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Published in final edited form as: Bone. 2024 Feb 13;181:117042. doi: 10.1016/j.bone.2024.117042

Abaloparatide is more potent than teriparatide in restoring bone mass and strength in type 1 diabetic male mice

Silvia Marino a,b, Serra Ucer Ozgurel c,d, Kevin McAndrews c,d, Meloney Cregor a,b,c,d, Alma Villaseñor e, Maricuz Mamani-Huanca e, Coral Barbas e, Arancha Gortazar f, Amy Y Sato a,b,c,d, Teresita Bellido a,b,c,d
PMCID: PMC13147481  NIHMSID: NIHMS1968372  PMID: 38360197

Abstract

This study investigated the efficacy of the two FDA-approved bone anabolic ligands of the parathyroid hormone receptor 1 (PTH1R), teriparatide or human parathyroid hormone 1–34 (PTH) and abaloparatide (ABL), to restoring skeletal health using a preclinical murine model of streptozotocin-induced T1-DM. Intermittent daily subcutaneous injections of equal molar doses (12 pmoles/g/day) of PTH (50 ng/g/day), ABL (47.5 ng/g/day), or vehicle, were administered for 28 days to 5-month-old C57Bl/6J male mice with established T1-DM or control (C) mice. ABL was superior to PTH in increasing or restoring bone mass in control or T1-MD mice, respectively, which was associated with superior stimulation of trabecular and periosteal bone formation, upregulation of osteoclastic/osteoblastic gene expression, and increased circulating bone remodeling markers. Only ABL corrected the reduction in ultimate load, which is a measure of bone strength, induced by T1-DM, and it also increased energy to ultimate load. In addition, bones from T1-DM mice treated with PTH or ABL exhibited increased ultimate stress, a material index, compared to T1-DM mice administered with vehicle. And both PTH and ABL prevented the increased expression of the Wnt antagonist Sost/sclerostin displayed by T1-DM mice. Further, PTH and ABL increased to a similar extent the circulating bone resorption marker CTX and the bone formation marker P1NP in T1-DM after 2 weeks of treatment; however, only ABL sustained these increases after 4 weeks of treatment. We conclude that at equal molar doses, ABL is more effective than PTH in increasing bone mass and restoring the cortical and trabecular bone lost with T1-DM, due to higher and longer-lasting increases in bone remodeling.

Key terms: PTH, Abaloparatide, diabetes, bone

1. Introduction

Type 1 diabetes (T1-DM) is a progressive autoimmune disease that destroys pancreatic β-cells causing a lifelong insulin deficiency [1]. In 2022, there were 8.75 million individuals worldwide diagnosed with T1-DM of which 17.0% were younger than 20 years, 64.0% were between 20 and 59 years, and 19.9% were aged 60 years or older [2]. High bone fragility and increased fracture risk are recognized severe public health complications in diabetic patients [3]. Up to 20% of patients who sustain a hip fracture die in the first 12 months, and less than 50% regain the previous level of function [4]. Importantly, diabetic patients exhibit higher mortality after a hip fracture as compared to normoglycemic individuals [5]. The mechanisms leading to bone deterioration in DM are complex. T1-DM patients present a decrease in bone mineral density (BMD) that does not explain the 5 times and 2 times increase in the risk for hip fracture and non-vertebral fracture, respectively [6]. The increase in bone fragility might result from a combination of reduced bone turnover, cortical and trabecular microarchitectural changes, chronic hyperglycemia, and oxidative stress that leads to a build-up of advanced glycation end products (AGEs) in bone collagen, reduction of glycosaminoglycan (GAG) and bone water content, and impaired Wnt signaling [7–11].

A major feature of the bone disease associated with diabetes is a marked decrease in bone formation rate (BFR). Thus, there is a strong rationale for the treatment of skeletal deterioration in DM with agents that stimulate bone formation. However, no clinical studies and/or post-hoc analyses support the use of osteoanabolic treatment in patients with T1-DM. Previous published work by our group demonstrated that the low bone mass and increased bone fragility exhibited by mice with established T1-DM were corrected by administration of PTH related protein (PTHrP)-derived peptides that interact with the same receptor as PTH and PTHrP (PTH1R), or the PTH-unrelated peptide PTHrP (107–111) [12]. The PTHrP-derived peptides exhibited additive effects in conjunction with mechanical stimulation in increasing bone formation and preventing osteocyte apoptosis in diabetic mice. More recently, we demonstrated that intermittent administration of the FDA-approved ligands of the PTH1R teriparatide/parathyroid hormone 1–34 (PTH) and abaloparatide/PTH-related peptide 1–34 (ABL) corrected the diabetic bone signature in an established model of T2-DM mice at the tissue, cell, and transcriptome levels [13].

