Maternal omega-3 fatty acid deficiency affects fetal thermogenic development and postnatal musculoskeletal growth in mice

Maternal omega-3 (n-3) polyunsaturated fatty acids (PUFAs) deficiency can affect offspring’s adiposity and metabolism by modulating lipid and glucose metabolism. However, the impact of n-3 PUFA deficiency on the development of fetal thermogenesis and its consequences is not reported. Using an n-3 PUFA deficient mice, we assessed fetal interscapular brown adipose tissue (iBAT), body fat composition, insulin growth factor-1 (IGF-1), glucose transporters (GLUTs), and expression of lipid storage & metabolic proteins in the offspring. The n-3 PUFA deficiency did not change the pups’ calorie intake, organ weight, and body weight. However, the offspring’s skeletal growth was altered due to excess fat to lean mass, reduced tibia & femur elongation, dysregulated IGF-1 in the mother and pups (p<0.05). Localization of uncoupling protein 1 (UCP1) in iBAT exhibited a reduced expression in the deficient fetus. Further, UCP1, GLUT1, GPR120 were downregulated while FABP3, ADRP, GLUT4 expressions were upregulated in the BAT of the deficient offspring (p<0.05). The deficiency decreased endogenous conversion of the n-3 LCPUFAs from their precursors and upregulated SCD1, FASN, and MFSD2A mRNAs in the liver (p<0.05). An altered musculoskeletal growth in the offspring is associated with impaired browning of the fetal adipose, dysregulated thermogenesis, growth hormone, and expression of glucose and fatty acid metabolic mediators due to maternal n-3 PUFA deficiency. BAT had higher metabolic sensitivity compared to WAT in n-3 PUFA deficiency. Maternal n-3 PUFA intake may prevent excess adiposity by modulating fetal development of thermogenesis and skeletal growth dynamics in the mice offspring. Highlight Maternal n-3 PUFA deficiency dysregulated the development of fetal adipose browning N-3 PUFA regulates fetal thermogenic development by altering UCP1 expression BAT had higher metabolic sensitivity compared to WAT in n-3 PUFA deficiency Increased fat mass and IGF-1 played a role in promoting adiposity in n-3 PUFA deficiency


