The mTORC1 complex in pre-osteoblasts regulates whole-body energy metabolism independently of osteocalcin

Abstract

Overnutrition causes hyperactivation of mTORC1-dependent negative feedback loops leading to the downregulation of insulin signaling and development of insulin resistance. In osteoblasts (OBs), insulin signaling plays a crucial role in the control of systemic glucose homeostasis. We utilized mice with conditional deletion of Rptor to investigate how the loss of mTORC1 function in OB affects glucose metabolism under normal and overnutrition dietary states. Compared to the controls, chow-fed Rptor _ob ^−/− mice had substantially less fat mass and exhibited adipocyte hyperplasia. Remarkably, upon feeding with high-fat diet, mice with pre- and post-natal deletion of Rptor in OBs were protected from diet-induced obesity and exhibited improved glucose metabolism with lower fasting glucose and insulin levels, increased glucose tolerance and insulin sensitivity. This leanness and resistance to weight gain was not attributable to changes in food intake, physical activity or lipid absorption but instead was due to increased energy expenditure and greater whole-body substrate flexibility. RNA-seq revealed an increase in glycolysis and skeletal insulin signaling pathways, which correlated with the potentiation of insulin signaling and increased insulin-dependent glucose uptake in Rptor-knockout osteoblasts. Collectively, these findings point to a critical role for the mTORC1 complex in the skeletal regulation of whole-body glucose metabolism and the skeletal development of insulin resistance.

References

[1] Ferron M et al. Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism. Cell, 2010, 142:296-308

[2] Fulzele K et al. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition. Cell, 2010, 142:309-319

[3] Rached MT et al. FoxO1 expression in osteoblasts regulates glucose homeostasis through regulation of osteocalcin in mice. J. Clin. Invest., 2010, 120:357-368

[4] Yoshizawa T et al. The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts. J. Clin. Invest., 2009, 119:2807-2817

[5] Wei J, Hanna T, Suda N, Karsenty G, Ducy P. Osteocalcin promotes beta-cell proliferation during development and adulthood through Gprc6a. Diabetes, 2014, 63:1021-1031

[6] Lee NK et al. Endocrine regulation of energy metabolism by the skeleton. Cell, 2007, 130:456-469

[7] Mizokami A et al. Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion. Bone, 2014, 69:68-79

[8] Ferron M, Hinoi E, Karsenty G, Ducy P. Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc. Natl Acad. Sci. USA, 2008, 105:5266-5270

[9] Ferron M, McKee MD, Levine RL, Ducy P, Karsenty G. Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice. Bone, 2012, 50:568-575

[10] Um SH, D’Alessio D, Thomas G. Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab., 2006, 3:393-402

[11] Bentzinger CF et al. Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab., 2008, 8:411-424

[12] Polak P et al. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. Cell Metab., 2008, 8:399-410

[13] Lee PL, Tang Y, Li H, Guertin DA. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease. Mol. Metab., 2016, 5:422-432

[14] Fitter S et al. mTORC1 plays an important role in skeletal development by controlling preosteoblast differentiation. Mol. Cell. Biol., 2017, 37:e00668-00616

[15] Chen J, Long F. mTORC1 signaling promotes osteoblast differentiation from preosteoblasts. PLoS One, 2015, 10

[16] Dai Q et al. mTOR/Raptor signaling is critical for skeletogenesis in mice through the regulation of Runx2 expression. Cell Death Differ., 2017, 24:1886-1899

[17] Riddle R et al. Tsc2 is a molecular checkpoint controlling osteoblast development and glucose homeostasis. Mol. Cell. Biol., 2014, 34:1850-1862

[18] Martin SK et al. mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis. Sci. Rep., 2018, 8

[19] Dirckx N et al. Vhl deletion in osteoblasts boosts cellular glycolysis and improves global glucose metabolism. J. Clin. Invest., 2018, 128:1087-1105

[20] Mosialou I et al. MC4R-dependent suppression of appetite by bone-derived lipocalin 2. Nature, 2017, 543:385-390

[21] Hara K et al. Measurement of the high-molecular weight form of adiponectin in plasma is useful for the prediction of insulin resistance and metabolic syndrome. Diabetes Care, 2006, 29:1357-1362

[22] Cawthorn WP et al. Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction. Cell Metab., 2014, 20:368-375

[23] Shah OJ, Wang Z, Hunter T. Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr. Biol., 2004, 14:1650-1656

[24] Rodda SJ, McMahon AP. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development, 2006, 133:3231-3244

[25] Um SH et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature, 2004, 431:200-205

[26] Harrington LS et al. The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J. Cell Biol., 2004, 166:213-223

[27] Carvalho E et al. Low cellular IRS 1 gene and protein expression predict insulin resistance and NIDDM. FASEB J., 1999, 13:2173-2178

