Prenatal stress increases the obesogenic effects of a high-fat-sucrose diet in adult rats in a sex-specific manner

Figures & data

Table I.  Schedule of variable stress during gestation.

	Time of day
Gestation day	Morning (09:00–12:00 h)	Mid-day (12:00–16:00 h)	Afternoon (16:00–20:00 h)
14	Restraint – 60 min	Swim – 10 min	Restraint – 60 min
15	Wet bedding – 12 h (08:00–20:00 h)
16	Swim – 10 min	Restraint – 60 min	Swim – 10 min
17	Noise – 6 h (09:00–15:00 h)	Fast overnight (15:00–09:00 h)
18		Swim – 10 min	Lights overnight
19	Swim – 10 min	Restraint – 60 min	Swim – 10 min
20		Noise – 6 h (12:00–18:00 h)
21	Restraint – 60 min	Swim – 10 min	Restraint – 60 min

Table II.  Body and food intake measurements in adult rats and statistical analysis of the groups.

	Mean ± SEM				Two-way ANOVA
Males	C	CE	HFS	HFSE	Diet	Fischer's	PNS	Fischer's	Interaction	Fischer's	DF
	n = 7	n = 8	n = 8	n = 8		F value		F value		F value
Body measurements
Initial weight (g)	301.6 ± 10.9	306.8 ± 9.5	307.9 ± 10.2	296.1 ± 7.7	ns	0.126	ns	0.226	ns	0.614	3, 31
Initial fat mass (g)	27.3 ± 2.3	28.2 ± 0.9	32.4 ± 2.5	29.1 ± 2.9	ns	1.804	ns	0.3	ns	0.908	3, 31
Initial lean mass (g)	232.8 ± 7.1	236.3 ± 8.1	234.8 ± 8.0	226.9 ± 6.6	ns	0.423	ns	0.2	ns	0.407	3, 31
Final weight (g)	388.0 ± 16.4	389.4 ± 12.7	451.8 ± 18.3	446.0 ± 16.2	< 0.001	17.859	ns	0.039	ns	0.004	3, 30
Final fat mass (g)	38.4 ± 3.6	39.8 ± 2.8	96.1 ± 9.8	100.9 ± 10.1	< 0.001	61.188	ns	0.166	ns	0.05	3, 30
Final lean mass (g)	294.4 ± 10.8	294.5 ± 9.4	302.8 ± 8.9	293.6 ± 10.3	ns	0.15	ns	0.213	ns	0.222	3, 30
Food intake (Kcal/day)	68.7 ± 0.3	69.5 ± 1.6	73.5 ± 0.9	79.6 ± 1.9	< 0.01	30.821	< 0.05	6.483	ns	3.965	3, 11
Energy efficiency (%)	1.9 ± 0.1	1.7 ± 0.1	3.7 ± 0.2	3.4 ± 0.3	< 0.001	68.319	ns	1.172	ns	0.11	3, 31
Females	C	CE	HFS	HFSE	Diet	Fischer's	PNS	Fischer's	Interaction	Fischer's	DF
	n = 8	n = 8	n = 8	n = 8		F value		F value		F value
Body measurements
Initial weight (g)	200.0 ± 4.9	199.5 ± 4.7	189.9 ± 6.2	203.3 ± 5.2	ns	0.369	ns	1.053	ns	1.746	3, 31
Initial fat mass (g)	19.1 ± 0.8	17.9 ± 1.1	17.6 ± 1.4	19.7 ± 1.4	ns	0.015	ns	0.139	ns	1.725	3, 31
Initial lean mass (g)	152.7 ± 3.8	153.1 ± 3.4	145.5 ± 5.6	154.5 ± 3.7	ns	0.488	ns	1.234	ns	1.049	3, 31
Final weight (g)	233.0 ± 6.1	234.1 ± 4.7	246.9 ± 9.1	267.1 ± 6.6	< 0.01	16.611	ns	2.639	ns	3.05	3, 31
Final fat mass (g)	23.1 ± 1.0^a	21.6 ± 1.6^a	41.1 ± 3.5^b	54.3 ± 4.7^c	< 0.001	69.526	ns	3.638	< 0.05	5.77	3, 31
Final lean mass (g)	175.1 ± 4.4	178.4 ± 3.5	166.8 ± 12.7	181.2 ± 3.0	ns	0.154	ns	1.566	ns	0.614	3, 31
Food intake (Kcal/day)	46.9 ± 0.4	54.6 ± 1.8	51.0 ± 2.0	56.5 ± 2.8	ns	2.379	< 0.05	11.102	ns	0.299	3, 11
Energy efficiency (%)	1.0 ± 0.1	0.9 ± 0.1	2.1 ± 0.1	2.0 ± 0.2	< 0.001	68.013	ns	0.491	ns	0.031	3, 31

