Fig. 1Mean weights, g, (n=12 to 15) of each treatment group over 14 weeks. Broadly, progeny were exposed to 0 mg/kg 2-aminoanthracene (2AA) diet (control), 50 mg/kg 2AA diet (low dose), and 100 mg/kg 2AA diet (high dose) in utero. Time seem to have an effect on weight gain, no statistical differences were noted with regards to 2AA treatment.
Fig. 2Histologic characterization (H&E stain, 100 µm) of adipose tissue of Sprague Dawley rats exposed to (A) 0 mg/kg- (control [C] pup), (B) 50 mg/kg- (low dose [LD] pup), and (C) 100 mg/kg-2-aminoanthracene (high dose [HD] pup) from gestation through of the postnatal period (H&E stain). Older pups were fed moderate high and regular diet for 6 weeks. Select images include: (D) control female regular diet (CFR); (E) control male regular diet (CMR); (F) control high fat diet (CFH); (G) control male high fat diet (CMH); (H) low dose male high fat (LMH); and (I) high dose male high fat diet (HMH). Small to moderate numbers of macrophages are scattered between adipocytes in all three groups of young pups. Fewer numbers of macrophages are present in all groups of older pups.
Fig. 3Numbers of CD68 positive cells (n=6 to 8) in adipose tissue of Sprague Dawley rats. (A) Intrauterine treatment to 2-aminoanthracene groups included: control (C) pup (0 mg/kg diet), low dose (LD) pup (50 mg/kg diet), and high dose (HD) pup (100 mg/kg diet). (B) In utero exposure was combined with dietary ingestions of regular diet (5M30 rodent diet; PMI Nutrition International) and high fat diet (Adjusted Fat Diet TD.96132) for 6 weeks 3 months postwean. Though the treated exposed rats indicated higher CD68 positive values, statistical inference showed they were not significant. CMH, control male high fat; CMR, control male regular diet; LMH, low dose male high fat; CFR, control female regular diet; LFR, low dose female regular diet; HFR, high dose female regular diet; HMH, high dose high fat.
Fig. 4Mean size of adipocytes (pixels) (n=5 to 6). Significant increment in the adipocyte size observed for the LMH and HMH animals. CMH, control male high fat; CMR, control male regular diet; LMH, low dose male high fat; CFR, control female regular diet; LFR, low dose female regular diet; HFR, high dose female regular diet; HMH, high dose high fat. aP<0.05.
Fig. 5Mean serum glucose concentration (mg/dL) of neonates exposed to 2-aminoanthracene (2AA) in utero. (A) Dams ingested 0 mg/kg (control [C]); 50 mg/kg (low dose [LD]); and 100 mg/kg (high dose [HD])-2AA from gestation through 2 weeks postnatal period; (B) control male high fat (CMH), low dose male high fat (LMH), and high dose high fat (HMH); (C) control female regular diet (CFR), low dose female regular diet (LFR), and high dose female regular diet (HFR); (D) control male regular diet (CMR), low dose male regular diet (LMR), and high dose male regular diet (HMR). Significant differences in serum glucose concentration were noted as aP<0.05, bP<0.01.
Fig. 6Relative expression (ΔCq) of adipose tissue genes involved in diabetic-related conditions. In utero exposure to 2-aminoanthracene (2AA) were 0 mg/kg 2AA diet (control [C]), 50 mg/kg diet (low dose [LD]), and 100 mg/kg diet (high dose [HD]) from gestation through 14 days postnatal. IL-6, interleukin-6; TNF-α, tumor necrosis factor α. Significant changes in differential gene expression is noted as aP<0.05, bP<0.01.
