nutrients Article Distinct Effects of a High Fat Diet on Bone in Skeletally Mature and Developing Male C57BL/6J Mice Dean S. Ross 1 , Tzu-Hsuan Yeh 1 , Shalinie King 1,2 , Julia Mathers 1 , Mark S. Rybchyn 1 , Elysia Neist 1 , Melissa Cameron 1 , Alexander Tacey 3,4 , Christian M. Girgis 5,6,7 , Itamar Levinger 3,4 , Rebecca S. Mason 1 and Tara C. Brennan-Speranza 1,8, *   Citation: Ross, D.S.; Yeh, T.-H.; King, S.; Mathers, J.; Rybchyn, M.S.; Neist, E.; Cameron, M.; Tacey, A.; Girgis, C.M.; Levinger, I.; et al. Distinct Effects of a High Fat Diet on Bone in Skeletally Mature and Developing Male C57BL/6J Mice. Nutrients 2021, 13, 1666. https://doi.org/10.3390/ nu13051666 Academic Editor: Jasminka Ilich-Ernst Received: 16 April 2021 Accepted: 8 May 2021 Published: 14 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Faculty of Medicine and Health, School of Medical Sciences, University of Sydney, Sydney 2006, Australia; dros8694@uni.sydney.edu.au (D.S.R.); cindy.th.yeh@gmail.com (T.-H.Y.); shalinie.king@sydney.edu.au (S.K.); juliamathers94@gmail.com (J.M.); m.rybchyn@unsw.edu.au (M.S.R.); ellyn@hotmail.com (E.N.); melissa.cameron@sydney.edu.au (M.C.); rebecca.mason@sydney.edu.au (R.S.M.) 2 Faculty of Medicine and Health, School of Dentistry, University of Sydney, Sydney 2006, Australia 3 Institute for Health and Sport (IHES), Victoria University, Melbourne 3011, Australia; alexander.tacey@live.vu.edu.au (A.T.); itamar.levinger@vu.edu.au (I.L.) 4 Australian Institute for Musculoskeletal Science (AIMSS), University of Melbourne and Western Health, St Albans 3021, Australia 5 Department of Diabetes and Endocrinology, Westmead Hospital, Sydney 2145, Australia; christian.girgis@sydney.edu.au 6 Department of Endocrinology, Royal North Shore Hospital, Sydney 2065, Australia 7 Faculty of Medicine and Health, University of Sydney, Sydney 2006, Australia 8 Faculty of Medicine and Health, School of Public Health, University of Sydney, Sydney 2006, Australia * Correspondence: tara.speranza@sydney.edu.au; Tel.: +61-2-9351-4099 Abstract: Increased risks of skeletal fractures are common in patients with impaired glucose handling and type 2 diabetes mellitus (T2DM). The pathogenesis of skeletal fragility in these patients remains ill-defined as patients present with normal to high bone mineral density. With increasing cases of glucose intolerance and T2DM it is imperative that we develop an accurate rodent model for further investigation. We hypothesized that a high fat diet (60%) administered to developing male C57BL/6J mice that had not reached skeletal maturity would over represent bone microarchitectural implications, and that skeletally mature mice would better represent adult-onset glucose intolerance and the pre-diabetes phenotype. Two groups of developing (8 week) and mature (12 week) male C57BL/6J mice were placed onto either a normal chow (NC) or high fat diet (HFD) for 10 weeks. Oral glucose tolerance tests were performed throughout the study period. Long bones were excised and analysed for ex vivo biomechanical testing, micro-computed tomography, 2D histomorphometry and gene/protein expression analyses. The HFD increased fasting blood glucose and significantly reduced glucose tolerance in both age groups by week 7 of the diets. The HFD reduced biomechanical strength, both cortical and trabecular indices in the developing mice, but only affected cortical outcomes in the mature mice. Similar results were reflected in the 2D histomorphometry. Tibial gene expression revealed decreased bone formation in the HFD mice of both age groups, i.e., decreased osteocalcin expression and increased sclerostin RNA expression. In the mature mice only, while the HFD led to a non-significant reduction in runt-related transcription factor 2 (Runx2) RNA expression, this decrease became significant at the protein level in the femora. Our mature HFD mouse model more accurately represents late-onset impaired glucose tolerance/pre-T2DM cases in humans and can be used to uncover potential insights into reduced bone formation as a mechanism of skeletal fragility in these patients. Keywords: high fat diet; preclinical; bone histomorphometry; osteoblasts; osteocytes; metabolism Nutrients 2021, 13, 1666. https://doi.org/10.3390/nu13051666 https://www.mdpi.com/journal/nutrients