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Under-carboxylated osteocalcin regulates glucose and lipid metabolism during pregnancy and lactation in rats

Abstract

Purpose

Under-carboxylated osteocalcin (UcOC), a bone-released hormone is suggested to regulate energy metabolism. Pregnancy and lactation physiological conditions that require high levels of energy. The current study attempts to examine whether UcOC is involved in regulating energy metabolism during these conditions using adult Wistar rats.

Methods and results

Insulin tolerance tests indicated insulin resistance during late pregnancy (day 19 of pregnancy; P19) and insulin sensitivity during early lactation (day 6 of lactation; L6). Gene expression analyses suggested that muscle glucose metabolism was downregulated during P19 and enhanced during L6. Concomitantly, circulatory UcOC levels were lower during pregnancy but higher during early lactation; the rise in UcOC levels was tightly linked to the lactation process. Altering endogenous UcOC levels pharmacologically with warfarin and alendronate in P19 and L6 rats changed whole-body insulin response and muscle glucose transporter (Glut4) expression. Glut4 expression can be increased by either UcOC or estrogen receptors (ERs), both of which act independent of each other. A high fat diet decreased UcOC levels and insulin sensitivity in lactating rats, suggesting that diet can compromise UcOC-established energy homeostasis. Gene expression of lipid metabolism markers and triglyceride levels suggested that UcOC suppression during early pregnancy is an essential step in maternal lipid storage.

Conclusion

Taken together, we found that UcOC plays an important role in energy homeostasis via regulation of glucose and lipid metabolism during pregnancy and lactation.

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Acknowledgements

AP and MR were involved in conception, design of experiments and data analysis. AP, MP, and TTC performed experiments. AP, and MR prepared the manuscript. AP, MR, NSA and HRK were involved in manuscript edits. The authors would like to thank Prof. N.G. Kondegowda, Assistant Professor, Department of Translational Research and Cellular Therapeutics, and C.J. Crook, Irell and Manella Graduate School of Biological Sciences and Department of Translational Research and Cellular Therapeutics, City of Hope, CA, for their valuable comments and edits to the manuscript. We thank MR laboratory trainees Ravi Pal, Raza Akhtar and Vijaya Verma for their assistance during experiments. All authors commented on and approved the final version of the manuscript. MR received grant support from the Department of Biotechnology, India (BT/PR21380/AAQ/1/678/2016) and AP received a senior research fellowship from the Council of Scientific and Industrial Research, India.

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Correspondence to M. Rudraiah.

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All procedures in rats were approved by the Institutional Animal Ethics Committee, Indian Institute of Science (Bangalore, India). This article does not contain any studies with human participants performed by any of the authors.

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Supplementary file1 (PDF 124 kb)

Supplementary file2 Supplementary Figure 1 Expression analysis of markers for insulin pathway, glycogenesis, and glycolysis in NPNL, pregnant, lactating, and forced wean rats. Soleus muscle tissue was collected during pregnancy, lactation, and after forced weaning for qPCR and western blotting. qPCR expression analysis of insulin pathway genes insulin receptor (A) and Foxo1 (B); insulin sensitivity markers Mcad (C), Nrf1 (D), and Pparα (E); glucose metabolism genes glycogenesis marker Gys1 (F) and gluconeogenesis marker Pck (G); and glucose transporter Glut4 (H) in muscle of NPNL, pregnant, lactating, and forced wean rats. Immunoblot analysis of insulin pathway molecules pPI3K (I), pPTEN (J), pPDK1 (K), pAKT/AKT (L), glycogenesis regulatory molecule pGSK3β (M) and glycolysis marker GAPDH (N) was performed. A representative immunoblot is presented in (O). β-tubulin was used as a loading control. Densitometric values are presented as mean ± SEM (n= 3 per group). NPNL analysis results were as set as 1-fold; analysis of other groups is expressed in relation to 1-fold and presented as mean ± SEM (n= 4 per group). Statistical significance was calculated by one-way ANOVA. *, **, and *** represent significantly different by p<0.05, 0.01, and 0.001. NPNL: nonpregnant nonlactating; P6, 12, and 19: day 6, 12, and 19 of pregnancy; L3, 6, 11, and 20: day 3, 6, 11, and 20 of lactation; F6: day 6 of forced weaning. (EPS 171 kb)

Supplementary file3 Supplementary Figure 2 qPCR expression analysis of insulin pathway genes in liver of NPNL, pregnant, lactating, and forced wean rats. qPCR expression analysis of insulin receptor (A), Foxo1 (B), insulin sensitivity marker Mcad (C), glycogenesis marker Gys2 (D), glycolysis markers Gck2 (E) and Pfk1 (F), gluconeogenesis marker Pck (G), glucose transporter Glut2 (H). NPNL analysis results were set as 1-fold and expression changes in other groups expressed in relation to 1-fold change of NPNL; results were presented as mean ± SEM (n=4 per group). Immunoblot analysis of insulin pathway molecules pPI3K (I), pPTEN (J), pPDK1 (K), pAKT/AKT (L), glycogenesis regulatory molecule pGSK3β (M) and glycolysis marker GAPDH (N) in liver of NPNL, pregnant, lactating, and forced wean rats. β-actin was used as the loading control. The densitometric value of each protein is represented as mean ± SEM (n= 3 per group). Statistical significance was calculated by one-way ANOVA. * and ** represent significant differences between groups by p<0.05 and 0.01, respectively. NPNL: nonpregnant nonlactating; P6, 12, and 19: day 6, 12, and 19 of pregnancy: L3, 6, 11, and 20: day 3, 6, 11, and 20 of lactation; F6: day 6 of forced weaning. (EPS 210 kb)

