We previously reported that after 2 weeks of the low-Ca diet (low-Ca group: 0.008% Ca) or control diet (control group: 0.90% Ca), no differences in serum glucose, corticosterone, or insulin levels were observed between the two groups. In adulthood, 1 rat month is comparable to 3 human years [
53]. The homeostasis model assessment of insulin resistance (HOMA-IR) has proved to be a robust tool for the assessment of insulin resistance [
68]. The low-Ca group rats showed higher values of HOMA-IR (
p < 0.05) and intact parathyroid hormone (
p < 0.05) and lower values of adiponectin (
p < 0.01). In the low-Ca group, the expression of hepatic
Hsd11b1 mRNA was up-regulated, and hepatic
Pck1 expression was down-regulated (
p < 0.001). The expression levels of
Nr3c1,
Ppara, and
Hsd11b2 showed a similar tendency. The 2-week Ca-deficient diet in rats was associated with the upregulation of the hepatic expression of
Hsd11b1 mRNA, which occurred before the animals developed obesity or overt features of metabolic syndrome [
69]. Over-activity of 11β-HSD1 is associated with increased intracellular active glucocorticoids [
57,
58,
59]. Rodent genetic studies have suggested that increased
Hsd11b1 expression or activity increases the risk of several components of metabolic syndrome [
60,
61]. In summary, a low-Ca diet alters glucocorticoid metabolism, which leads to hepatic upregulation of
Hsd11b1, and is possibly a key mechanism of the induction of metabolic complications caused by Ca deficiency [
69].
4. A Ca-Deficient Diet in Pregnant or Nursing Rats Affects the Offspring
Lillycrop et al. reported that pregnant rats on a protein-restricted diet developed hypomethylation and increased expression from the
Ppara and
Nr3c1 promoters in the liver of the offspring [
70,
71]. This demonstrates that maternal nutrition during pregnancy can affect the regulation of non-imprinted genes via the altered epigenetic regulation of gene expression, thereby inducing different metabolic phenotypes. A high-fat diet during pregnancy was reported to induce neonatal gender-specific hepatic fat accumulation by increased
pck1 expression and histone modification [
72].
4.1. The Methylation of Specific Cytosines within the 11β-Hydroxysteroid Dehydrogenase-1 Promoter in the Liver of the Offspring
We investigated the methylation of individual CpG dinucleotides in glucocorticoid-related genes in liver tissue of neonatal offspring from Ca-deficient rat dams. Female rats consumed either a Ca-deficient (0.008% Ca) or control (0.90% Ca) diet ad libitum from 3 weeks before conception to 21 days after parturition. Pups were allowed to nurse from their original mothers and were then sacrificed on day 21. The methylation of CpG dinucleotides in the
Pck1 [
73],
Ppara, Nr3c1,
Hsd11b1, and
Hsd11b2 promoters was measured in liver tissue by pyrosequencing [
65]. The methylation levels of all genes did not differ between groups, except for
Hsd11b1, which was significantly lower in the rats from the Ca-deficient dams (
p < 0.05). Serum corticosterone levels were higher in the male pups from the Ca-deficient dams than in those from the control dams (
p < 0.05). The expression levels of
Pck1 and
Nr3c1 were significantly lower in the Ca-deficient group than in the control group, whereas those of
Hsd11b1, Hsd11b2, and
Ppara did not differ significantly [
74].
Although the hepatic expression of
Hsd11b1 may have been initially up-regulated by epigenetic mechanisms in the offspring from Ca-deficient dams,
Hsd11b1 was likely down-regulated by other mechanisms during the early postnatal period. The methylation level of hepatic
Hsd11b1 was altered in the offspring as a consequence of the maternal dietary manipulation, but the epigenetic changes were not reflected in corresponding alterations in transcription. The nuclear receptor co-repressor complex is affected by environmental factors such as nutrients and hormones, which can lead to altered DNA methylation, acetylation, histone modification, other epigenetic changes, or some combination thereof; such epigenetic changes can and do alter the activity of DNA. These factors can also alter feedback loops involving nuclear receptors that normally regulate repression and maintain balance [
75].
