylrhodamine obese model, respectively [23, 24]. Also, obesity leads to changes in mitochondrial morphology in liver and increases Acknowledgements We thank Sergio Lozano-Rodriguez, M.D. of the Scientific Publications ER-mitochondria junctions [21], suggesting that MFN2 Support Coordination, School of Medicine, Universidad Autonoma de Nuevo regulation might influence susceptibility to gain more Leon, for his critical reading and comments on the manuscript. weight during growth, as shown previously and metabolic Funding dysfunction [9, 58]. It would be relevant to determine se- This work was funded by the National Council of Science and Technology in lective changes in mitochondrial dynamics in adulthood Mexico (CONACYT) (Grant number: 255317 and 261420) and the and identify whether there is a potential transgenerational IBRO-PROLAB 2016 for A.C. Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 15 of 16 Availability of data and materials 9. Frihauf JB, et al. 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Abstract

Background: Maternal overnutrition including pre-pregnancy, pregnancy and lactation promotes a lipotoxic insult leading to metabolic dysfunction in offspring. Diet-induced obesity models (DIO) show that changes in hypothalamic mitochondria fusion and fission dynamics modulate metabolic dysfunction. Using three selective diet formula including a High fat diet (HFD), Cafeteria (CAF) and High Sugar Diet (HSD), we hypothesized that maternal diets exposure program leads to selective changes in hypothalamic mitochondria fusion and fission dynamics in male offspring leading to metabolic dysfunction which is exacerbated by a second exposure after weaning. Methods: We exposed female Wistar rats to nutritional programming including Chow, HFD, CAF, or HSD for 9 weeks (pre-mating, mating, pregnancy and lactation) or to the same diets to offspring after weaning. We determined body weight, food intake and metabolic parameters in the offspring from 21 to 60 days old. Hypothalamus was dissected at 60 days old to determine mitochondria-ER interaction markers by mRNA expression and western blot and morphology by transmission electron microscopy (TEM). Mitochondrial-ER function was analyzed by confocal microscopy using hypothalamic cell line mHypoA-CLU192. Results: Maternal programming by HFD and CAF leads to failure in glucose, leptin and insulin sensitivity and fat accumulation. Additionally, HFD and CAF programming promote mitochondrial fusion by increasing the expression of MFN2 and decreasing DRP1, respectively. Further, TEM analysis confirms that CAF exposure after programing leads to an increase in mitochondria fusion and enhanced mitochondrial-ER interaction, which partially correlates with metabolic dysfunction and fat accumulation in the HFD and CAF groups. Finally, we identified that lipotoxic palmitic acid stimulus 2+ in hypothalamic cells increases Ca overload into mitochondria matrix leading to mitochondrial dysfunction. Conclusions: We concluded that maternal programming by HFD induces hypothalamic mitochondria fusion, metabolic dysfunction and fat accumulation in male offspring, which is exacerbated by HFD or CAF exposure after weaning, potentially due to mitochondria calcium overflux. Keywords: Maternal overnutrition, Diet induced obesity (DIO), Hypothalamus, Mitochondria, Mitochondria dynamics, Fusion, Fission * Correspondence: acm590@hotmail.com; alberto.camachomr@uanl.edu.mx Departmento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autonoma de Nuevo Leon, Monterrey, Mexico Unidad de Neurometabolismo, Centro de Investigación y Desarrollo en Ciencias de la Salud, Universidad Autónoma de Nuevo Leon, Monterrey, Mexico Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 2 of 16 Background weaning by selective diets might modulate mitochondria Maternal obesity in humans associates with an increased fusion and fission dynamics in male offspring leading to risk of obesity metabolic-related disorders in offspring [1, metabolic dysfunction. 2]. It is known that obesity and maternal overnutrition create changes in uterine milieu during pregnancy leading Methods to developmental alterations and defects in organ function Animals, diets and nutritional programming model in and metabolism in offspring [3, 4]. Also, maternal nutri- offspring by HFD, CAF and HSD exposure tional programming by hypercaloric diets exposure that All the experiments were first performed using 8– simulate Western diets, such as high fat diet (HFD), cafe- 10 weeks-old female Wistar rats, 200–250 g (n = 24). teria diet (CAF) or high sugar diet (HSD) in murine Animals were handled according to the NIH guide for models, change offspring metabolism leading to insulin the care and use of laboratory animals (NIH Publications resistance, type 2 diabetes mellitus (T2DM) [5–7], cardio- No. 80–23, revised in 1996), with approval of the local vascular diseases and hypertension [8], non-alcoholic liver Animal Care Committee (BI0002). All efforts were made diseases and steatohepatitis [9, 10]. Defective molecular to minimize the number of animals used and their suf- pathways related to maternal programming by nutrient fering. Rats were housed individually in Plexiglas style oversupply can lead to failure in mitochondria dynamics cages, maintained at 22–23 °C and 12-h light/dark cycle. including modifications in mitochondria fission and Water and food was available ad libitum. Animals were fusion, which are potentially linked to metabolic com- acclimated to the animal facility 7 days prior to diets ex- promise and disease susceptibility [11]. posure. Females rats were randomized into four dietary Diet-induced obesity murine models alter mitochondrial groups: Control diet, HFD diet, CAF diet and HSD function and dynamics in selective organs including mus- diet (Table 1). The formulation of diets was: Control that cles, adipose tissue, liver and brain [11]. In particular, ma- contained a caloric density of 3.35 kcal/g divided in 71% ternal obesity in rodents is associated with altered carbohydrates, 11% lipids and 18% proteins (Research mitochondria function, reactive oxygen species generation Diets, New Brunswick, NJ, Cat. D12450B). High-fat diet and an increased mtDNA and mitochondrial biogenesis in (HFD) contained a caloric density of 4.9 Kcal/g divided oocytes and zygotes [12]. Likewise, oocytes and blastocysts in 45% lipids, 20% proteins and, 35% carbohydrates. from obese mice show reduced mitochondrial membrane Cafeteria (CAF) diet was made of liquid chocolate, bis- potential, high levels of autophagy and reduced mtDNA cuits, bacon, fries potatoes, standard diet and pork pate and mitochondrial biogenesis [13, 14], potentially linked based on a 1:1:1:1:1:1:2 ratio, respectively; total calories to hepatic lipotoxic insult in fetuses of obese females [13]. 