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(LCPUFA) intake showed male-specific insulin-like growth factor-1 (IGF-1) changes in the offspring [3]. IGF-1 is a primary regulator for growth and body composition in fetal life and childhood, has a significant effect on the growth of the fetus and the placenta, as it is involved in the metabolism of muscle and fat tissue [4]. The concentration of IGF-1 in the mother's blood correlates with the newborn's Ponderal index [5]. However, the potential implications of n-3 fatty acid deficiency during pregnancy on IGF-1 concentration and its relationship in mother and offspring are unknown. The maternal, fetal, and neonatal LCPUFAs status can determine the metabolic outcome in infancy and later life.
Browning white adipose depots and activation of uncoupled respiration in brown fat lowers the fat accumulation. Brown fat is indispensable for maintaining the neonate's body temperature, and it helps in the burning of white fats for meeting the energy of the growing neonates. WAT sparsely intermingled with brown fat-like cells known as brite or beige adipocytes [25,26]. Uncoupling protein-1 (UCP1) is a prominent thermogenic protein involved in browning. The beige fat regulates glucose metabolism independent of UCP1, thus controlling systemic energy metabolism and glucose homeostasis [27]. Browning agents regulate energy homeostasis concerning thermogenesis. Enhancing brown adipogenesis prevents metabolic dysfunction in the offspring mice [28]. The functional ability of n-3 PUFA in activating brown fat thermogenesis is promising [11]. N-3 PUFA-induced thermogenesis is modulated by multiple mechanisms including gut-mediated energy expenditure [29], brownspecific microRNA (miRNA) upregulation [30], upregulation of thermogenesis biomarkers.
In contrast, these markers were not detectable levels in either the subcutaneous or visceral WATs [13]. The thermogenic response to β 3-adrenergic stimulation was enhanced in BAT and WAT by a diet rich in low n-6 to n-3 fatty acids [8]. N-3 PUFAs stimulate the beige and brown adipocyte differentiation by activating G-protein coupled receptor 120 (GPR120), which induces the growth hormone fibroblast growth factor 21 [31]. By regulating differentiation, n-3 PUFA can remodel fetal adiposity by balancing fat storage, and its oxidation [32]. Despite these data, the effects of n-3 PUFA deficiency in the browning of fetal adipose and its postnatal effects in adiposity are not studied.
Inadequate n-3 PUFA leads to docosahexaenoic acid (DHA) deficiency that could affect fetal and postnatal neurodevelopment [33], feto-placental changes in epigenetics [34], offspring growth and lipogenic capacity [35], and others. PUFA and its metabolites also regulate bone formation and resorption [17]. Diet rich in n-3 PUFA is reported to prevent bone loss [15]. However, limited data is available on maternal n-3 PUFA deficiency and its earliest changes on the skeletal growth in the offspring. India has the second-largest number of obese children globally [36], where fetuses are often nurtured in n-3 fatty acid deficiency due to its lower intake during pregnancy and lactation [37]. Maternal n-3 PUFA depletion during in utero development of adipose browning, its postnatal effects on musculoskeletal development, and IGF-1 of the offspring are not known yet. Moreover, the mechanism of n-3 PUFAs on the induction of browning response or increase in the activity or amount of UCP1 in developing adipose tissue is not clear.
We hypothesized that altered skeletal growth could be due to body fat changes and impaired thermogenic fat development during fetal adipogenesis. Using a recently developed n-3 PUFA deficient mouse model [34], we measured UCP1-mediated adipose browning in a fetus and the relationship of IGF-1 in mother-pups, expression of glucose, and lipid metabolic transporters in adipose and livers of offspring mice.

Diets, animals and sampling
All the procedures involved in the animal experiment were conducted in accordance with the guidelines of the committee for the control and supervision of experiments on animals (CPCSEA), Government of India. The study was approved by the institutional animal ethical committee of the National Institute of Nutrition, Hyderabad, India (No. NCLAS/IAEC/02/2017). The fetus and offspring examined in the present study are the continuity of our recently published report [34].
Weanling female (n=30 per group) Swiss albino mice were caged in pairs at 23±3°C, 55% ± 10% relative humidity with ~12h light/dark cycle and fed on n-3 PUFA sufficient and deficient diet ad libitum for five weeks before the introduction of an age-matched mating partner. Experimental diets were formulated as per the AIN93 diet with modification of fat composition. The feed's dietary and fatty acid composition are similar in the present study, as mentioned in our previously published work [34]. Diet consists 90% of base mixture [carbohydrate (54.5%), protein (25%), cellulose (5%), salt mixture (4%), vitamin mixture (1%), L-cystine (0.3%) and choline chloride (0.2%)] and 10% of fat source. Peanut and palmolein oils were used to reflect the Indian dietary fat intake since these are widely consumed oil sources, while linseed oil was used for the n-3 PUFA source. Two different blends of peanut oil, palmolein oil, and linseed oil were used as fat sources and mixed with base mixtures to obtain the n-6: n-3 PUFA ratios (50: 1 and 2:1) in the experimental diets.
The isocaloric diets were formulated to ensure that each mouse received the same calories and nutrients but different proportions of n-3 and n-6 fatty acids in the total mixture of PUFA.
Blended oils were flashed from time to time with nitrogen gas to prevent oxidation.
A line diagram of dietary intake and data collection time points of the study is presented in Supplementary Fig.1. ) mice (n=6) were euthanized by cervical dislocation, and uterine horns bearing implanted fetus were processed on ice to collect the fetus. Since BAT and bone development closely overlap with gD 14.5-17.5, a mouse fetus was investigated during this window [38]. Collected fetuses (n=6 fetus/each) were immediately stored in RNA later, formaldehyde (4%) solution and snap-frozen to carry out mRNA expression, immunohistology/fluorescence, and protein expression, respectively.
The remaining pregnant mice were allowed until parturition. Blood was collected from the retro-orbital plexus of the offspring and was euthanized to collect liver and fat (white and brown) tissues. The litter size of pups was normalized for each group. The pups' food intake and body weight were monitored throughout the study. Food intake of the offspring was calculated daily by subtracting leftover diet from the initial supplied and expressed in grams/week of each group. Feed efficiency (%) was calculated as (mean body weight gain *100)/energy intake, whereas energy intake (kcal/day) = mean food consumption x dietary metabolizable energy (kcal).