In the current study, we compared the effectiveness of equimolar doses (12 pmoles/g/day) of PTH and ABL on bone deterioration in skeletally mature C57BL/6J T1-DM male mice. We demonstrated that ABL was superior to PTH in restoring the bone lost with DM, increasing bone formation, improving bone microarchitecture, and restoring bone strength. This differential effect was due to a longer-lasting increase in bone remodeling accompanied by an increase in bone remodeling serum markers and osteoblastic/osteoclastic gene expression induced by ABL compared to PTH. Both ABL and PTH reversed the increase in the expression in bone of the Wnt antagonist SOST/sclerostin induced by T1-DM. Moreover, both agents increased the material property of ultimate stress, but only ABL was able to correct the defective mechanical properties of bone induced by T1-DM. PTH and ABL benefited the skeleton without correcting the diabetes status, as T1-DM mice remained hyperglycemic and glucose intolerant suggesting that bone protection by targeting the PTH1R is independent of the metabolic effects of diabetes.

2. Materials & methods

2.1. Experimental model

Male 11-week-old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were randomized based on spinal BMD. Mice were housed 5 mice/cage, received water ad-libitum and were exposed to a 12h light/dark cycle. T1-DM was induced by 5 daily injecting streptozotocin (STZ, 45mg/kg i.p. in 50 mM citrate buffer, pH 4.5) or buffer alone (controls, C) and confirmed by blood glucose values >250 mg/dL [12] 18 days after initiating the STZ injections (t1). The effects of DM on bone loss were followed for additional 4 weeks. At t2, control and diabetic mice were stratified based on BMD, glucose levels, and weight into treatment groups, and were injected s.c. five days a week with either vehicle (0.9% saline, 0.01 mM β-mercaptoethanol, 0.1 mM acetic acid), or human PTH (1–34) (50 μg/kg/day, Bachem, Torrance, CA) or ABL (47.5 μg/kg/day, Radius Pharmaceutical, Boston, MA). Endpoint measurements were performed 28 days after the first injection of the PTH1R ligands (t3), followed by euthanasia and tissue harvesting (Figure 1A). Previous studies by our and other laboratories showed that PTH exerts anabolic effect in mice at doses that range between 30 to 200 ng/g/day [14–16]. We have recently published that at a dose of 47.5 ng/g/day, ABL is effective in restoring bone loss [13]. Based on this evidence, we chose to administer equal molar doses (12 pmoles/g/day) of PTH or ABL equivalent to 50 ng/g/day PTH, to establish a direct comparison between the two agents.

Figure 1. At equimolar doses, ABL is more effective than PTH in increasing BMD under physiological and T1-DM conditions.

Figure 1.

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(A) Experimental scheme showing the T1-DM animal model, indicating the point in which the experiment started and STZ was injected (t0, 12 weeks, wk), when diabetes was established (t1, 15 weeks, wk), when PTH and ABL administration started (t2, 19 weeks, wk), and end of the experiment (t3, 23 weeks, wk). (B) Glucose levels and (C) total body mass of control, C, and T1-DM mice at t2. (D) Longitudinal analysis of total and femoral BMD. (E) Total and (F) femoral BMD before treatment (at t2, grey bars) and after treatment (at t3) with vehicle (white bars), PTH (green bars) or ABL (blue bars). Data are presented as box & whisker plots where each dot represents a mouse. ^p<0.05 versus control mice with the same treatment by unpaired and two-tailed Student’s t-test (D) or 2-way ANOVA followed by Bonferroni’s post hoc test; *p<0.05 versus respective vehicle-treated mice and Ϯp<0.05 versus PTH treated mice by 2-way ANOVA followed by Tukey’s post hoc; #p<0.05 versus t2, by one-way ANOVA with post hoc Tukey’s correction. C, control mice; n=8–10 mice per group.

2.2. Glucose Measurements and Glucose Tolerance Test (GTT)

Glucose measurements were performed with tail snipping for blood and measured by AlphaTrak 2 Blood Glucose Monitoring System (Zoetis Florham Park, NJ, USA). GTT was performed by the Center for Diabetes and Metabolic Diseases Core Facility, Indiana University. Mice were placed in cages with paper bedding, fasted overnight and administered 2 g/kg glucose according to their lean mass by intraperitoneal injection. Water was given ad libitum. Blood was collected 0, 10, 20, 30, 60, 90, 120 minutes post glucose injection for measurement of serum glucose. The tail tip (less than 1–2 mm) was snipped once with sterile scissors and all blood samples were taken from the same snip by removing the formed scab. Less than 5 μl was collected at each time point. Following blood collection, hemostasis was assured. Some STZ-administered mice from each treatment group injected with glucose had elevated glucose levels that were above the limits of detection (≥750 mg/dL), therefore, for statistical testing purposes, these values were recorded as 751 mg/dL.

