Hypergravity disruption of homeorhetic adaptations to lactation in rat dams include changes in circadian clocks

Abstract

Altered gravity load induced by spaceflight (microgravity) and centrifugation (hypergravity) is associated with changes in circadian, metabolic, and reproductive systems. Exposure to 2-g hypergravity (HG) during pregnancy and lactation decreased rate of mammary metabolic activity and increased pup mortality. We hypothesize HG disrupted maternal homeorhetic responses to pregnancy and lactation are due to changes in maternal metabolism, hormone concentrations, and maternal behavior related to gravity induced alterations in circadian clocks. Effect of HG exposure on mammary, liver and adipose tissue metabolism, plasma hormones and maternal behavior were analyzed in rat dams from mid-pregnancy (Gestational day [G]11) through early lactation (Postnatal day [P]3); comparisons were made across five time-points: G20, G21, P0 (labor and delivery), P1 and P3. Blood, mammary, liver, and adipose tissue were collected for analyzing plasma hormones, glucose oxidation to CO(2) and incorporation into lipids, or gene expression. Maternal behavioral phenotyping was conducted using time-lapse videographic analyses. Dam and fetal-pup body mass were significantly reduced in HG in all age groups. HG did not affect labor and delivery; however, HG pups experienced a greater rate of mortality. PRL, corticosterone, and insulin levels and receptor genes were altered by HG. Mammary, liver and adipose tissue metabolism and expression of genes that regulate lipid metabolism were altered by HG exposure. Exposure to HG significantly changed expression of core clock genes in mammary and liver and circadian rhythms of maternal behavior. Gravity load alterations in dam's circadian system may have impacted homeorhetic adaptations needed for a successful lactation.

Keywords: Circadian; Homeorhesis; Hypergravity; Lactation; Mammary; Pregnancy.

Fig. 1.. Dam activity during light (L) and dark (D) phases of 12h:12h LD cycles in control and hypergravity environments during mid and late gestation.

Values are mean ±SEM; to determine probability of difference, ANOVA was used with gravity, day and gravity × day as main effects; *indicates significant difference between treatments at p<0.05.

Fig. 2.. Plasma hormone concentrations during the periparturient period in control (open bars) and HG (black bars) treated rat dams.

Values are mean ±SEM; to determine probability of difference, ANOVA was used with gravity, day and gravity × day as main effects; *indicates significant difference between treatments at p<0.05. (A) Concentrations of PRL in plasma of rat dams during the periparturient period. (B) Concentrations of Cort in plasma in rat dams during the periparturient period. (C) Concentrations of INS in plasma in rat dams during the periparturient period.

Fig. 3.. Rate of glucose oxidation to CO₂ or incorporation into lipids in mammary, liver and adipose tissues from dams exposed to HG (black bars) or a control (open bars) environment during the periparturient period.

Tissue slices were incubated in Krebs-Ringer Bicarbonate (KRB) buffer in the presence of 1μCi/flask U-¹⁴C-glucose as a tracer. Glucose oxidation to CO₂ was measured and expressed as nmoles of glucose utilized per 100mg tissue/hr. Rate of glucose incorporation into lipids was measured and expressed as nmoles glucose incorporated into lipids/100mg tissue/hr. Values are expressed as mean ±SEM; to determine probability of difference, ANOVA was used with gravity, day and gravity × day as main effects; *indicates significant difference between treatments at p<0.05. (A) Glucose oxidation to CO₂ in mammary tissue. (B) Glucose incorporation to lipids in mammary tissue. (C) Glucose oxidation to CO₂ in liver tissue. (D) Glucose incorporation to lipids in liver tissue. (E) Glucose oxidation to CO₂ in adipose tissue; expressed as 1 hr per 1 million adipocytes. (F) Glucose incorporation to lipids in adipose tissue; expressed as 1 hr per 1 million adipocytes.

Fig. 4.. Effect of gravity and stage on relative expression levels of lipid metabolic and hormone receptor genes.

Total RNA was isolated from (A) mammary, (B) liver, and (C) adipose of late pregnant (G20) and early lactation (P1) control and HG treated dams. Expression of Acaca, Acyl, Lpl, GR and Prlr were measured using rt-q-PCR. Actb and B2m were used as reference genes and relative gene expression (RQ) was calculated using the delta-delta CT method with mean G20 control delta CT as normalizer. Values are expressed as mean ±SEM; to determine probability of difference, ANOVA was used with gravity, day and gravity × day as main effects; *indicates significant difference between treatments at p<0.05.

Fig. 5.. Effect of gravity and stage on relative expression levels of molecular core clock and clock regulatory genes.

Total RNA was isolated from (A) mammary and (B) liver of late pregnant (G20) and early lactation (P1) control and HG treated dams. Expression of Arntl, Npas2, Rora, Bhlh40e, Csnk1e, and Nr1d1 were measured using rt-q-PCR. Actb and B2m were used as reference genes in mammary and liver, respectively, and relative gene expression (RQ) was calculated using the delta-delta CT method with mean G20 control delta CT as normalizer. Values are expressed as mean ±SEM; to determine probability of difference, ANOVA was used with gravity, day and gravity × day as main effects; *indicates significant difference between treatments at p<0.05.