[28] Wang Y, Nishina PM, Naggert JK. Degradation of IRS1 leads to impaired glucose uptake in adipose tissue of the type 2 diabetes mouse model TALLYHO/Jng. J. Endocrinol., 2009, 203:65-74

[29] Flatt JP. Dietary fat, carbohydrate balance, and weight maintenance. Ann. N. Y. Acad. Sci., 1993, 683:122-140

[30] Solinas G, Borén J, Dulloo AG. De novo lipogenesis in metabolic homeostasis: More friend than foe? Mol. Metab., 2015, 4:367-377

[31] Wei J et al. Bone-specific insulin resistance disrupts whole-body glucose homeostasis via decreased osteocalcin activation. J. Clin. Investig., 2014, 124:1781-1793

[32] Zoch ML, Abou DS, Clemens TL, Thorek DLJ, Riddle RC. In vivo radiometric analysis of glucose uptake and distribution in mouse bone. Bone Res., 2016, 4:16004

[33] Sulston RJ et al. Increased circulating adiponectin in response to thiazolidinediones: investigating the role of bone marrow adipose tissue. Front Endocrinol., 2016, 7:128

[34] Pittas AG, Harris SS, Eliades M, Stark P, Dawson-Hughes B. Association between serum osteocalcin and markers of metabolic phenotype. J. Clin. Endocrinol. Metab., 2009, 94:827-832

[35] Singha UK et al. Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells. J. Cell Biochem., 2008, 103:434-446

[36] Kuo T-R, Chen C-H. Bone biomarker for the clinical assessment of osteoporosis: recent developments and future perspectives. Biomark. Res., 2017, 5

[37] Cao JJ, Sun L, Gao H. Diet-induced obesity alters bone remodeling leading to decreased femoral trabecular bone mass in mice. Ann. N. Y. Acad. Sci., 2010, 1192:292-297

[38] Yoshikawa Y et al. Genetic evidence points to an osteocalcin-independent influence of osteoblasts on energy metabolism. J. Bone Min. Res., 2011, 26:2012-2025

[39] Gillespie JR et al. GSK-3β function in bone regulates skeletal development, whole-body metabolism, and male life span. Endocrinology, 2013, 154:3702-3718

[40] Yao Q et al. Wnt/beta-catenin signaling in osteoblasts regulates global energy metabolism. Bone, 2017, 97:175-183

[41] Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes/Metab. Res. Rev., 1999, 15:412-426

[42] Perry RJ et al. Leptin mediates a glucose-fatty acid cycle to maintain glucose homeostasis in starvation. Cell, 2018, 172:234-248 e217

[43] Devlin MJ et al. Caloric restriction leads to high marrow adiposity and low bone mass in growing mice. J. Bone Min. Res., 2010, 25:2078-2088

[44] Bredella MA et al. Increased bone marrow fat in anorexia nervosa. J. Clin. Endocrinol. Metab., 2009, 94:2129-2136

[45] Cawthorn WP et al. Expansion of bone marrow adipose tissue during caloric restriction is associated with increased circulating glucocorticoids and not with hypoleptinemia. Endocrinology, 2016, 157:508-521

[46] Yamauchi T et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature, 2003, 423:762-769

[47] Liu Q et al. Adiponectin regulates expression of hepatic genes critical for glucose and lipid metabolism. Proc. Natl Acad. Sci., 2012, 109:14568-14573

[48] Zhang F, Xu X, Zhou B, He Z, Zhai Q. Gene expression profile change and associated physiological and pathological effects in mouse liver induced by fasting and refeeding. PLoS One, 2011, 6

[49] Ortega FJ et al. The gene expression of the main lipogenic enzymes is downregulated in visceral adipose tissue of obese subjects. Obesity, 2010, 18:13-20

[50] Jiang L et al. Leptin contributes to the adaptive responses of mice to high-fat diet intake through suppressing the lipogenic pathway. PLoS One, 2009, 4

[51] Roberts R et al. Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans. Diabetologia, 2009, 52:882-890

[52] McLaughlin T et al. Adipose cell size and regional fat deposition as predictors of metabolic response to overfeeding in insulin-resistant and insulin-sensitive humans. Diabetes, 2016, 65:1245-1254

[53] Herman MA et al. A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism. Nature, 2012, 484:333-338

[54] Fisher FM et al. FGF21 regulates PGC-1alpha and browning of white adipose tissues in adaptive thermogenesis. Genes Dev., 2012, 26:271-281

[55] Bostrom, P. et al. A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481, 463–468 (2012).