Note: Results are expressed by mean ± SEM. Different letters (a, b, and c) indicate statistical differences of at least p < 0.05 with post-hoc Bonferroni. Energy efficiency is calculated in percentage as the grams gained divided by the eaten kcal. C, control; CE, control stress; HFS, high-fat-sucrose; HFSE, high-fat-sucrose-stress; PNS, prenatal stress; DF, degrees of freedom.

Figure 1.  The effect of HFS diet intake and PNS on body fat mass gain in adult offspring. Results are group mean ± SEM. Different letters (a, b, and c) indicate significant differences p < 0.05, Post-hoc Bonferroni. (a) Males (n = 7–8), (b) Females (n = 8). C, Control; CE, Control stress; HFS, High-fat-sucrose; HFSE, High-fat-sucrose-stress; PNS, Prenatal stress; ns, not significant.

Figure 2.  The effect of HFS diet intake and PNS on different WAT depots. Results are group mean ± SEM. Different letters (a, b, and c) indicate significant differences p < 0.05, post-hoc Bonferroni. (a) Males (n = 8), (b) Females (n = 8); C, Control, n = 8; CE, Control stress, n = 8; HFS, High-fat-sucrose, n = 8; HFSE, High-fat-sucrose-stress, n = 8; WAT, White adipose tissue; Rp, Retroperitoneal; Epi, Epididymal; Peri, Periovarian; Mes, Mesenteric; Sc, Subcutaneous; PNS, Prenatal stress; ns, not significant.

Table III.  Biochemical measurements in serum in adulthood and statistical analysis of the groups.