Supplementary file4 Supplementary Figure 3. qPCR expression of insulin pathway markers insulin receptor (A) and Foxo1 (B), insulin sensitivity marker Mcad (C), glycolysis marker Gck2 (D), and glucose transporter Glut4 (E) from GWAT RNA of NPNL, pregnant, lactating, and forced wean rats. Gene expression from NPNL rats was set as 1-fold; analysis of other groups is expressed in relation to 1-fold and presented as mean ± SEM (n=4 per group). Statistical significance was calculated by one-way ANOVA. *, **, and *** represent significant differences by p<0.05, 0.01, and 0.001, respectively. GWAT: gonadal white adipose tissue; NPNL: nonpregnant nonlactating; P6, 12, and 19: day 6, 12, and 19 of pregnancy; L3, 6, 11, and 20: day 3, 6, 11, and 20 of lactation: F6: day 6 of forced weaning. (EPS 39 kb)

Supplementary file5 Supplementary Figure 4. Serum OC and CTx levels and histological changes in mammary glands of rats treated with either WF or ALN. Serum OC and CTx levels in WF-treated NPNL (A, E), WF-treated pregnant (B, F), WF-treated lactating (C, G), and ALN-treated lactating rats (D, H). Data are presented as mean ± SEM (n = 5 per group). Statistical significance was calculated by one-way ANOVA (A, D, E, H) and t-test (B, C, F, G). *, **, and *** represent significant differences by p<0.05, 0.01, and 0.01, respectively. Mammary gland sections from lactating, WF-treated (0.25 mg/kg BW), and ALN-treated (200 µg/kg BW) rats were stained with hematoxylin and eosin; images are presented at 40X magnification. Beneath epithelial tissue (dark purple), white fat depots can be seen. The red arrows indicate secretory parenchymal areas. OC: Osteocalcin; NPNL: nonpregnant nonlactating; VEH: vehicle; P6: day 6 of pregnancy receiving vehicle or 0.25 mg/kg BW/d WF treatment for three consecutive days; L6: day 6 of lactation receiving vehicle and ALN treatment for six weeks. (EPS 871 kb)

Supplementary file6 Supplementary Figure 5. Effects of HFD during lactation. Rats were maintained on regular laboratory chow diet or high-fat diet (ND and HFD, respectively) as described in Materials and Methods. Bodyweight of rats were monitored during lactation (A). Rats (n=3) were euthanized on day 6 of lactation. GWAT was collected and weighed (B). Blood was collected and serum analyzed for triglyceride levels (C). Liver sections were collected and stained with hematoxylin and eosin. Images are presented at 100, 200, and 400X magnification (D). Black arrows indicate lipid droplets; red arrows indicate infiltrated blood cells. H&E sections are representative of tissue sections from three rats. One group of rats was maintained during lactation and litter size (E) was monitored. Values are presented as mean ± SEM (n= 3). Statistical significance between the two groups was determined by two-way ANOVA (A, F) and t-test (B, C, E). * and ** represent significant differences by p<0.05 and 0.01, respectively. L6: day 6 of lactation; GWAT: gonadal white adipose tissue; TG: triglyceride; ND: regular laboratory chow diet; HFD: high-fat diet; L6: day 6 of lactation. (EPS 1767 kb)

Supplementary file7 Supplementary Figure 6. Gene expression analysis of lipid metabolism in adipose tissue of nonpregnant rats treated with or without ALN and WF. Adipose tissue was collected from both groups and total RNA was converted into cDNA. qPCR expression analysis was performed to examine expression of lipogenesis markers such as Lpl (A), Fatp1 (B), Fatp4 (C), Fabp (D), Acbp (E), Fasn (F), Mgat (G), and Dgat1 (H), and lipolysis enzymes such as Atgl (I), Hsl (J), and Mgll (K). Values are mean ± SEM (n= 4). Statistical significance between the two groups was determined by one-way ANOVA. *, **, and *** represent significant differences compared to NPNL by p<0.05, 0.01 and 0.001. ALN: alendronate; WF: warfarin; NPNL: nonpregnant nonlactating. (EPS 58 kb)

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Pandey, A., Khan, H.R., Alex, N.S. et al. Under-carboxylated osteocalcin regulates glucose and lipid metabolism during pregnancy and lactation in rats. J Endocrinol Invest (2020). https://doi.org/10.1007/s40618-020-01195-8

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Keywords

  • Under-carboxylated osteocalcin
  • Metabolism
  • Estrogen receptor
  • Pregnancy
  • Lactation