Influences of obese (ob/ob) and diabetes (db/db) genotype mutations on lumber vertebral radiological and morphometric indices: Skeletal deformation associated with dysregulated systemic glucometabolism

Background Both diabetes and obesity syndromes are recognized to promote lumbar vertebral instability, premature osteodegeneration, exacerbate progressive osteoporosis and increase the propensity towards vertebral degeneration, instability and deformation in humans. Methods The influences of single-gene missense mutations, expressing either diabetes (db/db) or obese (ob/ob) metabolic syndromes on vertebral maturation and development in C57BL/KsJ mice were evaluated by radiological and macro-morphometric analysis of the resulting variances in osteodevelopment indices relative to control parameters between 8 and 16 weeks of age (syndrome onset @ 4 weeks), and the influences of low-dose 17-B-estradiol therapy on vertebral growth expression evaluated. Results Associated with the indicative genotypic obesity and hyper-glycemic/-insulinemic states, both db/db and ob/ob mutants demonstrated a significant (P ≤ 0.05) elongation of total lumbar vertebrae column (VC) regional length, and individual lumbar vertebrae (LV1-5) lengths, relative to control VC and LV parameters. In contrast, LV1-5 width indices were suppressed in db/db and ob/ob mutants relative to control LV growth rates. Between 8 and 16 weeks of age, the suppressed LV1-5 width indices were sustained in both genotype mutant groups relative to control osteomaturation rates. The severity of LV1-5 width osteosuppression correlated with the severe systemic hyperglycemic and hypertriglyceridemic conditions sustained in ob/ob and db/db mutants. Low-dose 17-B-estradiol therapy (E2-HRx: 1.0 ug/ 0.1 ml oil s.c/3.5 days), initiated at 4 weeks of age (i.e., initial onset phase of db/db and ob/ob expressions) re-established control LV 1–5 width indices without influencing VC or LV lengths in db/db groups. Conclusion These data demonstrate that the abnormal systemic endometabolic states associated with the expression of db/db and ob/ob genomutation syndromes suppress LV 1–5 width osteomaturation rates, but enhanced development related VC and LV length expression, relative to control indices in a progressive manner similar to recognized human metabolic syndrome conditions. Therapeutic E2 modulation of the hyperglycemic component of diabetes-obesity syndrome protected the regional LV from the mutation-induced osteopenic width-growth suppression. These data suggest that these genotype mutation models may prove valuable for the evaluation of therapeutic methodologies suitable for the treatment of human diabetes- or obesity-influenced, LV degeneration-linked human conditions, which demonstrate amelioration from conventional replacement therapies following diagnosis of systemic syndrome-induced LV osteomaturation-associated deformations.

Recent reports suggest that the severity of lumbar vertebral (LV) osteodegeneration and osteoporosis is enhanced in both diabetics and obese patients exhibiting blatant systemic syndrome aberrations accompanied by chronic lower back discomfort or deformation [3][4][5]8,11]. In general, conventional pharmacotherapeutic replacement regimes fail to completely ameliorate progressive LV deterioration in afflicted patients [8,10,[31][32][33][34][35][36][37][38]. The lack of an identified experimental model that demonstrates progressive osteodegeneration following the expression of diabetes or obese states has impeded the evaluation of interventional therapeutic approaches focused on the alleviation of progressive LV osteodegeneration or destabilization [32][33][34][35][36][37][38][39][40]. The current studies were designed to evaluate the influences of diabetes (db/db) and obese (ob/ ob) genotype-mutations towards the induction of VC and LV osteo-retardation and degeneration, recognized as chronic human syndrome complications [8,37], and the progressive influences of the syndrome conditions on LV morphometric and radiological indices associated with the duration of mutation expression in the C57BL/-KsJ murine model.

Animals
Adult, female C57BL/ KsJ mice (Jackson Laboratory, Bar Harbor, ME derived), between 4 and 16 weeks of age were used in these studies. Littermate controls (designated as mixed +/+ and +/? normal phenotypes/genotypes), as well as littermate diabetes (db/db) and obese (ob/ob)-mutant (homozygous recessive) genotypes (Table 1), were pair matched for phenotype, tissue sampling and systemic metabolic indices analyses (i.e., blood glucose, serum insulin and triglyceride concentrations) comparisons during the course of these studies ( Table 2). All mice were housed five per cage, grouped according to genotype, under controlled environmental conditions (23 C), with an established photoperiod of 12 hr light/day (lights on: 0600 hr) [29]. Blood glucose levels (Ames Glucometer method), serum insulin and triglycerides levels [26,29] and body weights were monitored for each of the designated 8 and 16-week-old experimental age groups as previously described [26,29,41]. Animals exhibiting either lean (≤ 15 grams) or obese (≥ 25 grams) phenotypes (controls: ± 20 grams) and pronounced systemic hyperglycemia (≥ 200 mg/dl) relative to controls (≤150 mg/dl) by 8 weeks of age (Table 1) were considered as overt, Type 2 NIDDM obese (ob/ob) or diabetes (db/db) groups [16,36], Comparison between genotypic indices induced by the indicate mutations, relative to normal littermate controls, and the attributed protein expression deficits associated with metabolic syndrome expression complications when expressed on the C57BL/KsJ murine background strain. An age-related exacerbation of the severity of mutation-induced metabolic/structural complications occurs for each genotype mutation.
with the continued expression of these indices denoted through the chronic 16-week old ( Table 2) age group experimental periods.