3.72 kcal/g in 39% carbohydrates, 49% lipids, 12% pro- In fact, mitochondrial function in offspring after maternal teins and 513.53 mg of Sodium. High sugar diet (HSD) programming seems to be sex-dependent, showing insulin composition was Standard diet and condensed milk on a resistant and oxidative stress in males in compare with fe- 1:1.5 ratio, respectively; total calories 3.39 kcal/g in 69% males [15, 16]. Finally, in humans, placentas of obese carbohydrates, 18.5% lipids, 12.5% proteins and 228 mg mothers show a decrease in mtDNA and mitochondrial of Sodium. It is important to point out that these diets dysfunction which correlate with metabolic dysfunction in appear to be found in human population. After offspring [17]. randomization, female rats were fed for 9 weeks, includ- Mitochondrial functions are modulated by fission and fu- ing 3 weeks of pre-mating, mating, birth, and lactation. sion dynamic processes which assist to maintaining mito- Rats were mated with 12–14 weeks old Wistar males, chondria homeostasis [18]. Mitofusin 1 and 2 (MFN 1 and 300–350 g, during two days. Also, it is important to 2) and OPA1 and DRP1 modulate fusion and fission pro- highlight that body weight was quantified in these fe- cesses, respectively, helping to reduce cellular stress by join- males every week during pre-mating and gestation (See ing mitochondria or creating new mitochondria to assists Fig. 1a). We registered body weight of all offspring at deficient mitochondria [18]. Maternal programming by birth (approximately 15 rats/litter) and at the age of HFD exposure in rodent dams, decrease expression of 3 weeks we euthanized female offspring. Male offspring OPA1 and DRP1 proteins in skeletal muscles of female off- was grouped into 10–12 subjects per group and were al- spring and disrupt mitochondrial function in male offspring located into two groups: Group 1) exposure to Chow [19, 20], which might be transmitted across three genera- control diet to analyze maternal programming, including tions [20]. Furthermore, we and others have reported that three groups: Maternal HFD and offspring Chow control obese mice showed changes inMFN2expressioninhypo- diet (HFD-C), Maternal CAF diet and offspring Chow thalamus and other tissues [21–24], and importantly mito- control diet (CAF-C) and Maternal HSD and offspring chondria elongate in a Mfn1/2 dependent manner in Chow control diet (HSD-C); Group 2) exposure to the Agouti-related peptide neurons [25]. Here, we hypothesized same diet of their mothers until the age of 8 weeks, that maternal nutritional programming before and after resulting in three groups: Maternal HFD and offspring Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 3 of 16 Fig. 1 Effect of nutritional programming on dams and male offspring weight. a) Animal model. We fed female Wistar rats for 9 weeks according to the schedule to promote maternal programming or we fed offspring with hypercaloric diet exposure after weaning. b) Maternal body weight was followed for 6 weeks (pre-mating, mating and gestation) (Control n = 7, HFD n = 7, CAF n = 6 and HSD n = 4). c) Birth weight of offspring at day 0. d) Body weight after weaning for Control versus HFD-C, CAF-C, HSD-C for maternal programming groups, where HFD, CAF and HSD are maternal diet and C is control chow diet of offspring. e) Body weight after weaning for Control versus offspring fed with HFD, CAF and HSD after weaning (HFD-HFD, CAF-CAF, HSD-HSD groups). Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 HFD (HFD-HFD), Maternal CAF diet and offspring CAF Glucose tolerance and insulin tolerance test (GTT, ITT) diet (CAF-CAF) and Maternal HSD and offspring HSD assessments (HSD-HSD). We compared these groups with a group To test if maternal programming by selective hypercalo- where females and their offspring were fed with Chow ric diets leads to alterations in the tolerance of glucose Control diet. A total of seven experimental groups were and insulin, the GTT and ITT tests were carried out in included: Control, HFD-C, CAF-C, HSD-C, and the offspring. Males were 8 h–12 h fasted and then were HFD-HFD, CAF-CAF and HSD-HSD. Body weight, food intraperitoneally injected with 40% glucose body weight and calorie intake were quantified in offspring from or 1 U of insulin/100 g body weight. Blood glucose levels 3rd-7th week following by metabolic assessments as de- were quantified at 0 min, 15 min, 30 min, 45 min, scribed below (see Fig. 1a for details). The daily calorie 60 min, 90 min, and 120 min, as described previously intake was calculated from the weight of food consumed [26]. These tests were performed at the age of 7 weeks multiplied by the calories/gram of the food. for GTT and 8 weeks for ITT in each male rat. Table 1 Diet Composition Diet Percentage of macronutrients Ingredients Control Carbohydrates 71% Cystine 0.14 g, Coline 0.19 g, Vitamins 1.89 g, Celulose 2.89 g, Minerals 4.73 g, Soybean oil Lipids 11% 5.16 g, Starch 32.59 g, Dextrin 16.60 g, Sucrose 16 g, Casein 19.87 g per 100 g Proteins 18% Caloric density 3.35 kcal/g HFD Carbohydrates 35% Cystine 0.17 g, Coline 0.23 g, Vitamins 2.99 g, Celulose 1.72 g, Minerals 5.57 g, Inulin 1.72, Lipids 45% Soybean oil 5.75 g, Dextrin 29.46 g, Sucrose 10.35 g, Casein 24.14 g, Lard 17.72 g per 100 g Proteins 20% Caloric density 4.9 kcal/g CAF Carbohydrates 39% Lipids 49% Proteins 12% Chow diet 14.29 g, liquid chocolate 14.29 g, biscuits 14.29 g, bacon 14.29 g, fries potatoes Caloric density 3.72 kcal/g 14.29 g, pork pate 28.58 g 1:1:1:1:1:1:2 ratio per 100 g HSD Carbohydrates 69% Chow diet 40 g, condensed milk 60 g 1:1.5 ratio per 100 g Lipids 18.5% Proteins 12.5% Caloric density 3.39 kcal/g Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 4 of 16 Tissue samples collection particular interest were the following transitions: 16:0/36:4 Male rats were sacrificed by decapitation at 9 weeks of (872.8/599.6), 18:0/34:1 (878.8/577.5), 18:2/36:2 (900.9/ age. Blood samples were collected in 500 μL tubes 603.6) and 18:0/36:2 (904.9/603.6). Six