Skip to main content Accessibility help
×
Home

Information:

  • Access
  • Cited by 1

Figures:

Actions:

      • Send article to Kindle

        To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

        Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

        Find out more about the Kindle Personal Document Service.

        A post-weaning fish oil dietary intervention reverses adverse metabolic outcomes and 11β-hydroxysteroid dehydrogenase type 1 expression in postnatal overfed rats
        Available formats
        ×

        Send article to Dropbox

        To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

        A post-weaning fish oil dietary intervention reverses adverse metabolic outcomes and 11β-hydroxysteroid dehydrogenase type 1 expression in postnatal overfed rats
        Available formats
        ×

        Send article to Google Drive

        To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

        A post-weaning fish oil dietary intervention reverses adverse metabolic outcomes and 11β-hydroxysteroid dehydrogenase type 1 expression in postnatal overfed rats
        Available formats
        ×
Export citation

Abstract

Early life is considered a critical period for determining long-term metabolic health. Postnatal over-nutrition may alter glucocorticoid (GC) metabolism and increase the risk of developing obesity and metabolic disorders in adulthood. Our aim was to assess the effects of the dose and timing of a fish oil diet on obesity and the expression of GC-activated enzyme 11β-hydroxysteroid dehydrogenase type 1 (HSD1) in postnatal overfed rats. Litter sizes were adjusted to three (small litter (SL)) or ten (normal litter) rats on postnatal day 3 to induce overfeeding or normal feeding. The SL rats were divided into three groups after weaning: high-dose fish oil (HFO), low-dose fish oil (LFO) and standard-diet groups. After 10 weeks, the HFO diet reduced body weight gain (16 %, P<0·05), improved glucose intolerance and decreased hyperlipaemia levels (P<0·05) in SL rats, but the LFO diet did not have any effect on the same rats. Moreover, we chose postnatal week 3 (W3), 6 (W6) and 8 (W8) as the intervention time points at which to begin the 10-week HFO diet, and found that the HFO diet improved glucose utilisation and lipid metabolism at all time points. However, body weight of SL rats was reversed to normal levels by the post-weaning intervention (461 (sem 9·1) v. 450 (sem 2·0)). 11β-HSD1 mRNA expression in the adipose tissue (49 (sem 7·5) v. 161 (sem 18·3), P<0·05) and hepatic tissue (11 (sem 0·9) v. 16 (sem 1·5), P<0·05) was decreased by the HFO diet at W3, but not at W6 or W8 (P>0·05). In conclusion, the post-weaning HFO diet could reverse adverse outcomes and decrease tissue GC activity in postnatal overfed rats.

Obesity and related metabolic disorders have emerged as serious global health problems( 1 , 2 ). Unfortunately, many health problems in adulthood, such as type 2 diabetes, hyperlipaemia, obesity and other metabolic diseases, are associated with early-life nutrition( 3 ). Nutritional programming is defined as the process by which exposure to an abnormal nutritional environment during critical periods could permanently influence organ structure, function and genomic expression in the brain, adipose tissue, liver, pancreas and other organs( 4 6 ). Abnormal nutrition in early fetal life, infancy and adolescence can influence adipocyte proliferation, differentiation and energy homoeostasis in adulthood( 7 , 8 ). Therefore, early intervention for childhood obesity is considered the optimal strategy by clinicians.

Glucocorticoid (GC) play an essential role in adipocyte differentiation, lipolysis and insulin action( 9 11 ), and dysregulated GC action in tissues has been implicated in obesity, type II diabetes and other related diseases( 12 ). 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is highly expressed in adipose tissue, the liver and the brain, and it converts inactive cortisone into active cortisol( 13 ). The overexpression of 11β-HSD1 in visceral adipose tissue is positively correlated with obesity, dyslipidaemia, glucose intolerance and other metabolic disorders in rodents( 14 , 15 ) and humans( 16 , 17 ). Similarly, elevated hepatic 11β-HSD1 levels are correlated with insulin resistance and dyslipidaemia( 18 ). Notably, postnatal overfeeding induced by small litter (SL) rearing in rats can persistently increase 11β-HSD1 expression in peripheral tissues and aggravate the development of obesity and metabolic disorders in adults( 19 ). In addition, 11β-HSD1-inhibited or 11β-HSD1-knockout mice show resistance to diet-induced insulin resistance and hyperglycaemia( 20 , 21 ). Therefore, 11β-HSD1 is a potential contributor to the development of obesity and other metabolic diseases and may be a therapeutic target for the metabolic syndrome.

n-3 PUFA, which are particularly rich in fish oil, mainly include EPA (C20 : 5n-3) and DHA (C22 : 6n-3)( 22 ). They are important dietary elements that can modify the expressions of genes involved in obesity, hypertension, diabetes and other inflammatory conditions( 23 , 24 ). The decreasing n-3:n-6 PUFA ratio in modern diets contributes to the development of obesity, diabetes and other metabolic syndromes( 25 , 26 ). Conversely, increasing the consumption of n-3 PUFA can decrease adiposity, hypertriacylglycerolaemia and fatty liver disease, and improve insulin sensibility and glucose homoeostasis in rodents( 27 31 ) and humans( 32 ). 11β-HSD1, PPARγ and CCAAT/enhancer-binding protein α (C/EBPα) are involved in adipogenesis and lipogenesis and seem to be the key targets of n-3 PUFA( 33 35 ). However, the dose-dependent effect of the n-3 PUFA dietary intervention that accounts for the decreased risk of obesity and other metabolic disorders induced by overfeeding in early life remains unclear.

In addition, it is well known that childhood and adolescence are important periods for the development of adipose tissue( 7 , 36 , 37 ), and increased dietary n-3 PUFA levels during early critical windows of fat cell development limit adipose tissue growth, which may be a novel strategy for the prevention of childhood obesity( 38 ). A number of studies in humans have shown that early-life interventions may be beneficial for improving subsequent weight gain, depression disorder and some respiratory diseases in later life( 39 43 ). The aim of this study was to elucidate the effects of the doses and timing of a fish oil dietary intervention on reversing the adverse metabolic outcomes and expression of the GC-activated enzyme 11β-HSD1 in postnatal overfed rats.