Fatty acid profiles
Total lipids were extracted from plasma (100µl) as described before [39]. Samples were subjected to methylation at 70°C with 2% methanolic sulphuric acid (containing butylated hydroxytoluene 10 mg/L) for 4 hours. Methyl esters of fatty acids (FAME) were separated out of the fraction after cooling. Gas chromatography (Perkin Elmer Clarus 680) was performed using a flame ionization detector and a fused silica capillary column (#24019, Supelco, Bellefonte, PA, USA) to determine fatty acid composition of FAME fraction. To quantify total fatty acid content, C17:0 fatty acid was used as an internal standard and added to the samples before methylation. Individual fatty acid peaks were identified by comparison with the 63B standard (Nu-chek-Prep, USA).
2.3 Assessment of musculoskeletal development and lower limb analysis by dual-energy X-ray absorptiometry (DEXA) DEXA (Discovery, Hologic, Bedford, MA, USA) was used to evaluate the body composition i.e., bone mineral density (BMD), bone mineral content (BMC), body mass, and whole-body fat distribution of all mice. Prior to the DEXA scan, the mice were weighed and injected intraperitoneally using ketamine and xylazine at a final dosage of 80 and 10 mg/kg of body weight, respectively. The mice were lying flat on the scanning bed, with their limbs and tails protruding from their bodies. Total fat, fat percentage, lean body mass, bone mineral density (g/cm2), mineral content (g), were obtained. The lengths of the tibia and femur were quantified in pixels using DEXA radiographs using ImageJ 1.50i (NIH, USA).

Plasma insulin growth factor analyses
Plasma IGF-1 level was analyzed using ELISA assay (#E-EL-M3006, Elabscience, USA). Briefly, 100μl of standard and diluted samples were incubated in a pre-coated 96-well plate for 90 min. The samples were coated with a biotinylated detection antibody for 1h followed by incubation with HRP conjugate for 30min, and substrate reagent for 15min. The enzyme action was terminated by a stop solution (50μL) and OD was measured at 450nm. A four-parameter logistic curve was constructed to analyze the concentration of plasma IGF-1 as per the supplier's guideline.

Immunofluorescence of UCP1in fetal interscapular brown adipose tissue
Fetuses were collected from 14.5-17.5gD pregnant mice and fixed in 4% paraformaldehyde. After fixing, tissues were dehydrated and embedded in paraffin. Then sagittal sectioning of interscapular tissue was performed to obtain 4µm sections and coated in slides. The sections were de-paraffinized by placing the slides on a hot plate for 1h at 60°C.
De-paraffinized slides were immediately washed in xylene to prevent any further solidification. The slides were rehydrated stepwise with 95%, 75%, and 50% alcohol about 10 min each and finally washed with distilled water. The antigen retrieval step was performed by transferring the slides to 10mM citrate buffer (pH 6.0) in the microwave oven for 5 min. After two washes with 20mM PBS, the slides were blocked with 3% horse serum and kept in a humidified chamber for 90 min. Next, the slides were washed gently two times with 20mM PBS for 10 min and incubated overnight at 4°C with primary antibody (anti-UCP1, CST #14670s; 1:50 dilution), followed by incubation with Alexa fluor goat anti-rabbit IgG (#A11034, Invitrogen;1:200 dilution) for 1h in a humidified chamber. Slides were washed with 20 mM PBS twice after incubation and mounted with DAPI solution (#F6057, Sigma).
The images were captured under 40X magnification using a fluorescent microscope (Leica Microsystem, Germany) and quantified for total fluorescence with Image J software.
Corrected total cell fluorescence (CTCF) was calculated by formula; CTCF=Integrated density -(area of selected cell x mean fluorescence of background readings) after measuring fluorescence by ImageJ 1.50i (NIH, USA) and expressed in arbitrary units.
Tissues were homogenized using bead beater for 1-2 min (Mini Bead beater, Biospec) and spun at 12,000 g for 10 min to eliminate cell remnants (4ºC