	Mean ± SEM				Two-way ANOVA
Males	C	CE	HFS	HFSE	Diet	Fischer's	PNS	Fischer's	Interaction	Fischer's	DF
	n = 7	n = 8	n = 8	n = 8		F value		F value		F value
Biochemical measurements
Glucose (mg/dl)	104.0 ± 3.2	105.7 ± 1.8	119.8 ± 3.2	117.4 ± 3.5	< 0.001	21.028	ns	0.012	ns	0.45	3, 30
Triglycerides (mg/dl)	89.7 ± 10.3	96.6 ± 8.2	73.8 ± 7.1	84.6 ± 11.0	ns	2.282	ns	0.923	ns	0.046	3, 30
Cholesterol (mg/dl)	69.7 ± 3.9	74.9 ± 2.8	71.3 ± 4.4	70.5 ± 3.5	ns	0.148	ns	0.356	ns	0.64	3, 30
HDL (mg/dl)	21.8 ± 0.7	23.6 ± 0.8	21.0 ± 1.0	22.3 ± 0.9	ns	1.385	ns	3.206	ns	0.7	3, 30
FFA (mg/dl)	0.8 ± 0.0	0.9 ± 0.1	0.6 ± 0.1	0.6 ± 0.0	< 0.01	15.402	ns	0.932	ns	1.754	3, 30
Insulin (μU/ml)	7.3 ± 1.6	5.6 ± 1.1	9.7 ± 4.2	23.0 ± 5.6	< 0.05	4.625	ns	3.248	ns	2.285	3, 29
HOMA	30.0 ± 8.5	22.0 ± 3.3	33.0 ± 3.3	81.8 ± 19.7	< 0.05	5.059	ns	3.192	0.052	2.208	3, 29
Leptin (ng/ml)	3.2 ± 0.6	2.6 ± 0.3	15.7 ± 2.9	21.0 ± 2.7	< 0.001	55.043	ns	1.284	ns	1.988	3, 30
MCP1 (pg/ml)	213.8 ± 19.5	230.6 ± 22.5	194.9 ± 8.7	218.8 ± 21.7	ns	0.658	ns	1.153	ns	0.035	3, 30
Females	C	CE	HFS	HFSE	Diet	Fischer's	PNS	Fischer's	Interaction	Fischer's	DF
	n = 8	n = 8	n = 8	n = 8		F value		F value		F value
Biochemical measurements
Glucose (mmol/l)	87.2 ± 2.6	84.0 ± 3.7	96.4 ± 5.0	96.2 ± 2.9	< 0.01	8.476	ns	0.218	ns	0.169	3, 31
Triglycerides (mg/dl)	60.1 ± 10.1	76.9 ± 15.8	43.8 ± 3.5	44.5 ± 6.9	< 0.05	6.557	ns	0.739	ns	0.618	3, 29
Cholesterol (mg/dl)	48.6 ± 4.7	45.8 ± 4.9	41.4 ± 2.5	38.0 ± 2.4	0.059	3.868	ns	0.672	ns	0.004	3. 31
HDL (mg/dl)	18.4 ± 1.3	17.7 ± 1.5	15.9 ± 0.7	15.1 ± 1.1	< 0.05	4.707	ns	0.387	ns	0.001	3, 31
FFA (mg/dl)	0.9 ± 0.0	1.0 ± 0.1	0.7 ± 0.0	0.8 ± 0.1	< 0.01	8.735	ns	0.581	ns	0.233	3, 31
Insulin (μU/ml)	3.4 ± 1.2	0.2 ± 0.1	6.0 ± 0.5	3.5 ± 1.0	< 0.01	9.43	< 0.01	5.806	ns	0.097	3, 29
HOMA	7.6 ± 2.6	0.7 ± 0.3	12.0 ± 1.0	7.7 ± 2.9	< 0.05	5.468	< 0.05	5.325	ns	0.014	3, 25
Leptin (ng/ml)	1.3 ± 0.2	0.9 ± 0.1	3.8 ± 0.8	5.0 ± 0.7	< 0.001	36.504	ns	0.375	ns	2.203	3, 31
MCP1 (pg/ml)	160.6 ± 10.9	170.2 ± 15.5	168.8 ± 7.6	168.2 ± 11.1	ns	0.073	ns	0.149	ns	0.195	3, 31

Note: Results are expressed by mean ± SEM. HDL, high-density lipoprotein; FFA, free fatty acids; MCP1, monocyte chemoattractant protein 1; C, control; CE, Control stress; HFS, high-fat-sucrose; HFSE, high-fat-sucrose-stress; PNS, prenatal stress; DF, degrees of freedom; HOMA, homeostasis model assessment ([fasting plasma glucose (mM) fasting serum insulin (μU/ml)]/22.5).

Figure 3.  IPGTT conducted in female rats in the four experimental groups (n = 8 per group). Data are mean ± SEM, and were analyzed with RM-ANOVA. The results of each time point were analyzed with two-way ANOVA (Diet × PNS): When an interaction between two factors was observed (60 min point, p < 0.05; 90 min point, p < 0.01; 120 min point, p < 0.05), multiple comparisons were made with Student's t-test: #p < 0.05 versus C rats, €p < 0.05 versus HFS rats. C, Control; CS, Control stress; HFS, High-fat-sucrose; HFSE, High-fat-sucrose-stress; PNS, Prenatal stress, ns, not significant.Inset: AUC, area under the curve (n = 8 per group). Data are mean ± SEM, and were analyzed with two-way ANOVA (Diet × PNS).

Figure 4.  The effect of HFS diet intake and PNS on corticosterone concentrations in female rats. Results are expressed by mean ± SEM. (a) Serum corticosterone concentration (n = 8). Data were analyzed with two-way ANOVA (Diet × PNS); (b) Fecal corticosterone content (24 h collection; n = 5–6 per group). Data were analyzed with RM-ANOVA. C, Control; CE, Control stress; HFS, High-fat-sucrose; HFSE, High-fat-sucrose-stress; PNS, Prenatal stress; ns, not significant.