Methods

Animals

The animal protocols used in this study were approved by the University Committee on the Use and Care of Animals and were overseen by the Unit for Laboratory Animal Medicine at Nanjing Medical University (ID:20130102-01). Male Sprague–Dawley rats were used in this study. All animals were maintained on a 12 h light–12 h dark cycle under normal temperature (22±2°C) with free access to chow and tap water. The animals used in this study were housed in cages with three rats per cage after weaning.

Experimental design

On postnatal day 3, male pups were randomly assigned to normal litters (NL) or SL. The litter size in the NL group was adjusted to ten male pups to imitate normal feeding pattern, whereas the litter size in the SL group was adjusted to three male pups to simulate early postnatal overfeeding( 44 ). After postnatal day 21, rats from the NL or the SL were fed a standard diet and represented the control group (6 % dietary fat was soyabean oil, NL group or SL group). The SL rats were fed a low-dose fish oil diet (2 % dietary fat was fish oil, SL-LFO group) or a high-dose fish oil diet (6 % dietary fat was fish oil, SL-HFO group) and represented the intervention groups. The dietary nutrient compositions are shown in Table 1, and the diets (Slac, Shanghai, China) and the fatty acid compositions of the diets are shown in Table 2. The SL rats were fed the HFO or the LFO diet until postnatal week 13 (W13).

Table 1 Purified diet formula and composition (weight (%))

Table 2 Fatty acid profile of the diets (mg/100 mg)

LA, linoleic acid; AA, arachidonic acid; ALA, α-linolenic acid; DPA, docosapentaenoic acid.

At the beginning of the interventional experiment (Fig. 1), the SL rats were fed the HFO diet from postnatal week 3 (SL-HFOW3), week 6 (SL-HFOW6) or week 8 (SL-HFOW8) for 10 weeks. For male rats, postnatal week 3 is designated as the weaning period, postnatal week 6 is the puberty period and postnatal week 8 is the post-puberty period( 45 ). Body weight and food intake were monitored weekly throughout life. The rats were killed at W13, postnatal week 16 (W16) or postnatal week 18 (W18) after an overnight fast.

Fig. 1 Schematic overview of the experimental study design. Effects of the different doses of fish oil diets and timing of intervention on postnatal overfed rats. The small litter rats were fed the high-dose fish oil dietary intervention from postnatal week 3 (SL-HFOW3), week 6 (SL-HFOW6) or week 8 (SL-HFOW8) for 10 weeks. Postnatal week 3 is designated as the weaning period, postnatal week 6 is the puberty period and postnatal week 8 is the post-puberty period. NL, normal litter; SL, small litter; SL-LFO, SL rats fed low-dose fish oil; W3, week 3; W6, week 6; W8, week 8; W13, week 13; W16, week 16; W18, week 18.

Collection of blood and tissue samples

Rats were anaesthetised by an intraperitoneal injection of chloral hydrate (300 mg/kg body weight) after an overnight fast (12 h). Blood samples were collected from the right ventricle and centrifuged (2000 g for 15 min) to obtain serum fractions, which were promptly stored at −70°C until use. The epididymal and retroperitoneal white adipose tissues were dissected and weighed, and all tissue samples were snap-frozen in liquid N2 and stored at −70°C.

Analysis of adipose tissue histology

Adipose tissues were fixed in 10 % formaldehyde in PBS, pH 7·4, for 24–48 h at room temperature. After fixation, the tissues were dehydrated, cleared, embedded in paraffin blocks, cut into 8-μm-thick sections and stained with haematoxylin–eosin. The cross-sectional adipocyte area of rats was determined in at least three slices per adipose tissue sample and ten fields of vision per slice and was analysed with imaging software.

Intraperitoneal glucose tolerance test

The intraperitoneal glucose tolerance test (IPGTT) was performed as previously described( 46 ). In brief, rats were fasted overnight at W13, W16 and W18. A blood sample was then collected from the tail vein, and 2·0 g of d-glucose (50 % stock solution in saline)/kg of body weight was injected intraperitoneally. Blood samples were collected from the tail vein at 30-, 60- and 120-min intervals after glucose injection, and glucose levels were measured using a glucose meter (Accu-Chek; Roche).

Biochemical analysis

Total TAG, total cholesterol (TC) and HDL-cholesterol levels in the serum were measured using enzymatic colorimetric assays according to the protocols of the commercial clinical diagnostic kits (TCHOD-PAP reagent kit 20090 and GPO-PAP reagent kit 20080; BIOSINO BIO) using the Olympus AU400 analyser.

Serum fatty acid composition

Fatty acid profiles were determined by gas chromatography using the general technique reported by Bligh & Dyer( 47 ). In brief, fatty acid methyl esters were prepared using a 14 % boron trifluoride (BF3)/methanol reagent (Sigma). Lipid samples were heated at 90°C in glass tubes in a metal block for 30 min and allowed to stand for 10 min. The fatty acid methyl esters were analysed using an Agilent 7890Agas chromatography system. The peaks were identified by comparison with fatty acid standards (Nu-Chek-Prep), and the percentages of the areas for all resolved peaks were analysed using GC ChemStation software.

Quantitative real-time PCR

Total RNA was isolated from the liver and adipose tissue using TRIzol (Invitrogen), according to the manufacturer’s instructions, and quantified spectrophotometrically by OD260. Total RNA integrity was assessed using agarose gel electrophoresis, and complementary DNA were synthesised from 1·0 μg of the RNA sample using M-MLV RT (Promega) according to the manufacturer’s recommendations. The genes of interest, 11β-HSD1, C/EBPα and PPARγ, were analysed by real-time PCR using the SYBR Green ABI Prism 7500 sequence detector (Table 3). The expressions of the target genes were normalised to the expression of glyceraldehyde-3-phosphate dehydrogenase.

