Expression of Human NSAID Activated Gene 1 in Mice Leads to Altered Mammary Gland Differentiation and Impaired Lactation

Abstract

Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to altered milk quality and quantity.

Fig 1. Reduced survival and milk spots early in lactation.

A.) Percent pup survival to PND3 in pups born to WT and hNAG-1 dams. The data shown is average percent survival +/- SEM across 4 litters. B.) Qualitative analysis of milk spots scored on the day of birth through day PND2 and then averaged for each dam/litter. Data shown is average over 4 litters +/- SEM. Mann-Whitney statistical test was used to compare hNAG-1 transgenic dams to WT control. *, p<0.05.

Fig 2. Histological examination of mammary glands from WT and hNAG-1 mice.

Hematoxylin and eosin staining of WT and hNAG-1 mammary glands on L2 show that hNAG-1 (C&D) mice have reduced occupancy of the fat pad by mammary gland compared to WT (A&B) as denoted by the asterisk. Higher magnification shows that the acini in WT mammary glands (B) are lined with plump cuboidal cells while the hNAG-1 mammary glands (D) have low cuboidal cells and contain only small amount of secretion in their lumen (arrows). TUNEL staining (E-G) was done on L2 WT (E) and hNAG-1 (F&G) mammary glands. Representative images show DAB chromogen with hematoxylin counterstain.

Fig 3. Reduced pup growth and altered milk composition in hNAG-1 dams.

A.) On L2, Eight CD-1 pups were separated from their dam for 3 hours, weighed and placed back with the dam. Pups were allowed to nurse for 30 minutes and weighed again. Weight change due to increased milk intake was calculated. B-D.) Milk was isolated from WT (n = 4) and hNAG-1 (n = 6) mice on lactation day 2. ELISA to look at hNAG-1 concentrations present in milk (A), total triglycerides (B) or NEFA (C) in milk isolated from WT or hNAG-1 transgenic dams. Data shown is average +/- SEM. Mann-Whitney statistical test was used to compare hNAG-1 transgenic to WT dams. *, p<0.05 and ***, p<0.001.

Fig 4. Pup serum shows reduced triglycerides during mid-lactation (PND9) and at weaning (PND21) from hNAG-1 dams compared to WT dams.

CD-1 pups were cross-fostered with WT or hNAG-1 dams on PND2 so the pups are all WT CD-1 pups. Pup serum was examined for Triglycerides at PND 9 (A&B) or PND21 (C&D). Data shown is average +/- SEM. Mann-Whitney statistical test was done to compare each line to WT. *, p<0.05.

Fig 5. hNAG-1 mammary glands have reduced Cidea/CIDEA expression.

Mammary glands were isolated on L2 and RNA or protein was isolated from WT and hNAG-1 mice. A.) Quantitative real-time PCR was performed using primers specific for Cidea and data was normalized to cyclophilin B (Pip1b). Data shown is average +/- SEM. Mann-Whitney statistical test was used to compare each line to WT control. ***, p<0.001. B.) Whole cell lysates were run on a SDS polyacrylamide gel and then immunoblotted using antibodies specific for CIDEA and beta-actin, used as a loading control. Data shown are from independent animals, WT (n = 3) and hNAG-1 (n = 5).