species were present (Beckton Dickinson) and plasma fraction was isolated by at high concentrations, requiring quantification from the centrifugation at 4 °C and frozen at − 80 °C. Hypothal- [M + 1] isotopologue due to saturation of the detector by amus was dissected and divided into the left hemisphere the mono-isotopic ion. These were 16:1/34:2 (847.8/576.6), (for RNA extraction and gene expression analysis) and 18:1/32:1 (849.8/550.5), 18:1/32:0 (851.8/552.6), 18:2/34:1 right hemisphere (frozen immediately at 80 °C for west- (875.8/578.6), 16:0/36:2 (877.8/604.6) and 18:1/36:2 (903.9/ ern blot analysis). Also, liver and retroperitoneal white 604.6). The concentration of each species was calculated adipose tissue was measured and collected. using MultiQuant 3.0.1 software (AB SCIEX, Framingham, MA, USA) by relating the peak area of each species to that Plasma biochemistry determination of the internal standard. Glucose was determined by glucose strips and Accu-check ® (Roche, Cat. 05987270), insulin (Millipore Western blot analysis Inc., Cat. EZRMI-13 k) and leptin (Millipore Inc., Cat. Frozen hypothalamus was homogenized in 500 μllysis buf- EZML-82 K) were determined by Elisa kits according to fer as described previously with minor modifications [27]. manufacturers’ instructions. Samples were subjected to SDS–PAGE, nitrocellulose membranes blocked for 2 h at RT in TBS-T buffer (10 mM Mass spectrometric determinations of triglycerides Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5) containing 5% Triglycerides were extracted from 10 μL of plasma using a BSA (Santa Cruz Biotecnology, Inc., cat. sc2323) and incu- single phase extraction method as described in Fuller et al. bated overnight with primary antibodies at 4 °C in TBS-T (2015), with the exception that 100 pmol of glyceryl tri- Buffer 1% BSA: anti-MFN2 (Abcam, Product code heptadecanoate (TG (17:0/17:0/17:0); Sigma-Aldrich, Cat. ab56889, 1:1000), anti-Drp1 (Cell Signaling, Cat. 8570, T2151) was added to each sample as the internal standard. Dilution 1:2000) and B-actin (Cell Signaling, Cat. 8457, Extracted triglycerides were first separated on a RRHD Dilution 1:3000). Anti-mouse and anti-rabbit horseradish Eclipse Plus C18 column (2.1 × 150 mm; 1.8 μm) main- Peroxidase-conjugated were used as secondary antibody in tained at 40 °C. The samples were maintained at 16 °C TBS-Buffer 5% BSA (Cell Signaling, Cat. 7076S & Cat. with 3 μL injected into the mobile phase which was at a 7074P29, Dilution 1:3000). Proteins were detected by flow rate of 0.4 mL/min. Mobile phase A consisted of 60% chemiluminescence in the Chemidoc XRS+ System H O, 40% CH CN containing 10 mM NH COOH and (BioRad) using Clarity Western ECL Blotting Substrates 2 3 4 solvent B was 90% (CH ) CHOH, 10% CH CN containing (BioRad, Cat. No. 1705061). Images were quantified densi- 3 2 3 10 mM NH COOH. Mobile phase conditions were 90% tometrically with ImageJ Software 1.50i (Wayne Rasband, solvent A and 10% solvent B at injection, which was National Institutes of Health, Bethesda, MD, USA). linearly ramped to 50% by 6 min and then to 100% solvent B at 24 min. This was maintained for 3 min before return- RNA isolation and real time (RT)-PCR ing to 90% solvent A at 28 min, where the column equili- RNA extraction from hypothalamic samples was performed brated for 4 min prior to the next injection. as described previously [28]. RT-PCR was performed by For the first 4 min column flow was diverted to waste be- High-Capacity cDNA Reverse Transcription Kit (Applied fore being directed into the electrospray source (ES biosystems, Cat. 4,368,814) using random primers and 5500 V) of an AB SCIEX QTRAP 6500 triple quadrupole following standardized protocols. tandem mass spectrometer with an ion source temperature of 250 °C. Nitrogen was used for curtain gas, 25 units; colli- Quantitative PCR sion gas set at medium; nebulizer gas 1, 20 units and auxil- Based on that, obesity modulates ER stress and mitochon- iary gas 2, 40 units. Declustering potential was 120 V; dria fusion and fission dynamics markers in animal models, entrance potential, 8 V, collision energy, 36 V and collision we identified changes in mRNA of these markers in the off- exit potential, 26 V. Individual species of triglycerides were spring exposed to maternal programming. We performed a measured by multiple reaction monitoring in positive ion quantitative PCR using cDNA (10 ng), Light Cycler SBYR mode using the ammonium adduct and corresponding green 480 Master Mix (Roche LifeScience, Product No. fragment arising from the neutral loss of one fatty acid. As 04707516001) and the following primers: MFN2 Forward such, only the sum composition (i.e. total number of car- (5’-CCATGTGTCGCTTATCCTTCT-3′), Reverse (5’-TGA bons and double bonds) of the remaining two remaining CTCCAGCCATGTCCAT-3′); Itpr Forward (5’-CACCTA fatty acyl chains could be determined. Sixty three transi- TGACCACACTGTCTC-3′), Reverse (5’-AAGAACFCCA tions were monitored with 57 species detected in plasma. TGAGAGTGAC-3′); Hspa5 Forward (5’-CCAGTCAGA Fatty acyl chain lengths ranged from 14:0 to 22:6 and of TCAAATGTACCCA-3′), Reverse (5’-ATCAGCCCACCG Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 5 of 16 TAACAATC-3′); Tfam Forward (5’-GTACACCTTCCACT (i.e. threshold), to acquire two separate binary images. The CAGCTTT-3′)Reverse (5’AGCTAAACACCCAGATGCA proportion of the cytoplasm (stained with ER-Tracker A-3′); Drp1 Forward (5’-AACCCTTCCCATCAATACA Green) occupied by the mitochondrial network (stained TCC-3′)Reverse (5’-TCCAGAGAGGTAGATCCAGAT with TMRM) was then calculated from the area of both G-3′) and GAPDH like endogenous gen Forward (5’-G images, calculating mitochondrial volume fraction occu- TAACCAGGCGTCCGATAC-3′), Reverse (5’-TCTCTGC pancy of the cytosol. Images were analysed using the soft- TCCTCCCTGTTC-3′) (Integrated DNA Techologies, Inc.) ware program Image J. in LightCycler ® 480 Instrument II (Roche LifeScience, 2+ Product No. 05015278001). Mitochondrial Ca levels We assessed if lipotoxicity induced by palmitic acid pro- Transmission electron microscopy motes failure in mitochondrial calcium homeostasis and To elucidate changes in mitochondrial and ER morphology mitochondrial dysfunction linked to calcium-overload. Cells in the hypothalamus linked to hypercaloric diets exposure, were grown on 22 mm coverslips and pre-treated with indi- a TEM study was performed. Tissue samples