Table 3 Primer sequences used for mRNA quantification by real-time PCR

HSD1, hydroxysteroid dehydrogenase type 1; C/EBPα, CCAAT/enhancer-binding protein α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Statistical analysis

Data are expressed as mean values with their standard error of mean. Significant differences among the groups of rats were analysed using one-way ANOVA. Serum glucose levels during the IPGTT were analysed by one-way ANOVA with repeated measures. Statistical significance was accepted at P<0·05.

Results

Body weight and adipose tissue weight

Body weight and fat pad weight of SL rats were higher compared with NL rats at W13, W16 and W18 (P<0·05). After the 10-week intervention with the fish oil diet, body weight and fat pad weight of SL-HFOW3 rats were decreased compared with SL rats (P<0·05) and were similar to those of NL rats. Body weight and fat pad weight of SL-LFOW3 rats were not significantly different from SL rats (Table 4).

Table 4 Effects of the dose of the fish oil diet on body weight and adiposity (Mean values with their standard errors of mean, n 6 in each group)*

NL, normal litter; SL, small litter; SL-HFO, SL rats fed high-dose fish oil; SL-LFO, SL rats fed low-dose fish oil; RAT, retroperitoneal adipose tissue; EAT, epididymal adipose tissue.

a P<0·05 v. NL, b P<0·05 v. SL, c P<0·05 v. SL-HFO.

* Body weight, RAT weight and EAT weight were measured in rats fed the high-dose fish oil diet, low-dose fish oil diet or a standard diet at week 13. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant.

Body weight of SL-HFOW6 rats was less than that of SL rats (P<0·05), but higher than that of NL rats (P<0·05). Compared with SL rats, fat pad weight of SL-HFOW6 rats was slightly reduced. Body and fat pad weights of SL-HFOW8 rats were not significantly different from those of SL rats (Table 5).

Table 5 Effects of the timing of the fish oil diet on body weight and adiposity (Mean values with their standard errors of mean, n 6 in each group)*

NL, normal litter; SL, small litter; SL-HFO, SL rats fed high-dose fish oil; W3, postnatal week 3; RAT, retroperitoneal adipose tissue; EAT, epididymal adipose tissue; W6, postnatal week 6; W8, postnatal week 8.

a P<0·05 v. NL, b P<0·05 v. SL.

* Body weight, RAT weight and EAT weight were measured in rats fed the HFO diet for 10 weeks during different time points, and the starting points for the HFO intervention were W3, W6 and W8. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant.

Haematoxylin–eosin staining

As shown in Fig. 2, the average cross-sectional adipocyte area of SL rats in each field was larger than that of NL rats at W13, W16 and W18 (P<0·05, Fig. 2). The average cross-sectional adipocyte area in SL-HFOW3 rats was smaller than that of SL rats (P<0·05), and not different from that of NL rats. However, the average cross-sectional adipocyte area in SL-LFOw3 rats was not different from that of SL rats (Fig. 2(B)).

Fig. 2 Effects of the dose and timing of the fish oil dietary intervention on haematoxylin–eosin-stained sections (200×) and average cross-sectional adipocyte area in rat adipose tissue. Effects of different doses on haematoxylin–eosin-stained sections (A) and average cross-sectional adipocyte area (B) at week 13 (W13). Effects of different intervention start points on haematoxylin–eosin-stained sections (C) and average cross-sectional adipocyte area (D) at W13, week 16 (W16) and week 18 (W18). The starting points for the high-dose fish oil (HFO) intervention were week 3 (W3), week 6 (W6) and week 8 (W8), and the end points were W13, W16 and W18, separately. Values are means (n 3 in each group), with standard errors of mean. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant. a P<0·05 v. normal litter (NL), b P<0·05 v. small litter (SL), c P<0·05 v. SL rats fed high-dose fish oil (SL-HFO). SL-LFO, SL rats fed low-dose fish oil. , NL; , SL; , SL-HFO; , SL-LFO.

Moreover, the average cross-sectional adipocyte area in SL-HFOW6 rats was smaller than that in corresponding SL rats (P<0·05), but larger than that in NL rats (P<0·05). However, the average cross-sectional adipocyte area in SL-HFOW8 rats did not show any change compared with SL rats (Fig. 2(D)).

Glucose homoeostasis

The AUC for plasma glucose levels in SL rats was increased at W13, W16 and W18 compared with NL rats (P<0·05, Fig. 3(B) and (F)), indicating that glucose tolerance was impaired in SL rats. At W13, the AUC of SL-HFOW3 rats was decreased compared with SL rats (P<0·05) and recovered to a normal level. Glucose intolerance in SL-HFOW6 and SL-HFOW8 rats was also improved to the normal state (P<0·05, Fig. 3(F)). However, glucose intolerance was not altered in SL-LFOW3 rats (Fig. 3(B)).

Fig. 3 Effects of the dose and timing of the fish oil dietary intervention on intraperitoneal glucose tolerance test and AUC. Effects of different doses on intraperitoneal glucose tolerance test (A) and AUC (B) at week 13 (W13). Effects of different intervention start points on glucose homoeostasis (C, D, E) and AUC (F) at W13, week 16 (W16) and week 18 (W18). The starting points for the HFO intervention were week 3 (W3), week 6 (W6) and week 8 (W8), and the end points were, W13, W16 and W18, separately. Values are means (n 6 in each group), with their standard error of mean. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant. a P<0·05 v. normal litter (NL), b P<0·05 v. small litter (SL), c P<0·05 v. SL rats fed high-dose fish oil (SL-HFO). a: , NL; , SL; , SL-HFO; , SL-LFO; b, f: , NL; , SL; , SL-HFO; , SL-LFO; c: , NL; , SL; , SL-HFOW3; d: , NL; , SL; , SL-HFOW6; e: , NL; , SL; , SL-HFOW8.