Statistical analyses
The student's t-test was performed to compare two groups using Graphpad Prism v.8.
Statistical significance was considered when the p-value was less than 0.05. The experiments were performed independently with repeated times, as indicated in the text or figure legends.
The experimental values are expressed as a mean ± standard error of the mean (SEM).

Food intake, feed efficiency, and bodyweight of the offspring
The cumulative food intake (g) of the n-3 PUFA deficient and sufficient fed offspring did not differ significantly (week 1: n-3 def. vs n-3 suff. = 39.64 ± 5.41 vs. 40.12 ± 3.95 to week 9: 475.78±17.85 vs. 466.22 ±28.58, p>0.05) over a period of nine-weeks. No significant changes were observed in feed efficiency (%) between these groups (n-3 sufficient vs. n-3 deficient: 1.11 ± 0.22 vs. 1.17 ± 0.27, p>0.05). Both groups were comparable in overall and sex-specific body weight in the pups (p>0.05, Supplementary Table 2). There was no difference in the liver or adipose weights between these groups (data not presented).

Plasma fatty acid composition in mice offspring
Gas chromatography was used to analyze the fatty acids composition in the plasma of 21 d old mice ( Table 1). The arachidonic acid,20:4n-6 (ARA) levels were considerably increased by ~2 folds in n-3 deficient pups (p<0.05), but linoleic acid, 18:2n-6 (LA) levels remained unchanged in both, indicating increased conversion of LA to ARA in n-3 PUFA deficient mice compared to its counterpart. The presence of docosahexaenoic acid,22:6 n-3 (DHA) was significantly reduced by ~4.5 folds (p<0.05) in n-3 PUFA deficient mice' plasma.
The total n-3 PUFA content was substantially improved by ~4.8 folds in n-3 sufficient offspring (p<0.05). The overall proportion of n-6 to n-3 PUFAs in the pup's plasma from both groups was closely similar with placental n-6 to n-3 PUFAs ratio [34], indicating that dietary deficiency of n-3 PUFA was linearly transferred from mother to the pups, as evidenced by the plasma fatty acids composition.
The mRNA expression of the IGF-1 was significantly increased by ~1.7 folds (Fig.1d, p=0.003) in the n-3 deficient WAT of the 21d pups. N-3 PUFA deficiency significantly lowered the plasma IGF-1 concentration in dams during pregnancy [n-3 suff. vs. n-3 def.

Development of fetal brown adipose tissue in n-3 PUFA deficiency by scoring expression, and localization of uncoupling protein-1
To determine whether altered musculoskeletal growth and fat mass in n-3 deficient pups were due to impairment of brown adipogenesis during fetal development, we evaluated the expression and localization of a predominant brown adipose tissue marker, uncoupling protein 1 (UCP1) in tissues collected from the fetus's interscapular region (Fig.2a). Normalized UCP1 expression was calculated in terms of corrected total cell fluorescence (CTCF). The CTCF of UCP1 was significantly reduced by ~1.5 folds in the fetus of n-3 PUFA deficient mice (n-3 PUFA suff. vs. n-3 PUFA def.: 0.90 ± 0.083 vs. 0.57 ± 0.096, p=0.013, Fig.2

b-c).
Since the development and transition of fetal BAT are regulated through UCP1, its expression was measured longitudinally in the offspring BAT. The UCP1 expression was significantly decreased by ~2.9 folds (p<0.0001) in n-3 PUFA deficient 21d-offspring ( Fig.3 a-b). A significantly reduced expression of UCP1 in the fetus and offspring indicated an altered thermoregulation and energy metabolism during n-3 PUFA deficiency.