2.3. Analysis of skeletal phenotypes

Bone mineral density measurements.

Longitudinal study was performed at t0, t2 and t3 by dual-energy x-ray absorptiometry (DEXA) using a PIXImus densitometer (G.E. Medical Systems,Lunar Division, Madison, WI). BMD measurements included total BMD (excluding the head and tail), L1–L6 vertebra (spinal BMD), and entire femur (femoral BMD) [12]. Mice were stratified into the experimental groups based on BMD, glucose levels, and body weight measured at t2.

Bone microarchitecture analysis.

For microcomputed tomography (μCT) analysis, bones were dissected, cleaned of soft tissue, and stored in saline at −20°C. The femurs and L6 vertebrae were scanned in a μCT scanner (μCT35, Scanco Medical, Switzerland) at an isotropic voxel size of 10 μm. Medium-resolution scans were obtained (E = 55 kVp, I = 145 uA, integration time = 200 ms). For the trabecular analysis, a Gaussian filter (sigma = 1.2, support = 1) was applied and a threshold of 285 mg/cm3 was used. Femoral distal cancellous bone measurements were analyzed beginning 10 slices away from the growth plate to avoid the primary spongiosa for 260 slices.

L6 cancellous bone measurements were performed using a VOI spanning from the upper to the lower growth plate excluding primary spongiosa. Cortical bone was measured at a threshold of 260 mg/cm3. Femoral mid-diaphysis cortical analysis was performed for 20 slices region located at the calculated femoral midpoint. L6 cortical bone analysis was performed starting 10 slices away (towards caudal growth plate) from where the first spinous process attaches to the vertebral bod for 10 slices [17]. All nomenclature, symbols, and units adhered to guidelines [18].

Bone histomorphometric analysis.

To measure the dynamic histomorphometric indexes mineralizing surface to bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate normalized to bone surface (BFR/BS), bone fluorochrome labeling was performed by intraperitoneal injections with calcein green (30 mg/kg bw) and alizarin red (A, 50 mg/kg bw) administered 7 and 2 days, respectively, before the mice were sacrificed [19].

Left femurs were fixed in 10% buffered formalin, cut in half at the midshaft, then embedded undecalcified in methyl methacrylate. Thick cross-sections at the mid-diaphysis were prepared using a diamond-embedded wire saw (Histosaw, Delaware Diamond Knives, Wilmington, DE, USA) and grounded to a final thickness of 30–40 μm for periosteal and endosteal bone formation measurements [20]. Thin (4μm) longitudinal sections of the distal half of the femur were also prepared for cancellous bone measurements. Cancellous measurements were performed at 800 μm distance located 200–400 μm away from the growth plate. Histomorphometric analysis was performed using OsteoMeasure High Resolution Digital Video System (OsteoMetrics, Decatur, GA) interfaced to an Olympus BX51 fluorescence microscope (Olympus America Inc., Center Valley, PA) [20]. Terminology and units are those recommended by the Histomorphometry Nomenclature Committee of the ASBMR [21]. Analyses were performed in a blinded fashion.

2.4. Bone turnover markers

Blood was collected at t0, t1, t2, and t3 from the facial vein of 3-hour fasted mice. Serum was collected by utilizing BD Microtainer Tube with BD Microgard Closure SST Gel (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA). Procollagen type 1 N-terminal propeptide (P1NP), C-telopeptide fragments of type I collagen (CTX) and Osteocalcin (OCN) (Alpha Aesar., Ward Hill, MA, USA) were measured following the manufacturer’s instructions.

2.5. Biomechanical testing

Following microCT scanning, femurs were subjected to 3-point bending biomechanical analysis to assess the mechanical and material properties of femoral mid-diaphysis, using a 500 lbs Actuator (TestResources Inc., Shakopee, MN 55379, USA) as previously described [22]. Briefly, bones were thawed to room temperature, hydrated in 0.9% saline, placed with the posterior femoral surface lying on lower supports (8mm apart) and the left support immediately proximal to the distal condyles and loaded to failure at a rate of 2 mm/min (100P225 Modular Test Machine). Structural properties (energy to ultimate force, ultimate force, and stiffness) of the femur were derived from the load/displacement curves obtained during the three-point bending tests. Cross-sectional polar moment of inertia and anterior-posterior diameter were determined by μCT and were used to calculate apparent material properties.