Serum lipids

Total TAG levels in SL rats were higher compared with NL rats (P<0·05); total TAG levels in SL-HFOW3 rats were decreased (P<0·05), but not in SL-LFOW3 rats (Fig. 4(A)). Total TAG levels in SL rats with the HFO dietary intervention at all points were reduced to the normal levels (P<0·05, Fig. 4(D)). TC levels in SL-HFOW3 rats were not changed (P>0·05), but HDL-cholesterol levels were increased compared with NL and SL rats (P<0·05, Fig. 4(C)). TC levels in SL-HFOW6 rats were reduced compared with NL and SL rats (P<0·05, Fig. 4(E)). At W18, TC levels in SL rats were increased compared with NL rats, but TC levels in SL-HFOW8 rats were reduced to normal levels (P<0·05, Fig. 4(E)).

Fig. 4 Effects of the dose and timing of the fish oil dietary intervention on serum biological parameters in rats. Effects of different doses on serum levels of total TAG (A), total cholesterol (B) and HDL-cholesterol (C) at week 13 (W13). Effects of different intervention start points on serum levels of total TAG (D), total cholesterol (E) and HDL-cholesterol (F) at W13, week 16 (W16) and week 18 (W18). The starting points for the high-dose fish oil (HFO) intervention were week 3 (W3), week 6 (W6) and week 8 (W8), and the end points were W13, W16 and W18, separately. Values are means (n 6 in each group), with standard errors of mean. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant. a P<0·05 v. normal litter (NL), b P<0·05 v. small litter (SL), c P<0·05 v. SL rats fed high-dose fish oil (SL-HFO). , NL; , SL; , SL-HFO; , SL-LFO.

Serum fatty acid profile

As shown in Fig. 5, serum EPA and DHA levels were significantly increased in SL-HFOW3 rats compared with NL and SL rats (P<0·05) as well as SL-LFOW3 rats (P<0·05). Interestingly, SL rats that were fed the HFO or LFO diet exhibited a significant decrease in serum n-6 PUFA levels compared with NL and SL rats that were fed the standard diet (P<0·05).

Fig. 5 Effects of the three diets on serum fatty acid composition of rats at week 13 (W13). LA, linoleic acid; AA, arachidonic acid; ALA, α-linolenic acid; DPA, docosapentaenoic acid. The n-3 PUFA contain ALA, EPA, DPA and DHA, and n-6 PUFA contain LA and AA. Values are means (n 6 in each group), with their standard errors of mean. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant. a P<0·05 v. normal litter (NL), b P<0·05 v. small litter (SL), c P<0·05 v. (SL-HFO). , NL; , SL; , SL-HFO; , SL-LFO.

11β-Hydroxysteroid dehydrogenase type 1, CCAAT/enhancer-binding protein α and PPARγ mRNA expressions in adipose tissue

The expression of 11β-HSD1 mRNA in the retroperitoneal adipose tissue of SL rats was increased compared with NL rats at W13 and W16 (P<0·05), although this difference was not statistically significant at W18. Similar to 11β-HSD1, the expressions of C/EBPα and PPARγ mRNA in the retroperitoneal adipose tissue were increased in SL rats at W13 and W16 (P<0·05). The expressions of these mRNA were reduced to normal levels in SL-HFOW3 rats but not in SL-HFOW6 rats. The expressions of 11β-HSD1 and PPARγ mRNA in SL-HFOW8 rats were increased compared with NL or SL rats (P<0·05, Fig. 6(A) and (C)).

Fig. 6 Effect of the timing of the fish oil dietary intervention on 11β-hydroxysteroid dehydrogenase type 1 (HSD1) (A), CCAAT/enhancer-binding protein α (C/EBPα) (B) and PPARγ (C) mRNA expressions in the adipose tissue. The starting points for the high-dose fish oil (HFO) intervention were week 3 (W3), week 6 (W6) and week 8 (W8), and the end points were week 13, week 16 and week 18, separately. Values are means (n 6 in each group), with standard errors of mean. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant. a P<0·05 v. normal litter (NL), b P<0·05 v. small litter (SL). , NL; , SL; , SL-HFO.

11β-Hydroxysteroid dehydrogenase type 1 and CCAAT/enhancer-binding protein α mRNA expression in hepatic tissue

In hepatic tissue, the expression of 11β-HSD1 mRNA in SL rats was increased compared with NL rats (P<0·05), although this difference was not statistically significant at W16. The C/EBPα mRNA expression levels among the groups were similar to 11β-HSD1 expression levels. Importantly, the HFO dietary intervention was effective in reducing the liver expressions of 11β-HSD1 and C/EBPα at all time points, except at puberty (W6), where 11β-HSD1 expression was significantly lower than NL (Fig. 7(A)).

Fig. 7 Effect of the timing of the fish oil dietary intervention on 11β-hydroxysteroid dehydrogenase type 1 (HSD1) (A) and CCAAT/enhancer-binding protein α (C/EBP) (B) mRNA expressions in the liver. The starting points for the high-dose fish oil (HFO) intervention were week 3 (W3), week 6 (W6) and week 8 (W8), and the end points were week 13, week 16 and week 18, separately. Values are means (n 6 in each group), with standard errors of mean. Data were analysed by one-way ANOVA using the least square difference approach, and P<0·05 was considered to be statistically significant. a P<0·05 v. normal litter (NL), b P<0·05 v. small litter (SL). , NL; , SL; , SL-HFO.