Effect of maternal n-3 PUFA deficiency on the expression of energy metabolism mediators in pups' brown adipose tissue
Since brown adipose stores metabolic energy as triacylglycerol in lipid droplets (LDs), expression of LD-associated proteins such as perilipin 2 or ADRP, FABP4, FABP3 were measured in offspring's BAT. Expression of ADRP and FABP3 were significantly upregulated by ~4.1 folds (p<0.0001) and ~1.9 folds (p=0.047), while FABP4 expression was decreased by ~1.7 folds (p=0.002) in n-3 deficient BAT (Fig.3 a-b). Since the expression of glucose transporters (GLUTs) is associated with thermogenesis, the predominant GLUT mediators such as GLUT1 and GLUT4 were analyzed in the BAT. The GLUT1 expression was decreased significantly by ~5.9 folds (p<0.0001), whilst GLUT4 expression was increased by ~7.9 folds (p=0.0006) in the n-3 deficient BAT (Fig.3 c-d).
Dietary PUFAs and their metabolites may affect glucose and lipid metabolism in diverse ways including gene expression. Therefore, mRNA expression of tissue-specific functional genes (Supplementary Table 1

Expression of glucose transporters and fatty acid storage mediators in pup's white adipose tissue
To examine if pup's body fat accumulation was associated with the dysregulated expression of glucose transporters and lipid storage mediators, UCP1, ADRP, FABP3, FABP4, and GLUT1, GLUT4 were studied in WAT of 21-day-old pups. The UCP1 expression was undetected in the n-3 PUFA deficient WAT (data not shown). The ADRP expression was significantly increased by ~1.67 folds (p=0.002, Fig.5 a-b). However, the expression of FABP3 and FABP4 did not change between these groups (p>0.05). Like BAT, expression of GLUT1 was significantly decreased by ~10.2 folds (p=0.0002) in the n-3 PUFA deficient WAT, whilst the expression of GLUT4 did not change between these two groups

Effects of n-3 PUFA deficiency on gene expression in the liver
Since the n-3 PUFA deficiency led to an imbalance in the plasma n-6 and n-3 fatty acids, mRNA expression of desaturases, elongases, and fatty acid transporters were investigated in the liver. Expression of FASN (~1.34 folds), SCD1 (~1.74 folds), and MFSD2A (~2.0 folds) mRNAs were significantly increased (p<0.05), and FABP3 expression was significantly decreased in n-3 PUFA deficient fed liver ( Fig.6 a-e). However, changes in FABP4, PLIN2, PLIN3, FADS1, FADS2, ELOVL2, ELOVL5, and ELOVL6 mRNA expression were insignificant between these groups (p>0.05). Thus, an imbalance of n-6 and n-3 fatty acids in the plasma affected the expression of fatty acid transporter and metabolic genes in the liver as a result of n-3 PUFA deficient diet.