of hypothal- cated treatments and then were loaded with RM consisting amus of CAF-CAF and Control groups (n = 4) were fixed of: 156 mM NaCl;3 mM KCl; 2mMMgSO4;1.25mM with 2.5% glutaraldehyde in 0.1 M sodium cacodylate KH2PO4; 10 mM D-Glucose; 2 mM CaCl2; 10 mM Hepes; (pH 7.4) for 2 h at room temperature, post-fixed with 1% with pH 7.3–7.4, containing 20 μMRhod-2 (Molecular OsO4 in 0.1 M sodium cacodylate, and counterstained in Probes, Invitrogen, Cat. R1245), AM and 0.02% Pluronic 1% uranyl nitrate. Tissue samples were dehydrated through F-127 for 30 min at RT. The fluorescence intensity was de- a graduated acetone series and embedded in Epon 812 resin termined every 15 s. Images were obtained using a Zeiss for sectioning. Ultrastructural images of thin sections were Axiovert 100 M confocal microscope with a Plan-Neofluar observed under a transmission electron microscope × 63/1.25 oil immersion objective lens and equipped with a Carl-Zeiss EM 109, and collected with a bottom-mount helium-neon laser at RT. Fluorescence images labelled with film-based camera. Rhod-2 AM were collected using an excitation wavelength of 514 nm. Rhod-2, AM fluorescence was normalized and Measurements of mitochondrial mass, membrane plotted using the software Image J. potential (ΔΨm) and ER activity Obesity favours a lipotoxic environment with palmitic acid Statistical analysis being one of the lipids related to this response and known Statistical analysis was performed with GraphPad Prism to increase ER stress and mitochondrial dysfunction. To Software (Version 7.0a Graph Pad Software Inc., La Jolla, evaluate the effects of lipotoxicity on the mitochondria of CA). Data were expressed as Mean ± SD. The data pre- the hypothalamus, we used an in vitro model of hypothal- sented were analyzed using Two-way analysis of variance amic cells. Hypothalamic mHypoA-CLU192 cells were (Two-way ANOVA), analysis of variance (ANOVA) or grown on 22 mm coverslips in growth medium (1× Student’s t-test with post-hoc test of Dunnett’s multiple DMEM with 10% fetal bovine serum, FBS), 25 mM glu- comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p cose and 1% penicillin/streptomycin) and maintained at < 0.0001 were considered significant. 37 °C with 5% CO2 for 24 h. Cells were pre-treated with indicated treatments and then cells were loaded with re- Results cording medium (RM) consisting of; 156 mM NaCl; Selective nutritional programming alters pregnancy ratio, 3 mM KCl; 2 mM MgSO4; 1.25 mM KH2PO4; 10 mM body weight and food intake in offspring D-Glucose; 2 mM CaCl2; 10 mM Hepes; with pH 7.3–7.4, Our aim was to evaluate the effect of nutritional program- containing 25 nM TMRM (Tetramethylrhodamine, Mo- ming by maternal hypercaloric diets in mitochondrial dy- lecular Probes, Invitrogen Cat. T668) for ΔΨmand 1 μM namics in the hypothalamus of the offspring and evaluate if ER-Tracker Green (Molecular Probes, Invitrogen, Cat. this effect is exacerbated when offspring is exposed to these E34251) for 30 min at room temperature (RT) and were formula after weaning. We determined the effect of hyper- washed with saline solution containing 25 nM TMRM. caloric diets on body weight of female Wistar rats Images were acquired using a Zeiss Axiovert 100 M con- pre-mating, mating and during gestation (Fig. 1b). Female focal microscope with a Plan-Neofluar × 63/1.25 oil rats were fed with hypercaloric formula (HFD, CAF or immersion objective lens at RT. TMRM fluorescence was HSD) do not modify body weight before mating; however, excited at 543 nm and ER-Tracker Green excited at there was a significant decrease in female body weight dur- 488 nm wavelength laser. Images were analysed using the ing CAF exposure at 5–7 weeks and there were no changes software Image J. For each plane of the z-stack, the two during HFD and HSD exposure (Fig. 1b). While alterations channels ER-Tracker Green and TMRM were separated, in insulin and leptin levels have been found in mothers fed following the subtraction of the background fluorescence hypercaloric diets during pregnancy and lactation [29–31], Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 6 of 16 it is important to point out that in our murine model, ma- HSD-HSDexposurewedofinda significantdecreasein ternal hypercaloric diet intake does not develop obesity in body weight at week 5 which caught up to control values at mothers. Our main aim at this stage was to identify meta- week6(Fig. 1e). bolic changes in male offspringlinked tomaternal over nu- Next, we quantified the total food intake and body trition. To address this aim, pups (male and female) from weight every day in male offspring linked to diets expos- mothers were randomized at the age of 3 weeks in 7 experi- ure between 23 to 49 days of age. Subjects from the mental groups as previously described. Initially, we found HFD-C, CAF-C and HSD-C groups did not show change that nutritional programming by CAF diet decreased off- in food intake consumption or total kcal/day intake spring weight whereas HSD litters increases their body (Fig. 2a). By contrast, hypercaloric diet exposure after pro- weight at birth (Fig. 1c). Next, we assessed weight during gramming in the HFD-HFD group showed a decrease in 6–7 weeks of age of male offspring. The HFD-C group food intake and had less kcal/day intake when it was com- showed an increase in body weight at 6–7 weeks old when pared to Chow control group (Fig. 2b), this contrast with compared to control group, similar to their mothers (Fig. their weight where we did not found difference in com- 1d). Offspring exposed to CAF diet programming (CAF-C) pare with control group at age of 7 weeks (Fig. 2b, c, d showed a decreased in total body weight at age of 4– and Fig. 1e). Worthy of note, the CAF-CAF group had hy- 5 weeks old and caught up control body weight at 6 weeks perphagic behavior from day 36 to day 45 of age and de- (Fig. 1d). Also, hypercaloric challenged after weaning show- creased weight when compared with control group from ing in the HFD-HFD offspring group, decreased weight at 4week until 7week ofage (Fig. 2b, c, d and Fig. 1e). 