Discussion

Early postnatal life is critical for long-term programming of health, which might provide an opportunity to limit obesity and its metabolic consequences in later life( 48 ). In the present study, we explored the effects of the doses and timing of a fish oil dietary intervention on the adverse effects induced by postnatal overfeeding. We found that the HFO, but not LFO, dietary intervention could reduce weight gain and improve glucose intolerance, dyslipidaemia and local tissue 11β-HSD1 expression in postnatal overfed rats. A novel finding of this study was that the HFO prevented weight gain in SL rats and was more pronounced in the post-weaning period compared with an intervention that was implemented at puberty or post-puberty. These data suggest that dietary fatty acid composition and intervention timing may potentially interact with weight gain and metabolic regulation, and these effects may be partly involved in regulating tissue 11β-HSD1 expression levels.

It has been established that n-3 PUFA are not only a source of energy but also have protective effects against some metabolic diseases( 49 , 50 ). Some studies have postulated that the balance of dietary n-3:n-6 PUFA plays an important role in reducing the risk factors of the metabolic syndrome( 51 , 52 ). In adult Sprague–Dawley rats, an increase in the dietary ratio of n-3:n-6 PUFA to 1:1 may decrease body weight, hyperlipaemia and type II diabetes, whereas a decrease in the ratio of n-3:n-6 PUFA to 1:4 failed to induce these metabolic changes( 53 ). Therefore, we increased the dietary ratio of n-3:n-6 PUFA by replacing soyabean oil with fish oil without changing the energy content of the diets used in the dietary intervention for postnatal overfed rats. We found that a diet containing a higher ratio of n-3:n-6 PUFA effectively reduced body weight, visceral fat gain, adipocyte volume and blood lipids and improved insulin sensitivity to normal levels in SL rats. However, we did not observe any metabolic changes in SL rats that were fed a diet containing a lower ratio of n-3:n-6 PUFA. The serum fatty acid profile mirrors the dietary lipid composition and reflects the endogenous fatty acid metabolism to a certain extent( 54 ). As the main components of n-3 PUFA, EPA and DHA inhibited adipocyte differentiation, lipid droplet formation and improved glucose homoeostasis and insulin sensitivity( 55 58 ). Notably, the EPA and DHA levels in the circulation of SL-HFOW3 rats were increased compared with NL, SL and SL-LFOW3 rats, strengthening the beneficial anti-obesity effects of EPA and DHA on SL rats.

Developmental plasticity plays an important role in the aetiology of chronic diseases and is supported by the ‘developmental origins of health and disease’ hypothesis( 59 , 60 ). Thus, interventions that are implemented during developmental plasticity are considered the optimal strategy for treating chronic diseases. Perinatal mice fed a normal diet were resistant to high-fat diet-induced hyperphagia, obesity and type II diabetes( 61 ), but postnatal overfed rats still developed obesity in adulthood, even after being fed regular chow diet after weaning( 19 ). These observations indicate that the perinatal period represents a critical time frame during which metabolic regulatory set points may be modified( 34 ). Interestingly, a post-weaning n-3 PUFA diet could prevent adiposity, dyslipidaemia and other programmed outcomes in rats induced by a maternal and post-weaning, sucrose-rich diet( 62 ). In this study, we provided the HFO diet to SL rats at post-weaning, puberty or post-puberty periods, separately. We found that the most effective intervention was at weaning, and body weight and other metabolic indices of SL-HFOW3 rats were effectively recovered to normal levels, although the dietary intervention at puberty or post-puberty was useful in improving glucose intolerance and hyperlipaemia. In has been confirmed that the accumulation of adipose tissue includes increase in both adipose cell number and size( 7 , 63 ). In the present study, we found that the HFO diet at weaning was effective in reducing adipocyte size, and this effect was present partly at puberty and it disappeared completely at post-puberty. Therefore, the timing of the dietary intervention might be critical for regulating adipose tissue growth, particularly in postnatal overfed rats.

Furthermore, we observed 11β-HSD1 mRNA expression in the adipose tissues and the liver of rats. The results showed that the post-weaning HFO dietary intervention could decrease 11β-HSD1 mRNA expression levels in the adipose tissue of SL rats, which was consistent with the changes in the cross-sectional adipocyte area and adipose tissue weight. Many studies have indicated that increased 11β-HSD1 expression amplifies GC action and then promotes the differentiation of adipose stromal cell to mature adipocytes( 64 ) and increases visceral fat accumulation( 65 ), as well as the development of the metabolic syndrome( 66 ). In contrast, decreased 11β-HSD1 expression reduced fat accumulation and improved some metabolic diseases in rodent models and human clinical trials( 67 ). In addition, the expressions of C/EBPα and PPARγ, which are involved in pre-adipocyte differentiation( 68 , 69 ), and the modulation of 11β-HSD1 expression( 70 72 ) were also decreased to normal levels in SL-HFOW3 rats. All the genes studied, 11β-HSD1, PPARγ and CEBPα, were consistently expressed in the retroperitoneal adipose tissue, which could indicate their potential relevance in the regulation of GC metabolism in adipose tissue. Taken together, these results show that the post-weaning-to-puberty period could be a critical time to implement a dietary intervention to regulate GC activity, which may be an attractive therapeutic target in early nutrition programming.

Unlike 11β-HSD1 expression in the adipose tissue, the HFO dietary intervention could effectively reduce the expression of this mRNA in the liver of SL rats at all time points. 11β-HSD1 is expressed at high levels in the liver, and the high concentrations of cortisol in the liver could have important effects on hepatic insulin action( 73 ) and lipid metabolism( 74 ). An intervention with 11β-HSD1 inhibition led to attenuated GC action in the mouse liver, as well as improved insulin sensitivity and dyslipidaemia( 75 , 76 ). C/EBPα is an activator of 11β-HSD1 in the liver, and C/EBPα-deficient mice exhibit reduced 11β-HSD1 expression in hepatic tissue( 77 ). Similarly, the HFO diet decreased the expressions of both 11β-HSD1 and C/EBPα in the hepatic tissue of SL rats. These results might explain why hepatic 11β-HSD1 expression was reduced by the HFO dietary intervention, which contributes to the improvements in glucose intolerance and dyslipidaemia.