Discussion
The study, for the first time, showed that maternal n-3 PUFA deficiency dysregulated the development of fetal adipose browning, impaired body-fat distribution and growth hormone patterns in the offspring. The offspring at the fetal developmental stages showed a reduced UCP1 expression in thermogenesis sensitive iBAT, indicating impaired BAT development might lead to increased adiposity in the n-3 PUFA deficient offspring. In addition, maternal n-3 PUFA deficiency reduced femur and tibia elongation, increased ADRP, FABP3, GLUT4 expression in BAT, increased IGF-1 levels, decreased GLUT1, UCP1, and GPR120 expression in the offspring BAT. Despite isocaloric diets used in this study, n-3 PUFA deficiency reduced expression of UCP-1, resulting in lowered thermogenesis, which could lead to fatty acids' metabolic mobilization towards triglyceride storage. Imbalance in the n-6 to n-3 PUFA ratio in the diet and lower quantity of n-3 LCPUFAs in the n-3 PUFA deficient mice thus might promote an obese phenotype, since n-6 and n-3 fatty acids have opposing effects on adipogenesis and energy homeostasis. The increased fat mass and stored body fatIGF-1 levels, decreased expression of BAT thermogenesis mediators, altered expression of glucose transporters, and lipid metabolic mediators collectively contribute to the offspring's skewed musculoskeletal growth and development due to altered thermogenesis as a consequence of maternal n-3 PUFA deficiency.
In the present study, body fat accumulated in the n-3 PUFA deficient offspring is probably the result of inefficient thermogenesis. GPR120, a receptor or sensor of n-3 PUFA, is highly expressed in BAT [40] and stimulates brown fat activation by inducing FGF21 [31].
Lower induction of BAT activity and WAT browning could be due to decreased GPR120 and FGF21 expression observed in this study. EPA stimulates BAT thermogenesis in mice through GPR120-dependent epigenetic upregulation [9]. The absence of n-3 LCPUFAs, particularly EPA in n-3 PUFA deficient plasma (Table 1), observed in this study, indicate lower fatty acid oxidation and BAT thermogenesis in these mice. The n-3 PUFA deficiencyinduced fat accumulation could be due to destabilized somatomedin growth homeostasis through increased IGF-1 transcription and secretion with the simultaneous activation of the The n-3 PUFA may play a signalling role in thermogenesis by increasing BAT mass and fatty acid oxidation. A comparable n-6/n-3 PUFA (5:1) diet used previously showed a significant increase in the BAT mass might reflect a similar outcome with the n-3 sufficient (n-6/n-3 PUFA = 2:1) mice of the present study [20]. In the presence of dietary n-3 PUFA, adipocytes might acquire BAT-like phenotype within WAT and improved energy dissipation by enhancing fatty acid oxidation within this depot due to increased UCP1 expression in the fetus (Fig 3a)  increased in n-3 PUFA deficient fetal BAT (Fig 3c). The reduced UCP1 activity during n-3 PUFA deficiency could be due to lowered GLUT1 expression and increased insulin stimulative GLUT4 activity that might favor the conversion of BAT to WAT phenotype. In addition to energy homeostasis, BAT regulates glucose homeostasis via two different metabolic pathways. The active anabolic way in which glucose uptake is controlled by insulin and another is through thermogenesis by norepinephrine [50]. GLUT1 is highly expressed in BAT and positively associated with thermogenesis [51]. GLUT4 is also found in brown adipose tissue, and its localization is controlled by insulin in adipocytes [52]. As the principal player of thermogenesis, BAT can consume a significant amount of glucose from the 1 bloodstream in addition to free fatty acids. This consumption is mediated by stimulation of β 3adrenoceptors which comprises the cAMP-mediated increase in GLUT1 transcription and the de novo synthesis of GLUT1, and the mTOR complex 2-stimulated translocation of freshly synthesized GLUT1 to the plasma membrane leading to increased glucose uptake [53].
Increased expression of lipid droplet-associated protein ADRP in n-3 PUFA deficiency ( Fig.3a)  expression was increased while UCP1 expression was reduced in BAT (Fig.3a), probably due to compensatory response resulting from n-3 PUFA deficiency. Since FABP3 is highly expressed in muscle and BAT contains cell lineage of myoblast, increased FABP3 expression in n-3 PUFA deficiency could influence mesenchymal differentiation of BAT lineage towards pre-adipocyte precursor. In essence, these findings indicate inefficient burning of fatty acids during thermogenesis leading to skewed accumulation of lipids in BAT and altering the adiposity.
IGF-1 level indicates growth dynamics, which means the pups' growth trajectory. IGF-1 concentrations usually increase 20 th week onward during pregnancy. However, n-3 PUFA deficiency significantly lowered the IGF-1 levels in dams during pregnancy (Fig.1e). In contrast, IGF-1 expression and concentrations were significantly increased in n-3 PUFA deficient pups (Fig 1d and 1e). IGF1 signalling plays a decisive role in controlling brown fat development. Dysregulated IGF1, as observed in this study, could lead to defective thermogenesis and an increase in basal metabolic rate [59] to augment body fat accumulation due to impaired metabolism of fat and muscle tissue in the offspring [3,60]. A significant positive correlation exists between the mother's blood IGF-1 concentration and the ponderal index of the newborn [5]. The reduced lean-to-fat mass in the offspring observed in this study could be due to reduced IGF-1 levels in the mother in n-3 PUFA deficiency. However, gender-specific multigenerational growth hormonal effects need to be investigated.
The maternal diet with n-6 to n-3 PUFA ratio (2:1) used in the present study showed an increase in femur and tibia size ( Fig.1 c) without changing bone mineral content (BMC) in 1-mo offspring (data not presented). Earlier, a maternal diet with n-6 to n-3 PUFA (9:1) increased in femur and BMC in the 4-mo offspring [15]. Despite the differences in the n-6 to n-3 PUFA ratio and the study duration, increased femur elongation was reported in both cases. The study showed that a higher plasma DHA is positively correlated with lower bone resorption in piglets [16]. Reduced growth of femur and tibia in this study could be related to lower plasma DHA (Table 1) observed in n-3 PUFA deficient mice. The long-term n-3 LCPUFA intake, in particular EPA, improves the mechanical properties of cortical bone in mice [14]. However, EPA was present with undetectable levels in the n-3 PUFA deficient plasma offspring. Thus, maternal n-3 PUFA deficiency can dysregulate offspring's skeletal growth due to the absence of EPA and the lower presence of DHA.
The high n-6/n-3 fatty acid ratio (>20) affects adiposity in diverse ways, including a change in a systemic inflammatory response and lipid metabolic energy balance. However, the n-3 PUFA deficiency did not affect pro-inflammatory IL6 expression in this study (data not presented). The experimental data pointed out those distinct metabolites of PUFAs drive