2.6. Citrate content measurement

Bone citrate content as an index of hydroxyapatite nanocrystal stability, bone strength, and resistance to fracture [23]. Left ulnas were wrapped in saline and stored frozen before the analysis. The bone samples were individually pulverized using a cryoPREP Dry Impactor (Covaris®, Woburn, Massachusetts, USA). The bone was cooled in liquid nitrogen for 1 minute and was impacted consecutively by 2 impacts, one with #2 and the second with #6 (out of 6 levels) from the power-scale of the instrument. Then, the bone powder was weighted in an Eppendorf tube and 1 M HCI solution was added at 50 mg/ml of bone powder [24]. After vortexing for 1 minute, 2 metal balls were added to each sample, and samples were put under 1 cycle of 15 minutes at 50 Hz in the TissueLyser LT homogenizer (Qiagen, Germany). Samples were then incubated in an oven at 60 °C for 1 hour. This procedure provoked the extraction of citric acid from the hydroxyapatite matrix. Samples were tempered at room temperature for 15 minutes before neutralization, and then 10 M KOH was added to 100 μL/ml of HCI. Finally, samples were vortex for 1 minute and centrifuged at 4000 × g for 20 minutes at 20 °C. Supernatants were transferred to new tubes and stored at 4°C until the next step. Three blank samples were prepared in similar conditions as the experimental samples. Sample analysis was carried out in an Agilent 7100 CE system coupled with an electrospray source to an Agilent 6224 TOF Mass Spectrometer (Wilmington, USA), using an Agilent polyvinyl alcohol (PVA) coated capillary (total length, 125 cm; i.d., 50 μm). The separation buffer included 0.1 M formic acid prepared in 1% methanol at 75 mbar and 20 kV voltage. Data were acquired in negative ESI and full scan mode from 75 to 1000 m/z. The citric acid concentration in bone samples was quantified using a standard addition calibration curve for each sample. Four independent vials, 40 μL of the extracted bone sample were mixed with 10 μL of 0.6 mM MES (2-(N-Morpholino)-ethanesulfonic acid (Sigma Aldrich, Germany), used as internal standard, and 10 μL of water or 0.5, 1 and 2 mM citric acid standard (Sigma Aldrich, Darmstadt, Germany), respectively. This resulted in 4 levels of a standard addition calibration curve for each sample. Data files of samples were re-processed using MassHunter Quantitative Analysis B.09 for TOF software (Agilent, Santa Clara, CA, USA). Citric acid peak area was normalized by the MES area, and concentration in the homogenate was calculated by extrapolation of the curve. Finally, μmol/g of bone was calculated taking into account the dilution, solvent addition, and weight of the sample.

2.7. RNA extraction and quantitative PCR (qPCR)

Total RNA was extracted from mouse tibial diaphysis devoid of marrow or from the 4th lumbar vertebra using TRIzol (Invitrogen, Grand Island, NY, USA). These bones were chosen as representative of cortical bone and trabecular bone compartments, respectively. cDNA was synthesized using the high-capacity cDNA reverse transcription (Applied Biosystems Inc., Foster City, CA, USA). Gene expression was quantified by qPCR as described earlier [22] using sets of primers and probes designed using the Assay Design Center (Roche Applied Science, Indianapolis, IN, USA) or commercially available, carried out in an ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA). Relative mRNA expression was normalized to the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the ΔCt method. Ratios between genes of interest and housekeeping gene are expressed as fold change compared with mice receiving placebo.

2.8. Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.3.0 (GraphPad Software, Inc., CA, US). In case of equal variances, unifactorial (or one-variable) analysis was analyzed by unpaired and two-tailed Student’s t test (two independent groups), or one-way ANOVA test (multiple independent groups) followed by post hoc Tukey or Dunnett test (when p < 0.05). When variable “diabetes” was considered, a bifactorial analysis was performed by two-way ANOVA followed by Bonferroni’s post hoc test. Statistical details of each experiment (test used and value of n) can be found in Figure and Figure Legend sections. Data are presented as Box & whiskers plot where each dot represents a mouse. Statistically identified outliers for particular endpoints (value ± 2 times SD from the mean) were excluded.

2.9. Study approval

All animal protocols were approved by the Institutional Animal Care and Use Committee at Indiana University School of Medicine and animal care was carried out following institutional guidelines.