In conclusion, we have shown that the HFO, but not LFO, dietary intervention could reduce weight gain and improve glucose intolerance and dyslipidaemia in postnatal overfed rats. Moreover, the implementation of the HFO dietary intervention at the beginning of the post-weaning, puberty or post-puberty periods could improve glucose utilisation and dyslipidaemia in postnatal overfed rats, but only the intervention during the post-weaning period could significantly reverse obesity and down-regulate 11β-HSD1 expression both in adipose tissue and hepatic tissue. These characteristics may have potential therapeutic implications for appropriate dietary fatty acid compositions and interventional timing with respect to the development of adipose tissue and the prevention of obesity and the metabolic syndrome induced by postnatal overfeeding.

Acknowledgements

This study was supported by the Natural Science Foundation of China (81273064), the National Program on Key Basic Research Project (2013CB530604), the Key Program of Nanjing Public Health Bureau (ZKX14011) and the Jiangsu province Research Project (BE2015607).

The authors’ contributions are as follows: Y. D. and X. L. conceived and designed the experiment. Y. D., F. Y., N. Z., L. S., S. Z. and J. W. performed the experiments. Y. D. and X. L. analysed the data. Y. D. and X. L. wrote the manuscript. All the authors read and approved the final version of the manuscript.

The authors state that there is no conflicts of interest.

