Exercise Increases Bone in SEIPIN Deficient Lipodystrophy, Despite Low Marrow Adiposity

Abstract

Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation's most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via μCT was preserved in SEIPIN KO, though some quality parameters were attenuated. Running distance, speed, and time were comparable in KO and WT. Exercise reduced weight (-24% WT-E vs WT p<0.001) but not in KO. Notably, exercise increased trabecular BV/TV in both (+31%, KO-E vs KO, p=0.004; +14%, WT-E vs WT, p=0.006). The presence and distribution of BMAT in SEIPIN KO, though lower than WT, is unexpected and points to a uniqueness of this depot. That trabecular bone increases were achievable in both KO and WT, despite a difference in BMAT quantity/distribution, points to potential metabolic flexibility during exercise-induced skeletal anabolism.

Keywords: BSCL2; SEIPIN; anabolism; bone; bone marrow adipose tissue (BMAT); congenital lipodystrophy; endocrinology and metabolism; exercise.

Figure 1

Lipodystrohic SEIPIN KO run similar to WT despite lower body weight. Male SEIPIN KO and WT littermates allocated to voluntary running exercise (E) or non-exercise groups (KO, WT) at twelve weeks for a six-week experimental period (N=5-8). (A) Individual body weights throughout and (B) at harvest. (C) Voluntary wheel running metrics via cyclometer- average daily running distance (km/day), average running speed (km/h), and average daily running duration (min/day) in WT-E and KO-E. Data as individual values on box and whisker plots (min to max). Horizontal lines represent means. Significance via 2-way ANOVA with Tukey's post hoc (A, B) or unpaired t-test (C) Between-group * or ^#p < 0.05; **or ^##p < 0.01; ***or ^###p < 0.001.

Figure 2

Bone Marrow adipose tissue (BMAT) is extant, but reduced in SEIPIN KO Lipodystrophy. 12-week­ old male Bscl2 ^-/- KO allocated to exercise (E) vs non-exercise for 6 wk. compared to WT (A) Visualization of BMAT via 9.4 T MRI with advanced image analysis; each image is an average from superimposed individual images in the sagittal plane. Volumetric quantification of BMAT form N=5-7 MRI images is shown in (A). In (B–D), BMAT values are shown as % relative to WT mean. (B): Bone Marrow Adipose Tissue /Bone Volume (BMAT/BV). (C): Total Femoral Bone Marrow Adipose Tissue (BMAT). (D): Bone Volume (BV). Group differences in (B–D) were analyzed via one-way ANOVA with Tukey's post-hoc test, **p < 0.01. Due to insufficient number of specimens, WTE group was excluded. (E) plot of histomorphometric analysis: adipocyte area (1μm²) and number (1/mm²), via lmageJ, with X-axis representative of 2-3 experimental animals. For data in (E) there were no significant differences between groups by nested one-way ANOVA. (F): Whole tibia adiponectin (APN) mRNA via qPCR (n=5-8 I group) via 2-way ANOVA, Tukey's post hoc, between-group *p < 0.05. Data as individual values on box and whisker plots (min to max). Horizontal lines represent means.

Figure 3

Seipin KO exhibit reduced BMAT in the proximal femoral diaphysis. Twelve-wk.-old male SEIPIN KO allocated to exercise (E) vs non-exercise for 6 weeks, compared to wild type (WT) (n=5-8). BMAT quantified by 9.4T MRI with advanced image analysis to analyze regional distribution of BMAT relative to bone volume (BV). Data expressed as relative% BMAT to WT mean. (A) BMAT/BV assessed at the hip and the proximal diaphysis (p.dia). (B) BMAT/BV assessed at the distal diaphysis (d.dia), metaphysis (meta), and epiphysis (epi). Analyzed via one-way ANOVA with Tukey's post hoc, *p < 0.05. Data as individual values on box and whisker plots (min to max). Horizontal lines represent means. (C) Visualization planes for 9.4T MRI inboth Figures 2 and 3 . (D) Extended group mean slices obtained at indicated planes.

Figure 4

Higher bone quantity in exercising WT and lipodystrophic SEIPIN KO mice. Twelve-week-old male SEIPIN KO and WT allocated to exercise (E) vs non-exercise for 6 weeks (n=S-8). (A) Trabecular bone microarchitecture (BV/TV, Tb.N, Tb.Th, Tb.Sp) assessed at the proximal tibial metaphysis via 1JCT. (B) Cortical geometry (Ct.Ar, Tt.Ar, Ct.Ar/Tt.Ar) and biomechanical measures (lxx, lyy, pMOI) assessed at the mid-tibial diaphysis. Data plotted as individual values with means represented by horizontal lines. Significance assessed via 2- way ANOVA, Tukey's post hoc. Significance for between-group comparisons: *p < O.OS; **p < 0.01. Data as individual values on box and whisker plots (min to max). Horizontal lines represent means.

Figure 5

TRAP staining shows similar osteoclast numbers in WT and SEIPIN KO. Twelve-week-old male SEIPIN KO and WT were allocated to exercise (E) vs non-exercise controls for 6 weeks. Representative histologic sections of the distal femur were stained with Fast Green and TRAP, images via Olympus IX81.

Figure 6

SEIPIN global KO demonstrate higher liver Plin1 expression, consistent with lipodystrophy. (A) Liver mRNA (N=5) via qPCR shows relative expression of Plin1. (B), Representative photo of KO and WT demonstrating lipodystrophic liver phenotype in KO. (C) Whole Tibia mRNA (N=4-8) via qPCR for metabolism, bone markers. Box and whisker plot, min to max with means. Between group differences as *p < 0.05 vs. WT.