3. Results

3.1. ABL is more effective than PTH in increasing BMD under physiological and T1-DM conditions at equimolar doses.

T1-DM was induced in skeletally mature male C57BL/6J mice by injecting STZ (Figure 1A), and it was confirmed by blood glucose values > 250 mg/dL after 18 days, which remained elevated compared to non-diabetic control mice (C) throughout the experiment (Figure 1B). Body weight, lean, and fat mass were lower in T1-DM mice (Figure 1C and Supplementary Figure 1A), confirming the development of T1-DM. Longitudinal analysis showed that T1-DM mice exhibited reduced total and femoral BMD before treatment (at t2), but no changes in spinal BMD (Figure 1D). In control mice, treatment with PTH or ABL for 2 weeks did not induce detectable changes in total or spinal BMD, but ABL significantly increased femoral BMD (Supplementary Figure 1B-D). T1-DM mice treated with vehicle presented a reduction in both total and femoral BMD compared to control mice which was corrected by both PTH and ABL to a similar extent. No changes were detected in the spine. After 4 weeks of treatment (at t3), ABL, but not PTH, increased total (9.5%, p<0.05), femoral (11.6%, p<0.05), and spinal (10.6%, p<0.05) BMD in control mice; and reversed and further increased total (5%, p<0.05) and femoral (4%, p<0.05) BMD in T1-DM mice compared to the respective vehicle administered mice (Figure 1E and 1F and Supplementary Figure 1E, indicated by # and *, respectively). In contrast, PTH only reversed the reduction in total and femoral BMD induced by T1-DM (Figure 1E and 1F, compared to before the treatment started at t2 and indicated by #). Blood glucose remained elevated in T1-DM mice treated with PTH or ABL (Figure 2A); and T1-DM mice remained glucose intolerant compared to control mice, as evidenced by the impaired glucose clearance curves and the higher area under the curves in the glucose tolerance test (Figure 2B and 2C). Furthermore, neither treatment restored the low body weight of T1-DM mice (Supplementary Figure 2A-C).

Figure 2. T1-DM mice remained hyperglycemic and glucose intolerant after PTH and/or ABL treatment.

Figure 2.

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(A) Blood glucose measured after 4 weeks of treatment of PTH or ABL. (B) Glucose tolerance test (GTT) was performed 3 days before the harvest and the corresponding area under the curve (AUC) measurements. Data are presented as box & whisker plots where each dot represents a mouse. ^p<0.05 versus control mice by 2-way ANOVA followed by Bonferroni’s post hoc test. n=6–7 per group.

3.2. ABL is more effective than PTH in increasing cancellous and cortical bone and in restoring bone architecture and ultimate force in T1-DM.

T1-DM mice exhibited a reduction in BV/TV and trabecular thickness without changes in trabecular number quantified by μCT in the distal femur (Figure 3A). No effect of diabetes was seen in vertebral bone (Supplementary Table 1). Only ABL increased BV/TV, trabecular thickness and number, and connectivity density, and decreased trabecular separation, compared to PTH or vehicle-treated mice in both control and T1-DM conditions at both bone sites. The increased trabecular number and thickness by ABL might be due to increased resorption and formation, respectively, which together could explain the increase in connectivity. In addition, ABL-treated mice exhibited a reduction in structural model index (SMI, −39% in control mice and −14% in T1-DM mice), consistent with a change towards a plate-like microstructure predicting stronger bone [25], as well as in material density (MD) in trabecular bone of the distal femur (Figure 3). Similarly, in cancellous bone of the spine (L6), ABL treatment improved all trabecular indexes in both control and T1-DM mice (Supplementary Table 1).

Figure 3. At equimolar doses, ABL is superior to PTH in restoring and improving bone architecture and material properties, but both PTH and ABL increase the material properties of toughness in physiological conditions and ultimate stress in T1-DM conditions.

Figure 3.

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Analysis of bone microarchitecture by μCT in (A) cancellous (distal femur) and (B) cortical (femoral mid-diaphysis) bone. (C and D) Biomechanical parameters measured at the femoral mid-diaphysis by 3-point bending in C and T1-DM mice. C, control mice; BV/TV, bone volume/tissue volume; Tb.Th, trabecular thickness; Tb.Tn, trabecular number; BA/TA, bone area/total area; Ct.Th, thickness of cortical bone; BA, bone area. Data are presented as box & whisker plots where each dot represents a mouse. ^p<0.05 versus control mice with the same treatment by 2-way ANOVA followed by Bonferroni’s post hoc test; *p<0.05 versus respective vehicle-treated mice and by 2-way ANOVA followed by Tukey’s post hoc. n=8–10 mice per group.

Additionally, ABL increased BA/TA and cortical thickness compared to vehicle or PTH-treated mice and the polar moment of inertia (pMOI) compared to PTH-treated mice in both physiological and T1-DM conditions. PTH at this dose exhibited a minor, if any, effect in cancellous or cortical bone. At the trabecular level, the response to ABL was lower in T1-DM mice compared to control mice.

We next performed a 3-point bending assay to quantify the changes in structural and material properties. Ultimate force, a direct measure of bone morphology and bone material, was reduced in T1-DM mice compared to control (Figure 3C and Supplementary Table 2). ABL, but not PTH, increased ultimate force and energy to ultimate load in control and T1-DM mice. No differences in bone apparent material properties were detected in T1-DM bones (Figure 3D and Supplementary Table 2). However, bones from T1-DM mice treated with PTH or ABL exhibited increased ultimate stress in T1-DM mice, and ABL but not PTH also increased material toughness in control mice.