References

1. Haidar, YM & Cosman, BC (2011) Obesity epidemiology. Clin Colon Rectal Surg 24, 205210.
2. Jing, L, Binkley, CM, Suever, JD, et al. (2016) Cardiac remodeling and dysfunction in childhood obesity: a cardiovascular magnetic responance study. J Cardiovasc Magn Reson 18, 28.
3. Ojha, S, Saroha, V, Symonds, ME, et al. (2013) Excess nutrient supply in early life and its later metabolic consequences. Clin Exp Pharmacol Physiol 40, 817823.
4. Koletzko, B, Symonds, ME & Olsen, SF (2011) Programming research: where are we and where do we go from here? Am J Clin Nutr 94, 6 Suppl, 2036s2043s.
5. Buckley, AJ, Jaquiery, AL & Harding, JE (2005) Nutritional programming of adult disease. Cell Tissue Res 322, 7379.
6. Armitage, JA, Khan, IY, Taylor, PD, et al. (2004) Developmental programming of the metabolic syndrome by maternal nutritional imbalance: how strong is the evidence from experimental models in mammals? J Physiol 561, Pt 2, 355377.
7. Mostyn, A & Symonds, ME (2009) Early programming of adipose tissue function: a large-animal perspective. Proc Nutr Soc 68, 393400.
8. Habbout, A, Li, A, Rochette, L, et al. (2013) Postnatal overfeeding in rodents by litter size reduction induced major short- and long-term pathophysiological consequences. J Nutr 143, 553562.
9. Peckett, AJ, Wrigh, DC & Riddell, MC (2011) The effects of glucocorticoids on adipose tissue lipid metabolism. Metabolism 60, 15001510.
10. Gathercole, LL, Morgan, SA, Bujalska, IJ, et al. (2011) Regulation of lipogenesis by glucocorticoids and insulin in human adipose tissue. PLoS ONE 6, e26223.
11. Lee, MJ, Pramyothin, P, Karastergiou, K, et al. (2014) Deconstructing the roles of glucocorticoids in adipose tissue biology and the development of central obesity. Biochim Biophys Acta 1842, 473481.
12. Bujalska, IJ, Kumar, S & Stewart, PM (1997) Does central obesity reflect ‘Cushing’s disease of the omentum’? Lancet 349, 12101213.
13. Stomby, A, Andrew, R, Walker, BR, et al. (2014) Tissue-specific dysregulation of cortisol regeneration by 11betaHSD1 in obesity: has it promised too much? Diabetologia 57, 11001110.
14. Masuzaki, H, Paterson, J, Shinyama, H, et al. (2001) A transgenic model of visceral obesity and the metabolic syndrome. Science 294, 21662170.
15. Masuzaki, H, Yamamoto, H, Kenyon, CJ, et al. (2003) Transgenic amplification of glucocorticoid action in adipose tissue causes high blood pressure in mice. J Clin Invest 112, 8390.
16. Johansson, A, Andrew, R, Forsberg, H, et al. (2001) Glucocorticoid metabolism and adrenocortical reactivity to ACTH in myotonic dystrophy. J Clin Endocrinol Metab 86, 42764283.
17. Dube, S, Norby, BJ, Pattan, V, et al. (2015) 11β-Hydroxysteroid dehydrogenase types 1 and 2 activity in subcutaneous adipose tissue in humans: implications in obesity and diabetes. J Clin Endocrinol Metab 100, E70E76.
18. Chapagain, A, Caton, PW, Kieswich, J, et al. (2014) Elevated hepatic 11β-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia. Proc Natl Acad Sci U S A 111, 38173822.
19. Hou, M, Liu, Y, Zhu, L, et al. (2011) Neonatal overfeeding induced by small litter rearing causes altered glucocorticoid metabolism in rats. PLoS ONE 6, e25726.
20. Seckl, JR & Walker, BR (2004) 11beta-hydroxysteroid dehydrogenase type 1 as a modulator of glucocorticoid action: from metabolis to memeory. Trends Endocrinol Metab 15, 418424.
21. Harno, E, Cottrell, EC, Keevil, BG, et al. (2013) 11-Dehydrocorticosterone causes metabolic syndrome, which is prevented when 11β-HSD1 is knocked out in livers of male mice. Endocrinology 154, 35993609.
22. Poudyal, H, Panchal, SK, Diwan, V, et al. (2011) Omega-3 fatty acids and metabolic syndrome: effects and emerging mechanisms of action. Prog Lipid Res 50, 372387.
23. Rafiee, M, Sotoideh, G, Djalali, M, et al. (2016) Dietary ω-3 polyunsaturated fatty acid intake modulates impact of insertion/deletion polymorphism of ApoB gene on obesity risk in type 2 diabetic patients. Nutrition 32, 11101115.
24. Simopoilos, AP (2000) Human reguirement for n-3 polyunsaturated fatty acids. Poilt Sci 79, 961970.
25. Patterson, E, Wall, R, Fitzgerald, GF, et al. (2012) Health implications of high dietary omega-6 polyunsaturated fatty acids. J Nutr Metab 2012, 539426.
26. Ailhaud, G, Massiera, F, Weill, P, et al. (2006) Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity. Prog Lipid Res 45, 203236.
27. Buettner, R, Parhofer, KG, Woenckhaus, M, et al. (2006) Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Mol Endocrinol 36, 485501.
28. Weitz, D, Weintraub, H, Fisher, E, et al. (2010) Fish oil for the treatment of cardiovascular disease. Cardiol Rev 18, 258263.
29. Nobili, V, Bedogni, G, Alisi, A, et al. (2011) Docosahexaenoic acid supplementation decreases liver fat content in children with non-alcoholic fatty liver disease: double-blind randomised controlled clinical trial. Arch Dis Child 96, 350353.
30. Samane, S, Christon, R, Dombrowski, L, et al. (2009) Fish oil and argan oil intake differently modulate insulin resistance and glucose intolerance in a rat model of dietary-induced obesity. Metabolism 58, 909919.
31. Jelenik, T, Rossmeisl, M, Kuda, O, et al. (2010) AMP-activated protein kinase alpha2 subunit is required for the preservation of hepatic insulin sensitivity by n-3 polyunsaturated fatty acids. Diabetes 59, 27372746.
32. Su, HY, Lee, HC, Cheng, WY, et al. (2015) A calorie-restriction diet supplemented with fish oil and high-protein powder is associated with reduced severity of metabolic syndrome in obese women. Eur J Clin Nutr 69, 322328.
33. Martínez-Fernández, L, Laiglesia, LM, Huerta, AE, et al. (2015) Omega-3 fatty acids and adipose tissue function in obesity and metabolic syndrome. Prostaglandins Other Lipid Mediat 121, Pt A, 2441.
34. Hou, M, Ji, C, Wang, J, et al. (2009) The effects of dietary fatty acid composition in the post-sucking period on metabolic alterations in adulthood: can ω3 polyunsaturated fatty acids prevent adverse programming outcomes? J Endocrinol 215, 119127.
35. Stimson, RH & Walker, BR (2103) The role and regulation of 11β-hydroxysteroid dehydrogenase type 1 in obesity and the metabolic syndrome. Horm Mol Biol Clin Investig 15, 3748.
36. Budge, H, Sebert, S, Sharkey, D, et al. (2009) Session on ‘obesity’. Adipose tissue development, nutrition in early life and its impact on later obesity. Proc Nutr Soc 68, 321326.
37. Symonds, ME, Pope, M, Sharkey, D, et al. (2012) Adipose tissue and fetal programming. Diabetologia 55, 15971606.
38. Ailhaud, G & Guesnet, P (2004) Fatty acid composition of fats is an early determinant of childhood obesity: a short review and an opinion. Obes Rev 5, 2126.
39. Lloyd, LJ, Langley-Evans, SC & McMullen, S (2010) Childhood obesity and adult cardiovascular disease risk: a systematic review. Int J Obes (Lond) 34, 1828.
40. Whitlock, EP, O’Connor, EA, Williams, SB, et al. (2010) Effectiveness of weight management interventions in children: a targeted systematic review for the USPSTF. Pediatrics 125, e396e418.
41. VanDevanter, DR, Kahle, JS, O’Sullivan, AK, et al. (2015) Cystic fibrosis in young children: a review of disease manifestation, progression, and response to early treatment. J Cyst Fibros 15, 147157.