Conclusions
The study is limited to the fact that it could not determine if the maternal effects were solely responsible for the changes in the offspring, as they continued breast feeding whilst mother continued on n-3 PUFA deficient diet. Again, the study could not measure musculoskeletal development in mature offspring since the earliest changes were considered right after parturition. Nevertheless, our data shows that maternal n-3 PUFA regulates thermogenic development of the fetal adipose tissue by changing the expression of UCP1.
Impaired fatty acid oxidation could contribute to this process. BAT thermogenesis can be induced to combat obesity, and the roles of n-3 PUFAs could be important. Increased fat mass, IGF-1, and decreased UCP1 played an essential role in promoting adiposity and decreased WAT to BAT conversion affecting thermoregulation in n-3 PUFA deficiency. Individuals with a longer leg could have a greater musculoskeletal mass than a shorter counterpart at a given size. Skeletal muscle is the primary target for glucose disposal, necessary for glucose homeostasis. DHA level of visceral fat is affected the most compared to other organs in the body of the n-3 PUFA deficient rats [68]. Excessive body fat in Indian newborns compared to Caucasians of similar age could be due to chronic intake of n-3 fatty acid-deficient diet for several generations that might raise the endogenous n-6 fatty acid levels much higher than usual [62,69]. Maintaining the optimal n-6/n-3 fatty acids become challenging with high intakes of n-6 fatty acids. The n-3 PUFA deficiency affects the delivery of n-3 LCPUFAs to the fetus and decreases the efficiency of endogenous conversion of n-3 LCPUFAs from their precursors.
Present data suggest that maternal n-3 PUFA deficiency affect fetal development of thermogenesis sensitive fat tissue and growth hormone levels, adiposity, energy metabolism in the offspring. All these anomalies may carry subtle risks for developing obesity and impaired musculoskeletal health later.