Table IV.  Differentially expressed genes in Peri WAT related to obesity and glucocorticoid metabolism in adult female rats.

	Mean ± SEM				Two-way ANOVA							Variation (%)
Genes	C n = 8	CE n = 8	HFS n = 8	HFSE n = 8	Diet	Fischer'sF value	PNS	Fischer'sF value	Interaction	Fischer'sF value	DF	Diet	PNS
Adpn	1.1 ± 0.2^a	1.3 ± 0.1^a	1.3 ± 0.3^a	3.0 ± 0.5^b	< 0.01	8.271	< 0.01	8.376	< 0.05	5.154	3, 30	↑ 79	↑ 129
Apln	0.5 ± 0.1	0.6 ± 0.1	0.8 ± 0.2	1.0 ± 0.1	< 0.01	8.613	ns	1.13	ns	0.299	3, 31	↑ 68	–
Cdkn1a	1.6 ± 0.3	2.1 ± 0.5	1.0 ± 0.2	0.8 ± 0.2	< 0.01	7.913	ns	0.294	ns	1.448	3, 31	↓ 50	–
Fasn	2.7 ± 0.5	8.8 ± 0.6	1.7 ± 0.4	4.2 ± 0.9	< 0.01	7.695	< 0.001	17.646	ns	3.301	3, 30	↓ 51	↑ 197
Il6	1.1 ± 0.3	0.4 ± 0.2	0.7 ± 0.2	0.5 ± 0.1	ns	0.611	< 0.05	5.652	ns	2.142	3, 30	–	↓ 50
Lep	0.4 ± 0.1	0.3 ± 0.1	0.6 ± 0.1	0.7 ± 0.1	< 0.01	10.147	ns	0.113	ns	1.099	3, 31	↑ 105	–
Ppargc1a	1.9 ± 0.3^a	6.8 ± 2.2^b	2.5 ± 0.7^a	1.5 ± 0.4^a	ns	3.56	ns	2.383	< 0.05	5.868	3, 28	↑ 253	↓ 40
Scd1	0.6 ± 0.1	1.0 ± 0.2	0.7 ± 0.2	1.0 ± 0.2	ns	0.012	< 0.05	4.772	ns	0.011	3, 31	–	↑ 59
11β-hsd2	1.1 ± 0.3	1.3 ± 0.3	1.9 ± 0.4	2.7 ± 0.2	< 0.01	13.078	ns	2.535	ns	1.04	3, 30	↑ 92	–
11β-hsd1	1.0 ± 0.1	1.0 ± 0.1	1.3 ± 0.3	1.2 ± 0.2	ns	1.657	ns	0.061	ns	0.105	3, 30	–	–
GR	1.2 ± 0.2	0.9 ± 0.1	1.2 ± 0.2	1.2 ± 0.1	ns	0.967	ns	0.770	ns	0.328	3, 31	–	–
MR	0.9 ± 0.1	1.0 ± 0.1	1.0 ± 0.2	0.9 ± 0.1	ns	0.018	ns	0.002	ns	0.218	3, 31	–	–

Note: Results are expressed by mean ± SEM. Different letters (a, b) indicate significant differences p < 0.05, post-hoc Bonferroni. C, control; CE, control stress; HFS, high-fat-sucrose; HFSE, high-fat-sucrose-stress; PNS, prenatal stress; ↑ , increase in mRNA level; ↓ , decrease in mRNA level; Adpn, adiponutrin; Apln, apelin; Cdkn1a, cyclin-dependent kinase inhibitor 1A; Fasn, fatty acid synthase; Il6, Interleukin 6; Lep, leptin; Ppargc1a, peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; Scd1, stearoyl-coenzyme A desaturase 1; 11β-hsd2, 11β-hydroxysteroid dehydrogenase 2; 11β-hsd1, 11β-hydroxysteroid dehydrogenase 1; GR, glucocorticoid receptor; MR, mineralocorticoid receptor.