5–6 weeks and caught up control body weight at 7 weeks Lastly, we evaluated food efficiency by showing the kcal/ (Fig. 1e). Moreover, CAF-CAF diet exposure decreased weight ratio and observed that HFD maternal program- body weight from week 4 to week 7 of age and they did not ming does not affect food efficiency (Fig. 2e); however, fat recover to control values (Fig. 1e). Finally, during the hypercaloric surplus after programming in the HFD-HFD Fig. 2 Effect of nutritional programming and hypercaloric diet exposure after programming (offspring diet) on food consumption. a and d) Food was weighted daily for 28 days for the different groups both maternal programming and offspring diet. b and e) Kcal per day was calculated by group in this time frame. c and f) Food efficiency by week was calculated. Data are means ± SD. *p ≤ 0.05 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 7 of 16 group, displayed a significantly decrease in food efficiency increases substantially the plasma levels of the TG species: (Fig. 2f). These results suggest that maternal program- 16: 0/36:2 + 1, 16: 0/36:4, 18: 0/34:1, 18: 0/36:2, 18: 1/32:0 + ming by HFD and CAF exposure led to failure in their 1, 18: 1/36:2 + 1, 18: 2/34:1 + 1, 18: 2/36:2 and decrease the weight and energy homeostasis, and these alterations were 16: 0/36: 2 + 1, 16: 1/34: 2 + 1, 18: 1/32:0 + 1 and 18: 1/32:1 exacerbated in their offspring fed with HFD and CAF diets +1 (Fig. 4d). Also, maternal programming promotes in- after weaning. crease in 16: 0/36:4, 18: 0/36:2, 18: 2/36:2 and 16: 0/36:2 + 1, 18: 1/32:0 + 1, 18: 1/36:2 + 1 and 18: 2/34:1 + 1 for HFD Cafeteria diet exposure during pregnancy and lactation and HSD, respectively; and 18: 1/36:2 + 1, 18: 2/34:1 + 1 for disrupts glucose sensitivity in male offspring both programming groups (Fig. 4d). Male offspring tolerance to glucose and insulin was ana- lyzedat7and8weeks old by performing GTTand ITT, re- Nutritional programming by cafeteria diet modifies liver spectively, to determine whether hypercaloric diets disrupt and white adipose tissue weight in male offspring glucose sensitivity. Initially, we found no significant differ- Next, we sought to identify the effect of diets exposure ences in basal glucose levels in the seven experimental during pregnancy and lactation on liver and retroperi- groups during GTT test (Fig. 3a, d). Additionally, there toneal white adipose tissue weight. We identified that were no changes in glucose sensitivity evaluated during the maternal programming by HFD, CAF or HSD exposure GTT in maternal programming groups (HFD-C, HSD-C) did not change liver weight in offspring males (Fig. 5a). and in the offspring hypercaloric exposure after program- On the other hand, hypercaloric surplus after weaning in ming (HFD-HFD, HSD-HSD and CAF-CAF) (Fig. 3b, c, e, the HFD-HFD group showed a decrease in liver weight f). Besides, nutritional programing by CAF-C exposure de- when compared to control group (Fig. 5b). Also, we creased plasma glucose concentration and showed a signifi- found that maternal metabolic programming by fat in cant decrease in AUC (area under the curve) during the the HFD-C group led to an increase of retroperitoneal GTT (Fig. 3b, c). Next, during the ITT the HFD-C group white adipose tissue weight when it was compared to displayed an increase in basal glucose plasma levels when control group (Fig. 5c). Finally, hypercaloric exposure by compared to Chow control group (Fig. 3g). Also, ITT fat or sugar intake after maternal programming, in showed reduced insulin sensitivity in HFD-C and CAF-C HFD-HFD, HSD-HSD and CAF-CAF groups, increased groups during versus Control (Fig. 3h, i) and significant in- retroperitoneal white adipose tissue weight with respect crease in AUC for HFD-C group (Fig. 3i). There were no to the Chow control group (Fig. 5d). changes in insulin sensitivity in HSD-C, CAF-CAF and HSD-HSD groups (Fig. 3j,k,l). These results suggest that Hypercaloric diet exposure during pregnancy and maternal programing by HFD and CAF exposure might lactation promotes hypothalamic mitochondria fusion affect glucose homeostasis in male young offspring. and ER stress response in male offspring Afterwards, we determined leptin and insulin plasma We sought to identify the effect of nutritional programming levels associated to maternal diet and hypercaloric diets in by hypercaloric diet exposure on hypothalamic mitochon- offspring after weaning. We found that maternal program- dria dynamics evidenced by changes in expression of pro- ming by HFD and CAF or HSD (HFD-C and CAF-C or teins involved in mitochondria fusion (MFN 2, Opa 1) and/ HSD-C) increase plasma insulin and leptin concentration, or fission (DRP 1). We found that HFD-C, HFD-HFD and respectively (Table 2). Also, the plasma insulin and leptin HSD-HSD diets exposure led to a significant decrease in concentration were found increased in the groups the hypothalamic DRP1 protein expression in offspring HFD-HFD and CAF-CAF when they were compared to (Fig. 6a and c). By contrast, HFD-C, HFD-HFD, CAF-CAF Chow control group (Table 2). and HSD-HSD diets showed an increase in the protein ex- Finally, total triglycerides (TG) and selective species were pression of MFN2 in hypothalamus (Fig. 6b and c). These identified by lipidomic approach in the offspring. We ana- data suggest that hypercaloric diet exposure during preg- lyzed 57 selective TG species and we found that CAF diet nancy potentially promoted positive hypothalamic mito- exposure was the most selective formula to show robust chondrial fusion. Next, based on the results of metabolism TG changes in plasma. On this context, maternal program- and protein expression found in the groups of HFD-C, ming by HFD, CAF or HSD does not change total TG HFD-HFD and CAF-CAF, we quantified their mRNA ex- levels in offspring (Fig. 4a); however, CAF exposure during pression of MFN2, Opa 1 and DRP 1 genes. We identified programming and after weaning in the CAF-CAF group that HFD-C, HFD-HFD or CAF-CAF groups showed de- promotes increase in total TG species (Fig. 4b). Also, ma- crease expression of MFN 2 mRNA (Fig. 6d). We did not ternal programming by CAF promotes increases in the se- find significant differences in the mRNA expression of lective TG species: 16: 0/36:4, 18: 2/34:1 + 1, and a decrease DRP1 and Opa 1 genes (Fig. 6e and f). We also analyzed in 16: 0/36: 2 + 1, 16: 1/34: 2 + 1, 18: 1/32:0 + 1 and 18: 1/ the expression of Ip3r1, which is related to calcium homeo- 32:1 + 1 (Fig. 4c). Of note, CAF diet exposure after weaning stasis between ER and mitochondria and it has been Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 8 of 16 Fig. 3 Effect of nutritional programming and offspring diet on glucose homeostasis. a, d, g and j) Basal glucose was measured for all groups. b, e, h and k) GTT and ITT Tests were performed at 15, 30, 45, 60, 90, and 120 min. c and f) Area Under Curve for GTT. i and l) Area Under Curve for ITT. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 9 of 16 Table 2 Concentrations of insulin and leptin Insulin Experiment Group Concentration ng/μLSD P value Maternal programming Control 0.0782 0.0033 N/A HFD-C 0.163 0.055 0.0009*** CAF-C 1.234 0.4971 0.6189 HSD-C 0.074 0.0142 0.9617 Maternal programming plus diet offspring Control 0.0782 0.0033 N/A HFD-HFD 0.2478 0.0808 0.0001**** CAF-CAF 0.2644 0.0542 0.0001**** HSD-HSD 0.08941 0.0071 0.951 Leptin Experiment Group Concentration ng/μLSD P value Maternal programming Control 0.0671 0.0191 N/A HFD-C 0.063 0.0099 0.7709 CAF-C 17.026 3.983 0.0001**** HSD-C 0.0526 0.0031 0.0355* Maternal programming plus diet offspring Control 0.0671 0.0191 N/A HFD-HFD 0.1036 0.0189 0.0007*** CAF-CAF 0.09779 0.0202 0.0002*** HSD-HSD 0.05895 0.00496 0.5173 Fig. 4 Lipidomic profile in plasma samples. Lipids were extracted from plasma samples following standard protocols and were analyzed as described in Methods. The concentration of each species was calculated using MultiQuant 3.0.1 software (AB SCIEX, Framingham, MA, USA) by relating the peak area of each species to that of the internal standard. Total plasma TG levels in maternal nutritional programming (a) and maternal and hypercaloric diet exposure after weaning (b). Selective plasma TG species in maternal nutritional programming (c) and maternal and hypercaloric diet exposure after weaning (d). Concentrations are expressed as the mean ± SEM with Chow and HFD, CAF or HSD. n =10–12. *p <0.05, **p <0.01 and ***p <0.001 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 10 of 16 Fig. 5 Effect of nutritional programming and offspring diet on liver and adipose tissue weight. Liver (a, b) and adipose tissue (c, d) were weighted after dissection from all groups. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 Fig. 6 Effect of nutritional programming and offspring diet on mitochondrial dynamics in hypothalamus. Relative densitometry for protein levels of DRP1 (a, c)or MFN 2(b, c) n =8 per group. (c) Pictures are representative of Western Blot for DRP 1, MFN2 and Actin as loading control. mRNA expression levels of MFN2 (d), DRP 1 (e), Opa 1 (f) and Ip3r1 (g) n = 7 per group. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 11 of 16 reported upregulated in obesity or lipotoxicity [21]. Off- physiological role of ER-mitochondria. It has been re- spring fed with HFD or CAF after maternal programming ported that positive energy balance during obesity pro- (HFD-HFD and CAF-CAF) increased the mRNA expres- motes enhanced ER-mitochondria contacts, modulated sion of Ip3r1 gene (Fig. 6g). mitochondria physiology by calcium fluxes [21]. We ana- Additionally, we identified morphological alterations in lyzed in an in vitro system whether the lipotoxicity insult ER and mitochondria in CAF-CAF, and Control experi- induced by the saturated fatty palmitic acid modulates cal- mental groups by using TEM. We chose CAF-CAF be- cium fluxes from ER to mitochondria triggering mito- cause is one of the groups with the greatest difference in chondrial dysfunction. Hypothalamic cells were loaded metabolism and mitochondrial dynamics. We observed with TMRM, treated with the indicated conditions and that nutritional programming and offspring intake of were quantified as described using confocal microscopy to hypercaloric diet after weaning in the CAF-CAF group determine changes in ΔΨm(Fig. 8a). Palmitic acid stimu- promotes evident hypothalamic ER disorganization, with lation during 1 and 3 h to hypothalamic cells induced an unstacked cisternae rims, which were extremely distended increase of ΔΨm, however after 6 h of treatment we found and surrounding mitochondria and bigger mitochondria a decrease of ΔΨm, this effect was exacerbated at 12 h (Fig. 7b and d). These results indicated an increase in the (Fig. 8b), showing that palmitic acid induces mitochon- ER-mitochondria contacts and confirm our previous find- drial dysfunction. ings at molecular level showing that CAF-CAF intake lead To assess mitochondrial mass and ER function, cells to mitochondrial fusion and ER dysfunction. were co-loaded with TMRM to label mitochondria and with ER-Tracker Green, which aid to label the cytosol, Lipotoxicity of saturated fatty palmitic acid induced allowing measurement of the volume occupancy of the decrease of ER signal, mitochondrial mass, membrane mitochondrial network within the dimensions of the potential (ΔΨm) and mitochondrial calcium overload cytosol and ER (Fig. 8b). At 1, 3 and 6 h after palmitic Based on our previous results showing that hypercaloric acid treatment we identified a significant decrease in diet exposure promoted an increase in mitochondrial fu- mitochondrial mass (Fig. 8b), which was more evident at sion and in endoplasmic reticulum Ip3r1 mRNA expres- 12 h of treatment with palmitic acid. Similarly, we ob- sion in hypothalamus, and, that CAF diet exposure after served a decrease in the ER-Tracker Green fluorescence weaning (CAF-CAF group) increases mitochondrial fusion intensity when hypothalamic cells were treated for 1 and and ER contacts in offspring, we sought to determine the 3 h and an increase at 6 h. However, at 12 h we observed Fig. 7 Effect of nutritional programming of CAF diet plus offspring CAF diet on hypothalamus ultrastructure. a) Control, b) CAF-CAF groups. Note that CAF-CAF diet promote appearance of bigger mitochondria and mitochondria-ER interactions in CAF-CAF (d, 3000×) in compare with Control (c, 3000×). Unstacked and extremely distended endoplasmic reticulum cisternae rims extended (* asterisk) around mitochondria (▼ arrow head) was observed in CAF-CAF group. N, nucleus; G, Golgi apparatus Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 12 of 16 Fig. 8 Lipotoxicity of palmitic acid induced decrease in mitochondrial mass, membrane potential (ΔΨm) and in ER signal. (a) Representative confocal microscopy images of ΔΨm, which was measured by the retention of TMRM (red), and ER-Tracker Green to define the cytosol. (b) Quantification of mitochondrial membrane potential. Mitochondrial mass, which was calculated from the images stained with TMRM and ER-Tracker Green. ER-Tracker Green fluorescence intensity. Data are mean ± SEM, and values are from three independent experiments. *p < 0.01 compared to control group. Scale bar = 10 μm again a decrease similar to the one observed at 1 and formula which has low fat or carbohydrates percentage. 