Fig. 7Relative expression (ΔCq) of adipokine and cytokines involved in diabetic-related conditions. (A) In utero exposure of 0 mg/kg 2-aminoanthracene diet (control), 50 mg/kg diet (low dose), and 100 mg/kg diet (high dose) from gestation through 14 days postnatal was combined with dietary ingestions of regular diet (5M30 rodent diet; PMI Nutrition International) and high fat diet (Adjusted Fat Diet TD.96132) for 6 weeks 3 months postwean. (A) Control male regular diet (CMR), low dose male regular diet (LMR), and high dose male regular diet (HMR). (B) Control female regular diet (CFR), low dose female regular diet (LFR), and high dose female regular diet (HFR). (C) Control male high fat (CMH), low dose male high fat (LMH), and high dose high fat (HMH). IL-6, interleukin-6; TNF-α, tumor necrosis factor α. Significant differences (aP<0.05, bP<0.01) in adiponectin and IL-6 mRNA expression were noted.
Fig. 8(A-D) Serum adiponectin levels in rats exposed to 2-aminoanthracene (2AA) in utero and moderate high fat 3 months postwean. Changes in adiponectin amounts were significant (P<0.01) in neonatal rats but not significantly altered in the regular dietary female animals. Both the male regular and high fat dietary group showed significant (P<0.05) reduction of adiponectin in 2AA treated rats. C, control; LD, low dose; HD, high dose; CMH, control male high fat; LMH, low dose male high fat; HMH, high dose high fat; CFR, control female regular diet; LFR, low dose female regular diet; HFR, high dose female regular diet; CMR, control male regular diet; LMR, low dose male regular diet; HMR, high dose male regular diet. aP<0.05, bP<0.01.
Table 1Nucleotide sequences designed as forward and reverse primers of each specific gene
Gene name |
Forward & reverse primer sequence |
Adiponectin |
|
Forward |
5' CCGCTTACATGTATCACTC 3' |
Reverse |
5' ATACTGGTCGTAGGTGAAGA 3' |
CD68 |
|
Forward |
5' AAGTCCTAGTCCAAGCTCTA 3' |
Reverse |
5' AGGACACATTGTATTCCACT 3' |
CD14 |
|
Forward |
5' CTCAGAATCTACCGACCA 3' |
Reverse |
5' ATAGATTGAGCGAGTTTAGC 3' |
IL-6 |
|
Forward |
5' GGAGTTTGTGAAGAACAACT 3' |
Reverse |
5' CTAGGGTTTCAGTATTGCTC 3' |
Leptin |
|
Forward |
5' CTGTCGTGACTGACTCTATG 3' |
Reverse |
5' GCTAAGTGATTTCTCATTCC 3' |
TNF-α |
|
Forward |
5' GAACACCCTGGTACTAACTC 3' |
Reverse |
5' TAGATAAGGTACAGCCCATC 3' |
Table 2Changes in animal weights (g), (n=12 to 15) after administration of low and high doses of 2AA in combination to regular rat chow diet and moderate high fat diet fed Sprague Dawley rat progeny for 5 weeks
Diet |
2AA |
Week 1 |
Week 2 |
Week 3 |
Week 4 |
Week 5 |
High fat |
|
|
|
|
|
Control, g |
445.42±120.55 |
465.37±126.87 |
479.30±130.88 |
498.25±133.16 |
504.82±135.11 |
Low dose, g |
481.72±130.37 |
499.17±140.44 |
511.37±144.91 |
527.82±146.90 |
533.45±151.32 |
High dose, g |
421.13±120.89 |
434.06±123.43 |
451.46±132.95 |
468.4±137.05 |
472.88±140.97 |
Regular diet |
|
|
|
|
|
Control, g |
465.74±123.15 |
467.57±130.19 |
459.08±127.62 |
466.05±124.51 |
470.85±131.89 |
Low dose, g |
501.71±133.56 |
503.28±140.47 |
503.77±138.18 |
510.63±144.35 |
512.71±143.22 |
High dose, g |
437.82±100.41 |
448.52±111.47 |
438.70±105.08 |
448.36±107.06 |
466.32±109.52 |