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. 2022 Aug 1;17:101609.
doi: 10.1016/j.bonr.2022.101609. eCollection 2022 Dec.

Skeletal manifestations in a streptozotocin-induced C57BL/6 model of Type 1 diabetes

Affiliations
Free PMC article

Skeletal manifestations in a streptozotocin-induced C57BL/6 model of Type 1 diabetes

Jennifer M Hatch et al. Bone Rep. .
Free PMC article

Abstract

Diabetes Mellitus is a metabolic disease which profoundly affects many organ systems in the body, including the skeleton. As is often the case with biology, there are inherent differences between the sexes when considering skeletal development and disease progression and outcome. Therefore, the aim of this study was to develop a protocol to reliably induce diabetes in both sexes of the C57BL/6 mouse utilizing streptozotocin (STZ) and to characterize the resulting bone phenotype. We hypothesized that destruction of the β-cells in the pancreatic islet by STZ would result in a diabetic state with downstream skeletal manifestations. Beginning at 8 weeks of age, mice were injected for 5 consecutive days with STZ (65 mg/kg males, 90 mg/kg females) dissolved in a citrate buffer. The diabetic state of the mice was monitored for 5 weeks to ensure persistent hyperglycemia and mice were euthanized at 15 weeks of age. Diabetes was confirmed through blood glucose monitoring, glucose and insulin tolerance testing, HbA1c measurement, and histological staining of the pancreas. The resulting bone phenotype was characterized using microcomputed tomography to assess bone structure, and whole bone mechanical testing to assess bone functional integrity. Mice from both sexes experienced loss of β-cell mass and increased glycation of hemoglobin, as well as reduced trabecular thickness and trabecular tissues mineral density (TMD), and reduced cortical thickness and cortical bone area fraction. In female mice the change area fraction was driven by a reduction in overall bone size while in male mice, the change was driven by increased marrow area. Males also experienced reduced cortical TMD. Mechanical bending tests of the tibiae showed significant results in females with a reduction in yield force and ultimate force driving lower work to yield and total work and a roughly 40 % reduction of stiffness. When tissue level parameters were estimated using beam theory, there was a significant reduction in yield and ultimate stresses as well as elastic modulus. The previously reported mechanistic similarity in the action of STZ on murine animals, as well as the ease of STZ administration via IP injection make this model is a strong candidate for future exploration of osteoporotic bone disease, Diabetes Mellitus, and the link between estrogen and glucose sensitivity.

Keywords: Bone; GTT; HbA1c; Hyperglycemia; ITT; Mechanics.

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Differences in mass, tibial length and HbA1c following reduction of β-cell area. A. STZ-treated mice gained significantly less mass over the duration of the study in both sexes. Percent change of mass was calculated as the final mass at 15 weeks of age divided by the initial mass at 8 weeks of age, multiplied by 100. B. Tibial length in STZ-treated females was shorter compared with control mice while no difference was noted in males. C. STZ-treated mice had significantly higher levels of glycated hemoglobin in the blood compared to control mice. D. STZ-treated females had significantly lower β-cell population in the pancreas compared to healthy mice. Data presented as mean +/− SD.
Fig. 2
Fig. 2
STZ-treated mice lack endogenous insulin yet retain the ability to utilize exogenous insulin. A. Control mice responded to a bolus glucose injection within 10 min, returning close to pre-injection levels after 1 h. STZ males showed no signs of metabolizing glucose until the 45-min mark, while STZ females did not begin to metabolize glucose within the first hour after injection. STZ mice did not recover to pre-injection BG levels during the time-course of the observation. B. Comparison of the areas under the curves generated from GTT data indicate a significant impairment in the ability of STZ-treated mice to process the injected glucose. C. Insulin tolerance test results normalized to the BG level at time = 0 show that mice in all groups responded similarly to injection of exogenous insulin. D. Comparison of the areas over the curves generated from ITT data show no significant change in the ability of STZ-treated mice to process injected insulin. E. There was no significant difference measured fAGEs in the bones of STZ treated mice versus control. Data presented as mean +/− SD.
Fig. 3
Fig. 3
A. Schematic Representations of Mechanical Data. Bones from STZ-treated female displayed lower yield and ultimate form versus those from control animals leading to reductions in work to yield and total work. B. Control bones withstood higher stresses than bones from STZ-treated mice. Data presented as mean +/− SD, n = 15.

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