3 h, which indicates ER stress (Fig. 8b). These results Diet might be the major driving force behind changes in showed that lipotoxic insult promotes time-dependent parameters as microbiota [32] and also CAF diet is con- hypothalamic mitochondria and ER dysfunction. sidered a robust model of metabolic syndrome when it is Next, we sought to identify time-dependent changes in compared to HFD [33]. In this context, we identified that 2+ mitochondrial Ca levels during lipotoxicity of saturated selective maternal overnutrition and hypercaloric expos- fatty palmitic acid stimulation. After 3 h of treatment with ure in male offspring after weaning using the HFD or palmitic acid, cells were incubated with the mitochondrial CAF diets led to body weight alterations and increase in 2+ Ca indicator Rhod-2 AM. Palmitic acid-treated cells ex- glucose, insulin, leptin and TG levels. Also, we found that hibited a slight increase in Rhod-2 fluorescence, indicating HFD maternal programming is effective to promote hypo- 2+ elevated mitochondrial Ca levels (Fig. 9). thalamic mitochondria fusion as well as the consumption of the HFD, CAF and HSD diets by the offspring after ma- Discussion ternal programming. Obesity and maternal overnutrition during pregnancy lead Ourinitial resultsshowedthatdamssubjected to CAF for- to several metabolic changes that trigger chronic related mula weight significantly less before delivery when compare diseases including Type 2 diabetes mellitus (T2MD) in to control, HFD or HSD diets. In offspring, we found that offspring [7, 13, 14, 20]. We sought to identify whether CAF diet decreased birthweight and whereas HSD increased hypercaloric diets exposure including HFD, CAF and birthweight. It is known that maternal overnutrition and HSD, during pregnancy and lactation selectively modu- obesity in females might impact survival and normal devel- lates body metabolic parameters and hypothalamic mito- opment in offspring [13, 14] and also increase body weight chondrial dynamics in male offspring after weaning. We in rats and in non-human primates with diets like HSD [31, compared the hypercaloric formula to standard chow diet 34, 35]. In fact, there is evidence that maternal HFD Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 13 of 16 2+ Fig. 9 Lipotoxicity of palmitic acid induced changes in mitochondrial Ca levels. a) Hypothetical model of calcium over flux from ER to 2+ mitochondria and metabolic complications during lipotoxicity. b) Mitochondrial Ca after 3 h of treatment with palmitic acid and the normalised values of Rhod-2 AM fluorescence are shown in the histogram. Data are mean ± SEM, and values are from three independent experiments exposure in rats does not promote changes in offspring body food intake at adulthood to define time dependent effects weight at birth [36]. A recent meta-analysis study showed of hypercaloric exposure before and after weaning. that maternal HFD increased birth weight in male mice, and Our data showed that maternal programming by CAF-C there was a trend in male rats to show low birth weight [37]. decreases plasma glucose levels during the GTT analysis, We hypothesized that no correlation from our results com- whereas the HFD- C group showed an increase in plasma pared with previous studies might be potentially due to the glucose during ITT test. These results partially agree with murine strain, age and time of diet exposure, as reported previous reports showing maternal HFD effect on glucose previously [36, 38, 39]. increase at 6 weeks of age in offspring [31, 43] and with the Next, we identified the effect of nutritional program- fact that maternal HFD reduced insulin tolerance [42]. In ming after weaning in body weight of offspring. At this addition, CAF diet exposure increases plasma glucose levels stage, we modulated hypercaloric surplus in offspring by in male and female offspring at the age of 3 months [44]. exposure to Chow control diet or keep them with the No correlations between our results and the reported by hypercaloric mother’s diet. We found that maternal HFD Desai et al. 2014 and Melo et al. 2014, might be related to exposure (HFD-C group) increases body weight whereas the age of the subjects, 24 weeks versus 8 weeks from our HFD-HFD and CAF-CAF decrease body weight at 5th– studies [31, 43]. However, we do report that nutritional pro- 6th and 4th–7th weeks of age respectively, compared with gramming by HFD or CAF decrease insulin sensitivity and Control group. Also, CAF exposure after maternal pro- enhance glucose metabolism during the ITT and GTT, re- gramming (CAF-CAF group) had an increase in food in- spectively. Also, hypercaloric stimuli by HDF or CAF ex- take at 6th week. Supporting our findings, CAF formula posure after maternal programming lead to an increase in decreases rate of growth shortly after its exposure [40], plasma insulin, leptin and TG lipid species levels compared and on the other hand, maternal programming by HFD to control, which correlates with an increase in retroperi- exposure leads to body weight increase in mice offspring toneal white adipose tissue weight in these groups as well [31] tentatively associated to hyperphagia from 5 to as a decrease in liver weight in HFD-HFD and CAF-CAF. 6 weeks of age [41, 42]. In this context, we did not find These results agree with previous data reported where in- difference between HFD-C and Control group in food in- creased leptin and insulin concentrations were found in take; however, the HFD-HFD group where dams and off- young rats and obese rats on HFD [45–47]. Our data also spring were fed HFD displayed a substantial decrease in show that compare to HFD and HSD which also show glu- food intake, disruption in food efficiency and body weight cose, leptin and insulin deregulation, the consumption of versus Control group. These results suggest that develop- CAF diet after weaning in offspring seems to be the most mental nutritional programming might set up a threshold significant nutritional formula to promote changes in total to increase the susceptibility to metabolic dysfunction. In TG plasma concentrations and in selective TG species. The any case, it would be potentially important to