The down-regulation of
Hsd11b1 suggests that a compensatory mechanism may diminish cortisol production in the liver. Reduced hepatic glucocorticoid exposure also represents a compensatory mechanism that limits the metabolic complications of insulin resistance. In our study, no significant difference in serum 11β-HSD1 levels was found among the offspring groups; however, this may have been due to tissue-specific differences between serum and liver. Whether glucocorticoids modulate
Hsd11b1 expression is unknown, and
Hsd11b1 expression differs greatly between the liver and other tissues [
76,
77,
78]. Obese rodents exhibit tissue-specific dysregulation of 11β-HSD1; it is usually up-regulated in adipose tissue and down-regulated in the liver [
79,
80]. In both obese Zucker rats and obese humans, 11β-HSD1 activity is high in adipose tissue but low in the liver [
77,
78,
81]. In adipose tissue and smooth muscle cells, glucocorticoid induces
Hsd11b1 mRNA expression, but contradictory results have been obtained in the liver [
78,
81].
In summary, a Ca-deficient diet during pregnancy and nursing induced hypomethylation of specific CpG dinucleotides in the
Hsd11b1 promoter in the liver tissue of neonatal offspring. These changes in
Hsd11b1 expression likely contribute to marked increases in glucocorticoid hormone action in liver tissue [
67] and potentiate the induction of insulin resistance during adult life [
56].
4.2. A Ca-Deficient Diet in Dams during Gestation Increases Insulin Resistance in Male Offspring
The offspring rats of the same experimental methods as described in the previous section were raised to adults. Pups were allowed to nurse from their original mothers until weaning, when they were fed a control diet. The offspring were then sacrificed at an age of 180 days. The mean levels of insulin and glucose as well as the HOMA-IR values were higher only in the male offspring from the Ca-deficient dams than in those from the control dams (
p < 0.01) [
82]. In all offspring, the serum leptin levels were correlated with the serum insulin levels, and they were inversely correlated with the levels of ionized Ca.
A Ca-deficient diet in dams during gestation and early nursing may alter the glucocorticoid metabolism of her offspring, resulting in higher intracellular glucocorticoid concentration in the hepatic cells of the offspring; this abnormal glucocorticoid metabolism may induce the metabolic complications associated with Ca deficiency. Dietary Ca restriction in dams during pregnancy alters postnatal growth, the expression of Hsd11b1, and insulin resistance in a sex-specific manner.
4.3. Osteocalcin in the Offspring from a Ca-Deficient Dams
Osteocalcin (OC), or bone γ carboxyglutamic acid (Gla) protein, is the most abundant non-collagenous bone matrix protein [
83]. OC is specifically expressed in osteoblast lineage cells and secreted from bone into the bloodstream [
84]. OC is subjected to post-translational carboxylation by a vitamin K-dependent carboxylase to yield carboxylated (Gla-OC) and undercarboxylated (Glu-OC) molecules [
85]. Glu-OC acts directly on pancreatic β-cells to increase insulin secretion, as well as insulin sensitivity and glucose tolerance [
86,
87,
88]. The offspring rats of the same experimental methods as described in the previous section were raised to adults [
82]. The mean levels of Glu-OC in Ca-deficient female offspring were higher than those in control female offspring and control male offspring. The mean levels of Gla-OC were higher in Ca-deficient female offspring than those in control female offspring, whereas no significant difference was observed in these measures between the two groups in male offspring. The effects of Glu-OC on glucose homeostasis have been reported to differ by sex [
89]. Increased Glu-OC could contribute to lower insulin resistance in female Ca-deficient offspring, and therefore might be beneficial for glucose metabolism. Consequently, only male Ca-deficient offspring may acquire insulin resistance.