Declaration of competing interest
The authors declare no conflict of interest

Acknowledgments
The study was partly sponsored by the fund received from the Department of Biotechnology,  a  p  a  t  a  J  ,  G  a  l  l  a  r  d  o  A  ,  R  o  m  e  r  o  C  ,  V  a  l  e  n  z  u  e  l  a  R  ,  G  a  r  c  i  a  -D  i  a  z  D  F  ,  D  u  a  r  t  e  L  ,  e  t  a  l  .  n  -3  p  o  l  y  u  n  s  a  t  u  r  a  t  e  d  f  a  t  t  y  a  c  i  d  s  i  n  t  h  e  r  e  g  u  l  a  t  i  o  n  o  f  a  d  i  p  o  s  e  t  i  s  s  u  e  b  r  o  w  n  i  n  g  a  n  d  t  h  e  r  m  o  g  e  n  e  s  i  s  i  n  o  b  e  s  i  t  y  :  P  o  t  e  n  t  i  a  l  r  e  l  a  t  i  o  n  s  h  i  p  w  i  t  h  g  u  t  m  i  c  r  o  b  i  o  t  a  .  P  r  o  s  t  a  g  l  a  n  d  i  n  s  ,  L  e  u  k  o  t  r  i  e  n  e  s  a  n  d  E  s  s  e  n  t  i  a  l  F  a  t  t  y  A  c  i  d  s  .  2  0  2  2  ;  1  7  7  :  1  0  2  3  8  8  .  [  3  0  ]  F  a  n  R  ,  T  o  n  e  y  A  M  ,  J  a  n  g  Y  ,  R  o  S  H  ,  C  h  u  n  g  S  .  M  a  t  e  r  n  a  l  n  -3  P  U  F  A  s  u  p  p  l  e  m  e  n  t  a  t  i  o  :  o  l  a  m  h  o  s  s  e  i  n  i  S  ,  N  e  m  a  t  i  p  o  u  r  E  ,  D  j  a  z  a  y  e  r  y  A  ,  J  a  v  a  n  b  a  k  h  t  M  H  ,  K  o  o  h  d  a  n  i  F  ,  Z  a  r  e  e  i  M  ,  e  t  a  l  .  ω  -3  f  a  t  t  y  a  c  i  d  d  i  f  f  e  r  e  n  t  i  a  l  l  y  m  o  d  u  l  a  t  e  d  s  e  r  u  m  l  e  v  e  l  s  o  f  I  G  F  1  a  n  d  I  G  F  B  P  3  i  n  m  e  n  w  i  t  h  C  V  D  :  a  r  a  n  d  o  m  i  z  e  d  ,  d  o  u  b  l  e  -b  l  i  n  d  p  l  a  c  e  b  o  -c  o  n  t  r  o  l  l  e  d  s  t  u  d  y  .  N  u  t  r  i  t  i  o  n  .  2  0  1  5  ;  3  1  :  4  8  0  -4  .  [  6  1  ]  G  a  i  l  l  a  r  d  D  ,  N  e  g  r  e  l  R  ,  L  a  g  a  r  d  e  M  ,  A  i  l  h  a  u  d  G  .  R  e  q  u  i  r  e  m  e  n  t  a  n  d  r  o  l  e  o  f  a  r  a  c  h  i  d  o  n  i  c  a  c  i  d  i  n  t  h  e  d  i  f  f  e  r  e  n  t  i  a  t  i  o  n  o  f  p  r  e  -a  d  i  p  o  s  e  c  e  l  l  s  .  T  h  e  B  i  o  c  h  e  m  i  c  a  l  j  o  u  r  n  a  l  .  1  9  8  9  ;  2  5  7  :  3  8  9  -9         Swiss albino mice fed with n-3 PUFA deficient (n-3 def) and n-3 PUFA sufficient (n-3 suff.)  Σ LC n-6 PUFA is the sum of C20:4 n-6 and C22:4 n-6 2 Σ LC n-3 PUFA is the sum of C20:5 n-3 and C22:6 n-3 Student's t-test between n-3 deficient and n-3 sufficient group; Mean ± SEM, * p<0.05; *** p< 0.0005;