determine change in the profiles of triglycerides, ceramides and dhCer Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 14 of 16 have been linked to insulin resistance and obesity [48]and effect, as has been reported in other animal models [20]. alterations in the expression of ceramide synthase and dihy- Overall, our data propose that hypercaloric nutritional pro- droceramide desaturase increasing lipotoxicity [49, 50]. graming leads to exacerbation of mitochondria fusion in Additionally, our data correlate with negative effects on hypothalamus, which correlates, with metabolic comprom- normal organ weight during hypercaloric nutritional pro- ise in offspring. gramming [31]. Overall, we speculate that initial metabolic, Finally, we tested the hypothesis whether increase in hormonal and organ weight alterations in offspring at mitochondria fusion and ER-mitochondria contacts are 8 weeks, found in our experimental models, might com- potential negative modulators of mitochondria metabolism. promise basal metabolic settings and potentially increase It has reported that the onset of mitochondrial dysfunction 2+ the susceptibility to glucose imbalance in the adulthood. is secondary to ER stress and Ca release [59, 60]. We ini- Metabolic programming during maternal overnutrition tially identified that HFD-C decreases expression of Hspa5 and obesity leads to ER stress activation [13, 14, 31, 51, 52], and HFD-C, HFD-HFD and CAF-CAF manipulation in- and mitochondrial dysfunction in oocytes and embryos [14, creases Ip3r1 expression, two ER calcium markers. We 20], and impairs hypothalamic glucose metabolism in male found that palmitic acid stimulation to hypothalamic cells offspring [53]. It is known that mitochondrial dynamics is leads to ER stress, which correlates with a decrease in both regulated by cellular bioenergetic demands [54]. For in- mitochondrial membrane potential and mitochondrial mass stance, during high energy demand mitochondrial fusion at 6 h. Of note, palmitic acid stimulation increases mito- 2+ results in extended mitochondrial networks which provides chondrial Ca levels at earlier time-course such as 3 h after advantage to cell homeostasis; however, disruption of mito- treatment. This supports the hypothesis that lipotoxic 2+ chondrial fusion has been shown to result in energy failure stimulation with palmitic acid promotes initial Ca release and mitochondrial dysfunction [54]. Here we added new from ER leaking to mitochondria matrix and tentatively evidence by showing that HFD-C, HFD-HFD, CAF-CAF promote mitochondrial dysfunction. These results also and HSD-HSD exposure decrease the mitochondrial fission agree with the increase of the expression of Ip3r1 in mater- 2+ marker DRP1 and increase the fusion MFN 2 protein nal hypercaloric diets, given its role on Ca flux from ER marker expression in the hypothalamus of offspring. Of im- to mitochondria [21, 59, 61]. portance, mitochondrial dysfunction shows a lower expres- sion of the mitochondrial fusion marker protein OPA1, Conclusions which correlates with increased expression of the mito- Maternal programming by HFD, CAF or HSD increase chondrial fission marker protein DRP1 [54]. In our case, we insulin and leptin plasma levels, respectively, however, HFD found a negative feedback caused by maternal program- maternal programming exposure is potentially effective in ming and offspring diet that increase MFN 2 and decrease promoting hypothalamic mitochondrial fusion and ER stress in DRP 1 protein levels, which potentially suggest positive in the offspring. Positive mitochondria fusion is replicated in mitochondria fusion. We have not found correlation be- male offspring programmed by HFD, CAF and HSD and ex- tween mRNA expression and total protein levels of MFN 2 posure to HFD, CAF and HSD after weaning, which correl- and DRP1, our observations by TEM suggest an enhance in ate with failure in glucose, leptin and insulin sensitivity and mitochondria-ER contacts in the offspring of CAF-C group fat accumulation for the HFD and CAF nutrient exposure. and positive hypothalamic mitochondria fusion, bigger We suggest that lipotoxic insults related to lipid overload mitochondria and enhance mitochondria-ER contacts in might lead to mitochondrial dysfunction linked to calcium the CAF-CAF group. Hypothalamic DRP1 regulates ROS overload by the ER-mitochondria crosstalk. signaling in glucose sensing [55] and also modulates leptin Abbreviations sensitivity in POMC neurons [56]. DRP1 ablation leads to C: Control; CAF: Cafeteria diet; DIO: Diet induced obesity; DRP1: Dynamin- higher ROS production and dysfunctional mitochondria related protein 1; ER: Endoplasmic reticulum; GTT: Glucose tolerance test; HFD: High Fat Diet; HSD: High Sugar Diet; Ip3R1: Inositol 1,4,5-Trisphosphate [57]. On the other hand, others and we have reported up Receptor Type 1; ITT: Insulin tolerance test; MFN2: Mitofusin 2; regulation of hypothalamic MFN2 protein levels of obese mRNA: Messenger RNA; OPA1: Optic Atrophy type 1; mice [21, 22], and defective MFN2-expression in POMC or POMC: Proopiomelanocortin; ROS: Reactive Oxigen Species; T2DM: Type 2 diabetes mellitus; TEM: Transmission electron microscopy; Agrp neurons promotes or prevents obesity in a rodent TMRM: Tetramethylrhodamine obese model, respectively [23, 24]. Also, obesity leads to changes in mitochondrial morphology in liver and increases Acknowledgements We thank Sergio Lozano-Rodriguez, M.D. of the Scientific Publications ER-mitochondria junctions [21], suggesting that MFN2 Support Coordination, School of Medicine, Universidad Autonoma de Nuevo regulation might influence susceptibility to gain more Leon, for his critical reading and comments on the manuscript. weight during growth, as shown previously and metabolic Funding dysfunction [9, 58]. It would be relevant to determine se- This work was funded by the National Council of Science and Technology in lective changes in mitochondrial dynamics in adulthood Mexico (CONACYT) (Grant number: 255317 and 261420) and the and identify whether there is a potential transgenerational IBRO-PROLAB 2016 for A.C. Cardenas-Perez et al. Nutrition & Metabolism (2018) 15:38 Page 15 of 16 Availability of data and materials 9. Frihauf JB, et al. 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Nutrition & MetabolismSpringer Journals

Published: Jun 5, 2018

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