[56] Brun J et al. Bone regulates browning and energy metabolism through mature osteoblast/osteocyte PPARgamma expression. Diabetes, 2017, 66:2541-2554

[57] Fulzele K et al. Osteocyte-secreted Wnt signaling inhibitor sclerostin contributes to beige adipogenesis in peripheral fat depots. J. Bone Min. Res, 2017, 32:373-384

[58] Liu W et al. Osteocyte TSC1 promotes sclerostin secretion to restrain osteogenesis in mice. Open Biol., 2019, 9:180262

[59] Goodpaster BH, Sparks LM. Metabolic flexibility in health and disease. Cell Metab., 2017, 25:1027-1036

[60] De Pergola G et al. Fuel metabolism in adult individuals with a wide range of body mass index: effect of a family history of type 2 diabetes. Diabetes Nutr. Metab., 2003, 16:41-47

[61] Ukropcova B et al. Family history of diabetes links impaired substrate switching and reduced mitochondrial content in skeletal muscle. Diabetes, 2007, 56:720-727

[62] Guridi M et al. Alterations to mTORC1 signaling in the skeletal muscle differentially affect whole-body metabolism. Skelet. Muscle, 2016, 6:13

[63] Umemura A et al. Liver damage, inflammation, and enhanced tumorigenesis after persistent mTORC1 inhibition. Cell Metab., 2014, 20:133-144

[64] Tremblay F, Marette A. Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J. Biol. Chem., 2001, 276:38052-38060

[65] Chang GR et al. Rapamycin protects against high fat diet-induced obesity in C57BL/6J mice. J. Pharm. Sci., 2009, 109:496-503

[66] Liu Y et al. Rapamycin-induced metabolic defects are reversible in both lean and obese mice. Aging, 2014, 6:742-754

[67] Chang GR et al. Long-term administration of rapamycin reduces adiposity, but impairs glucose tolerance in high-fat diet-fed KK/HlJ mice. Basic Clin. Pharm. Toxicol., 2009, 105:188-198

[68] Lamming DW et al. Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science, 2012, 335:1638-1643

[69] Houde VP et al. Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue. Diabetes, 2010, 59:1338-1348

[70] Moayeri A et al. Fracture risk in patients with type 2 diabetes mellitus and possible risk factors: a systematic review and meta-analysis. Ther. Clin. Risk Manag., 2017, 13:455-468

[71] Vestergaard P. Discrepancies in bone mineral density and fracture risk in patients with type 1 and type 2 diabetes-a meta-analysis. Osteoporos. Int., 2007, 18:427-444

[72] Oei L et al. High bone mineral density and fracture risk in type 2 diabetes as skeletal complications of inadequate glucose control: the Rotterdam Study. Diabetes Care, 2013, 36:1619-1628

[73] Leite Duarte ME, da Silva RD. Histomorphometric analysis of the bone tissue in patients with non-insulin-dependent diabetes (DMNID). Rev. Hosp. Clin., 1996, 51:7-11

[74] Boronat S, Barber I, Thiele EA. Sclerotic bone lesions in tuberous sclerosis complex: a genotype-phenotype study. Am. J. Med. Genet., 2017, 173:1891-1895

[75] Longo KA et al. The 24-hour respiratory quotient predicts energy intake and changes in body mass. Am. J. Physiol.-Regulatory, Integr. Comp. Physiol., 2010, 298:R747-R754

[76] Srinivas S et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol., 2001, 1:4

[77] Kraus D, Yang Q, Kahn BB. Lipid extraction from mouse feces. Bio-Protoc., 2015, 5

[78] Mehlem A, Hagberg CE, Muhl L, Eriksson U, Falkevall A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat. Protoc., 2013, 8:1149

[79] Fitter S et al. Plasma adiponectin levels are markedly elevated in imatinib-treated chronic myeloid leukemia (CML) patients: a mechanism for improved insulin sensitivity in type 2 diabetic CML patients? J. Clin. Endocrinol. Metab., 2010, 95:3763-3767

[80] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25:402-408

[81] Ward, C. M., Thu-Hien, T. & Pederson, S. M. ngsReports: a bioconductor package for managing FastQC reports and other NGS related log files. Bioinformatics 36, 2587–2588 (2019).

[82] Schubert M, Lindgreen S, Orlando L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res Notes, 2016, 9:88

[83] Dobin A et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2012, 29:15-21

[84] Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 2013, 30:923-930

[85] Zhou Y et al. A statistical normalization method and differential expression analysis for RNA-seq data between different species. BMC Bioinforma., 2019, 20

[86] Law CW, Chen Y, Shi W, Smyth GK. Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol., 2014, 15

[87] McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res., 2012, 40:4288-4297

[88] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 2010, 26:139-140

[89] Smyth, G. K. Limma: Linear Models for Microarray Data. In: Gentleman R., Carey V. J., Huber W., Irizarry R. A., Dudoit S. (eds) Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Statistics for Biology and Health. Springer, New York, NY (2005).

Funding

Diabetes Australia

Australia Postgraduate Award

Department of Health | National Health and Medical Research Council (NHMRC)(APP1109207)

Department of Education and Training | Australian Research Council (ARC)(DP160100454)