3.3. ABL is superior to PTH in restoring and increasing bone remodeling rate in both physiological and T1-DM diabetes conditions, and both agents reversed the increase in Sost expression induced by T1-DM.

PTH or ABL increased to a similar extent the circulating bone resorption marker CTX (48% and 40%, respectively; P < 0.05), the bone formation marker P1NP (81% and 105%, respectively; P < 0.05), and the remodeling marker OCN (179% and 210%, respectively; P < 0.05) in T1-DM mice after 2 weeks of treatment, whereas only the effect of ABL persisted after 4 weeks (Figure 4A and 4B, respectively). In addition, only ABL was effective under physiological conditions in increasing all the markers.

Figure 4: ABL promoted osteoblastic and osteoclastic gene expression, and increased bone formation and bone resorption markers for a longer duration and superior to PTH treatment, but both agents prevented the Sost increase induced by T1-DM.

Figure 4:

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Serum C-telopeptide fragment of type 1 collagen (CTX) and procollagen type 1 N-terminal pro-peptide (P1NP) and plasma osteocalcin (OCN) measured after 2 weeks of treatment(A) and at the end of the study (B); n = 7–10 per group. (C) Osteoblastic genes alkaline phosphatase (Alp) and Osterix measured in 4th lumbar vertebra. (D) Expression of Sost measured in the tibial diaphysis devoid of marrow. (E) Osteoclastic genes Rankl, Opg, Rankl/Opg, Cathepsin K measured in 4th lumbar vertebra. n=3–10 per group. Data are presented as box & whisker plots where each dot represents a mouse. ^p<0.05 versus control mice with the same treatment by 2-way ANOVA followed by Bonferroni’s post hoc test; *p<0.05 versus respective vehicle-treated mice and by 2-way ANOVA followed by Tukey’s post hoc.

Consistent with our earlier study [12], Sost mRNA expression was increased in bones from T1-DM mice, whereas diabetic mice treated with PTH or ABL did not exhibit Sost upregulation (Figure 4C). Bones from control or T1-DM mice treated with ABL, but not with PTH, exhibited increased mRNA expression of the osteoblastic markers Alkaline phosphatase and Osterix1 (Figure 4D) and osteoclastic markers RANKL and Cathepsin K (Figure 4E). OPG mRNA expression was increased by ABL treatment in control mice and decreased by both PTH and ABL in T1-DM mice, which resulted in an increase in the ratio RANKL/OPG by ABL in both control and T1-DM conditions.

Taking together, these findings indicate that at equimolar doses PTH and ABL increase bone resorption and formation, and that the effect of ABL persists longer.

3.4. ABL is superior to PTH in restoring and increasing bone formation rate in both physiological and T1-DM diabetes conditions.

Bones from T1-DM mice exhibited reduced cancellous mineralizing surface normalized to bone surface (MS/BS, −20%; P < 0.05), mineral apposition rate (MAR, −22%; P < 0.05), and bone formation rate (BFR, −38%; P < 0.05) and periosteal MAR and BFR (−37% and −49%, respectively; P < 0.05) (Figure 5A and D). PTH increased MAR and BFR (62% and 92%, respectively; P < 0.05) only in the cancellous bone of T1-DM mice, whereas ABL increased all the bone formation indexes in both trabecular (MS/BS 22%, MAR 39% and BFR 70% vs control; MS/BS 46%, MAR 99% and BFR 194% vs T1-DM; P < 0.05) and periosteal (MS/BS 68%, MAR 30% and BFR 110% vs control; MS/BS 93%, MAR 130% and BFR 372% vs T1-DM; P < 0.05) surfaces under physiologic and T1-DM conditions. In addition, the effect of ABL in diabetic mice was superior to that of PTH. Neither T1-DM nor the agents affected bone formation on the endocortical surface, except that ABL increased MAR and BFR (51% and 78%, respectively; P < 0.05) under physiologic conditions. Taking together these findings indicate that at equimolar doses while PTH partially restored cancellous BFR, ABL restored both cancellous and periosteal BFR in T1-DM.

Figure 5: PTH restored cancellous BFR in T1-DM mice, while ABL restored and increased both cancellous and cortical BFR in both physiological and T1-DM conditions.

Figure 5:

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Dynamic bone histomorphometric analysis of (A) cancellous bone of the distal femur, (B) periosteal surface, and (C) endosteal surface of the cross-section of the femoral midshaft and representative images. Scale bar =50 μm. Data are presented as box & whisker plots where each dot represents a mouse. ^p<0.05 versus control mice with the same treatment by 2-way ANOVA followed by Bonferroni’s post hoc test; *p<0.05 versus respective vehicle-treated mice and by 2-way ANOVA followed by Tukey’s post hoc. C, control mice; MS/BS, mineralized surface/bone surface; MAR, mineral apposition rate; BFR, bone formation rate. n=6–9 per group.