42. von Berg, A, Filipiak-Pittroff, B, Schulz, H, et al. (2016) Allergic manifestation 15 years after early intervention with hydrolyzed formulas – the GINI study. Allergy 71, 210219.
43. Khan, A, McCormack, HC, Bolger, EA, et al. (2015) Childhood maltreatment, depression, and suicidal ideation: critical importance of parental and peer emotional abuse during developmental sensitive periods in males and females. Front Psychiatry 6, 42.
44. Plagemann, A, Harder, T, Schellong, K, et al. (2012) Early postnatal life as a critical time window for determination of long-term metabolic health. Best Pract Res Clin Endocrinol Metab 26, 641653.
45. Tirelli, E, Laviola, G & Adriani, W (2003) Ontogenesis of behavioral sensitization and conditioned place preference induced by psychostimulants in laboratory rodents. Neurosci Biobehav Rev 27, 163178.
46. Chen, H, Simar, D, Lambert, K, et al. (2008) Maternal and postnatal overnutrition differentially impact appetite regulators and fuel metabolism. Endocrinology 149, 53485356.
47. Bligh, EG & Dyer, WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911917.
48. Patel, MS & Srinivasan, M (2011) Metabolic programming in the immediate postnatal life. Ann Nutr Metab 58, 1828.
49. Kremmyda, LS, Tvrzicka, E, Stankova, B, et al. (2011) Fatty acids as biocompounds: their role in human metabolism, health and disease: a review. Part 2: fatty acid physiological roles and applications in human health and disease. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155, 195218.
50. Flachs, P, Horakova, O, Brauner, P, et al. (2005) Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat. Diabetologia 48, 23652375.
51. Russo, GL (2009) Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to clinical implications in cardiovascular prevention. Biochem Pharmacol 77, 937946.
52. Simopoulos, AP (2006) Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother 60, 502507.
53. Liu, HQ, Qiu, Y, Mu, Y, et al. (2013) A high ratio of dietary n-3/n-6 polyunsaturated fatty acids improves obesity-linked inflammation and insulin resistance through suppressing activation of TLR4 in SD rats. Nutr Res 33, 849858.
54. Bertrand, C, Pignalosa, A, Wanecq, E, et al. (2013) Effects of dietary eicosapentaenoic acid (EPA) supplementation in high-fat fed mice on lipid metabolism and apelin/APJ system in skeletal muscle. PLOS ONE 8, e78874.
55. Pereira, SL, Leonard, AE, Huang, YS, et al. (2004) Identification of two novel microalgal enzymes involved in the conversion of the omega3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid. Biochem J 384, Pt 2, 357366.
56. Flachs, P, Rossmeisl, M & Kopecky, J (2014) The effect of n-3 fatty acids on glucose homeostasis and insulin sensitivity. Physiol Res 63, Suppl 1, S93S118.
57. Manickam, E, Sinclair, AJ & Cameron-Smith, D (2010) Suppressive actions of eicosapentaenoic acid on lipid droplet formation in 3T3-L1 adipocytes. Lipids Health Dis 9, 57.
58. Siriwardhana, N, Kalupahana, NS, Fletcher, S, et al. (2012) n-3 and n-6 polyunsaturated fatty acids differentially regulate adipose angiotensinogen and other inflammatory adipokines in part via NF-kappaB-dependent mechanisms. J Nutr Biochem 23, 16611667.
59. Victora, CG, Adair, L, Fall, C, et al. (2008) Maternal and child undernutrition: consequences for adult health and human capital. Lancet 371, 340357.
60. Gluckman, PD, Hanson, MA, Cooper, C, et al. (2008) Effect of in utero and early-life conditions on adult health and disease. N Engl J Med 359, 6173.
61. Gallou-Kabani, C, Vigé, A, Gross, MS, et al. (2007) Resistance to high-fat diet in the female progeny of obese mice fed a control diet during the periconceptual, gestation, and lactation periods. Am J Physiol Endocrinol Metab 292, 10951100.
62. Chicco, A, Creus, A, lllesca, P, et al. (2016) Effect of post-suckling n-3 polyunsaturated fatty acids: prevention of dyslipidemia and liver steatosis induced in rats by a sucrose-rich diet during pre- and post-natal life. Food Funct 7, 445454.
63. Spalding, KL, Arner, E, Westermark, PO, et al. (2008) Dynamics of fat cell turnover in humans. Nature 453, 783787.
64. Bujalska, IJ, Kumar, S, Hewison, M, et al. (1999) Differentiation of adipose stromal cells: the roles of glucocorticoids and 11beta-hydroxysteroid dehydrogenase. Endocrinology 140, 31883196.
65. Shively, CA, Register, TC & Clarkson, TB (2009) Social stress, visceral obesity, and coronary artery atherosclerosis: product of a primate adaptation. Am J Primatol 71, 742751.
66. Tomlinson, JW & Stewart, PM (2007) Modulation of glucocorticoid action and the treatment of type-2 diabetes. Best Pract Res Clin Endocrinol Metab 21, 607619.
67. Chapman, K, Holmes, M & Seckl, J (2013) 11beta-Hydroxysteroid dehydrogenases: intracellular gate-keepers of tissue glucocorticoid action. Physiol Rev 93, 11391206.
68. Sun, J, Wang, Y, Li, Y, et al. (2014) Downregulation of PPARγ by miR-548d-5p suppresses the adipogenic differentiation of human bone marrow mesenchymal stem cells and enhances their osteogenic potential. J Transl Med 12, 168.
69. Hollenberg, AN, Susulic, VS, Madura, JP, et al. (1997) Functional antagonism between CCAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma on the leptin promoter. J Biol Chem 272, 52835290.
70. Bruley, C, Lyons, V, Worsley, AG, et al. (2006) A novel promoter for the 11beta-hydroxysteroid dehydrogenase type 1 gene is active in lung and is C/EBPalpha independent. Endocrinology 147, 28792885.
71. Berger, J, Tanen, M, Elbrecht, A, et al. (2001) Peroxisome proliferator -activated receptor-gamma ligands inhibit adipocyte 11beta -hydroxysteroid dehydrogenase type 1 expression and activity. J Biol Chem 276, 1262912635.
72. Vagnerová, K, Loukotová, J, Ergang, P, et al. (2011) Peroxisome proliferator-activated receptor-γ stimulates 11β-hydroxysteroid dehydrogenase type 1 in rat vascular smooth muscle cells. Steroids 76, 577581.
73. Dube, S, Norby, B, Pattan, V, et al. (2014) Hepatic 11beta-hydroxysteroid dehydrogenase type 1 activity in obesity and type 2 diabetes using a novel triple tracer cortisol technique. Diabetologia 57, 14461455.
74. Paterson, JM, Morton, NM, Fievet, C, et al. (2004) Metabolic syndrome without obesity: hepatic overexpression of 11beta-hydroxysteroid dehydrogenase type 1 in transgenic mice. Proc Natl Acad Sci U S A 101, 70887093.
75. Harno, E, Cottrell, EC, Keevil, BG, et al. (2013) 11-Dehydrocorticosterone causes metabolic syndrome, which is prevented when 11beta-HSD1 is knocked out in livers of male mice. Endocrinology 154, 35993609.
76. Park, JS, Rhee, SD, Jung, WH, et al. (2012) Anti-diabetic and anti-adipogenic effects of a novel selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor in the diet-induced obese mice. Eur J Pharmacol 691, 1927.
77. Williams, LJ, Lyons, V, MacLeod, I, et al. (2000) C/EBP regulates hepatic transcription of 11beta-hydroxysteroid dehydrogenase type 1. A novel mechanism for cross-talk between the C/EBP and glucocorticoid signaling pathways. J Biol Chem 275, 3023230239.