4. Discussion

In the current study, we used a murine model of T1-DM that mimics the main features of the diabetic bone disease of patients to investigate whether the anabolic ligands of the PTH1R, PTH and ABL, reverse the reduced formation and low bone remodeling state of the disease. We found that ABL was more effective than PTH in increasing bone formation and remodeling and it had longer-lasting effect. Our findings are consistent with previous reports using chemically-induced and transgenic preclinical T1-DM models showing the efficacy of targeting the PTH1R to restore bone mass and strength in T1-DM mice via improving osteoblast survival and differentiation, and osteocyte survival, respectively as well as attenuating resorption-induced by T1-DM mice [12, 26–30]. Further, our study is the first to compare and contrast the effectiveness of PTH and ABL in the context of T1-DM. Similarly to the current findings for T1-DM, we recently reported that ABL was more potent than PTH in repairing bone deterioration in a model of established T2-DM [13]. The mechanisms underlying the superior anabolic effect of ABL over PTH remain unknown. The more favorable effects of ABL on the skeleton have been previously attributed to the limited bone resorptive response compared to PTH [35–41]. However, in the current study as well as our previously published T2-DM study [13], we observed that ABL increased bone resorption at similar levels to PTH or even more potently.

We chose the STZ administration model to male mice, as it is well documented in the field that male mice are more susceptible to diabetes development than female mice (although the causes remain uncertain) [31], and that STZ administered to mice fed a standard rodent diet induces Type 1 diabetes whereas STZ administered to mice fed a high fat diet induces Type 2 diabetes [12, 13, 32, 33]. In addition, despite the limitations of every pre-clinical model, these manipulations mimic closely the glucose and insulin changes observed in patients of T1 or T2 diabetes [1, 34].

We have previously demonstrated that PTH treatment, at the optimal dose of 100 ng/g/day for 28 days, increases cancellous and cortical bone mass, mineral apposition rate (MAR) and bone formation rate (BFR) in both euglycemic and T2-DM mice [13, 15]. In this study, although PTH did not exert its full anabolic effect, we found that the suboptimal dose of 50 ng/g/day PTH increased cancellous but not periosteal MAR and BFR, suggesting that the action of PTH depends on the bone surface and the dose. Conversely ABL, at the same dose increased cancellous and exerted a more profound and favorable effect on modeling bone formation on the periosteal surface of the cortical bone, resulting in increased cortical bone area, polar moment of inertia, and structural/apparent material properties. Recently in a model of estrogen-deficient osteoporosis, using similar doses of ABL and PTH, Lv et al. [35] demonstrated that ABL and PTH affect the cortical osteocyte transcriptome differently and that several pathways in osteocytes are uniquely regulated by ABL. This may explain, at least in part, the more beneficial changes and possibly the stronger cortical response following ABL treatment. Furthermore, the more pronounced effects of ABL on the cortical bone compartment could also explain the greater reduction in nonvertebral fractures achieved by ABL compared to PTH in osteoporotic patients [36]. The increase in bone fragility in T1-DM mice, as measured by the reduction in the structural property index ultimate load, was accompanied by a reduction in the bone content of citrate, an innate component of hydroxyapatite crystals that affects nanocrystal stability, bone strength, and resistance to fracture [23]. Importantly, previous studies demonstrated that osteocytes cultured in high glucose conditions did not increase citrate production in response to mechanical stimulation [37]. Saito et al. [38, 39] reported that bone volume is the major determinant of ultimate load while toughness and stiffness were mostly related to enzymatic and AGEs cross-link collagen content. Consistent with the improvements in cortical bone parameters, ABL treatment restored and further increased ultimate load and energy to ultimate load in the femurs of both T1-DM and control mice. Although PTH1R activation did not correct the reduction in citrate content, both PTH and ABL increase ultimate stress and toughness. Our study suggests that both PTH and predominantly ABL not only promote bone accrual but also improve bone material properties that are associated with increased bone strength. More studies are required to understand the mechanism(s) underlying these effects of PTH1R activation in the frame of diabetes.

Increased level of circulating sclerostin has been observed in both T1 and T2-DM patients and may represent one of the major contributors to low bone turnover and increased fracture risk [10, 40, 41]. Moreover, we and others have shown that the detrimental effects of hyperglycemia on osteocytes were counteracted by the administration of sclerostin-neutralizing antibodies, which corrected the damaging effects on bone and improved fracture outcomes in preclinical models of T1- and T2 DM [13, 42] and in clinical studies with osteoporotic patients [43, 44]. In agreement with our previous study [12], we observed an increase in Sost expression in the bones of T1-DM mice that was corrected by both PTH and ABL treatment.

In conclusion, the present study demonstrates the value of increasing bone formation to restore the damaging effects of T1-DM on bone; and it shows that an equal molar dose of ABL is more effective than PTH in increasing bone mass in both physiological and T1-DM conditions, due to higher and longer-lasting increase in bone remodeling. The superior osteoanabolic and protective effects of ABL in diabetes are likely due to the enhanced bone formation at the trabecular as well as cortical compartment.

Supplementary Material

1
2

Supplementary Table 1. Femoral and vertebral bone structural parameters determined by microCT of T1-DM mice treated with PTH or ABL. Cancellous and cortical bone phenotype of C and T1-DM male mice treated with PTH or ABL. μCT analysis was performed in the femur and L6 vertebra. BV/TV Bone Volume/Total Volume; Tb. Trabecular; Th Thickness; N Number; Sp Separation; Conn D. Connective Density; SMI Structural Model Index; MD Material Density; BA/TA cortical bone area; Ct Cortical; B. Bone; M. Medullary; T Total; pMOI Polar moment of inertia.; *p<0.05 vs C with the same treatment; Data are presented as mean ± SD. *p < 0.05 vs. respective vehicle administered mice and Ϯ p<0.05 versus PTH treated animals by 2-way ANOVA followed by Tukey’s multiple comparison test. ^p<0.05 vs. control mice with same treatment by 2-way ANOVA followed by Bonferroni’s multiple comparison test. n=6–10 mice per group.

Supplementary Table 2. Mechanical properties of the femoral mid-diaphysis from T1-DM diabetic mice treated with PTH or ABL. Structural properties, apparent material properties and Citrate Content measured in the femoral mid-diaphysis by 3-point bending testing. n = Data are presented as mean ± SD. *p < 0.05 vs. respective vehicle administered mice and Ϯ p<0.05 versus PTH treated animals by 2-way ANOVA followed by Tukey’s multiple comparison test. ^p<0.05 vs. control mice with same treatment by 2-way ANOVA followed by Bonferroni’s multiple comparison test. n=6–10 mice per group.

Highlights:

  1. Bone fragility associated with diabetes causes substantial morbidity and mortality.

  2. Data regarding the efficacy of anabolic therapies in T1-DM patients are scarce.

  3. T1-DM bone disease is characterized by a marked decrease in bone formation.

  4. Standard of care with antiresorptives stops bone loss but does not increase bone formation.

  5. PTH1R activation rebuilds bone, improves structure, and restores strength in T1-DM.

ACKNOWLEDGEMENTS

This research was supported by the National Institutes of Health (R01-AR059357 to TB; T32-AR065971 to SUO), American Society of Hematology (ASH scholar award to SM), and the Veterans Administration (I01 BX002104 and IK6BX004596 to TB). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. Abaloparatide (ABL) was provided by Radius Health, Inc.

Footnotes

Declarations of interest: none

Authors have no conflict of interest.

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Associated Data

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Supplementary Materials

1
NIHMS1968372-supplement-1.pdf (284.7KB, pdf)
2

Supplementary Table 1. Femoral and vertebral bone structural parameters determined by microCT of T1-DM mice treated with PTH or ABL. Cancellous and cortical bone phenotype of C and T1-DM male mice treated with PTH or ABL. μCT analysis was performed in the femur and L6 vertebra. BV/TV Bone Volume/Total Volume; Tb. Trabecular; Th Thickness; N Number; Sp Separation; Conn D. Connective Density; SMI Structural Model Index; MD Material Density; BA/TA cortical bone area; Ct Cortical; B. Bone; M. Medullary; T Total; pMOI Polar moment of inertia.; *p<0.05 vs C with the same treatment; Data are presented as mean ± SD. *p < 0.05 vs. respective vehicle administered mice and Ϯ p<0.05 versus PTH treated animals by 2-way ANOVA followed by Tukey’s multiple comparison test. ^p<0.05 vs. control mice with same treatment by 2-way ANOVA followed by Bonferroni’s multiple comparison test. n=6–10 mice per group.

Supplementary Table 2. Mechanical properties of the femoral mid-diaphysis from T1-DM diabetic mice treated with PTH or ABL. Structural properties, apparent material properties and Citrate Content measured in the femoral mid-diaphysis by 3-point bending testing. n = Data are presented as mean ± SD. *p < 0.05 vs. respective vehicle administered mice and Ϯ p<0.05 versus PTH treated animals by 2-way ANOVA followed by Tukey’s multiple comparison test. ^p<0.05 vs. control mice with same treatment by 2-way ANOVA followed by Bonferroni’s multiple comparison test. n=6–10 mice per group.

NIHMS1968372-supplement-2.pdf (147.6KB, pdf)

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