Radiographic and morphometric analysis of lumbar vertebral column (VC) and individual vertebrae (LV) maturation variances
At 8 and 16 weeks of age, each designated genotype group was subjected to light methoxyflurane (Parke-Davis, Detroit, MI) inhalation (45 sec exposure) anesthesia prior to radiographic imaging analysis of lumbar vertebral column (VC) regional length measurements, as well as individual lumbar vertebrae (LV) length and width measurements. A complete lumbar vertebral radiographic (1/30-1/24 sec exposure at a 40 KV/200 amp setting) image was captured on radiographic (Kodak, Rochester, NY) plate film, developed and the respective measurements of VC and individual LV 1-5 length and width indices determined using an Olympus flat-bed light-optics graphics recording unit connected to a data processing computer for statistical tabulation and analysis of morphometric measurement parameters [42]. All bone measurements were determined by enhanced digital image analysis of individual radiographs utilizing identified lumbar vertebral column location landmarks at both 8 and 16 weeks of age for all control and genotype-mutation designated groups. Morphometric data were collected, tabulated, analyzed and compared for intergroup differences relative to specified group mutation type ( Table 1), age (duration) of mutation expression (Table 2) as well as LVC and LV variances.

Estradiol treatments (E2-HRx)
17-B-estradiol (E2: 1 ug/ 3.5 days) was dissolved in sesame oil (0.1 ml) for subcutaneous injections (HRx: indicated as Day 0 of 3.5 day intervals) initiated at 3 weeks (21 days: weaning) of age. The oil (sesame: Sigma) vehicle (0.1 ml) served as the sham-control injection procedure as previously described [18,24]. These temporal dose regimes were selected based on previous studies indicating the restoration of diestral (i.e., control baseline) systemic ovarian steroid concentrations in hypogonadal db/ db mutants following initiation of the E2-HRx therapies prior to the overt onset of the diabetes-associated syndrome [17,18].

Statistical analysis
Values for body weights, bone morphometrics and systemic endometabolic indices were expressed as group means (± SEM) for the designated genotype groups. Intergroup and intra-group differences were determined using the Student's T-test exam, with a p ≤ 0.05 accepted as representing statistical differences for the specified parameter.

Genotype (mutation)-related influences on body mass and systemic endometabolic indices
Between 8 and 16 weeks of age, control groups demonstrated stable body weights in association with systemic euglycemia, normoinsulinemia and basal circulating triglyceride (triacylglycerol) levels ( Table 2). In contrast, both db/db and ob/ob mutation expressions induced significant increases in body masses and systemic endometabolic indices between 8 and 16 weeks of age relative to parameters (Table 2).

Radiographic and morphometric analysis of LVC and LV 1-5 maturation variances associated with genotype mutation expression
Radiographic ( Figure 1) and morphometric ( Figure 2) analysis of total LVC length and average LV 1-5 length and width indices demonstrated variances in VC maturation and growth indices associated with db/db and ob/ob mutation expressions in C57BL/KsJ groups. Compared to control growth indices (Figure 2), all genotype mutation groups demonstrated significant increases in average lumbar VC length measurements relative to control indices at 1386  (Table 2). By 16 weeks of age, the progressive and cumulative influences of the db/db and ob/ob mutation syndromes promoted exaggerated VC and LV lengths, but diminished individual LV 1-5 width expression, relative to control groups ( Figure 2).

Influences of 17-B-estradiol (E2) therapy (HRx) on VC and LV 1-5 growth indices in diabetes (db/db) genotype mutant groups
The therapeutic (0.1 ug/0.1 ml oil vehicle: sc injection @ 3.5 day intervals) re-establishment of normoglycemia (Table 3) by the administered E2-HRx regime following expression of the db/db syndrome did not influence total VC length indices, or individual LV 1-5 length parameters, in the mutation group or in controls ( Figure 3) at 8 weeks of age (i.e., 4 weeks post-onset of E2-HRx). In addition, at 16 weeks of age, the average LV 1-5 lengths of both oil-and E2-HRx db/db groups remained elongated relative to littermate control indices. In contrast, E2-HRx promoted a significant increase in average LV 1-5 width in the db/db mutant groups, relative to oil-HRx (sham vehicle injections) db/db mutants, and comparable to littermate controls (Figure 3) at both 8 and 16 weeks of age.

Discussion
The results of the current studies demonstrate that the expression of diabetes and obese metabolic syndromes in Representation of average lumber vertebral column (VC) lengths, as well as individual lumber vertebrae (LV) length and width indices, are denoted for control (+/?), obese (ob/ ob) and diabetes (db/db) mutant genotypes as groups means (± SEM) at 8 and 16 weeks of age. Significant (P ≤ 0.05) intergroup differences (i.e., control v.s. genotype mutant type) at each designed age are denoted by asterisks (*). Age-dependent (i.e., 8 v.s. 16 week values for the same genotype) intragroup differences are denoted by #.
Radiological comparisons between the lumbar vertebral seg-ments (LV 1-5) for control, obese (ob/ob) and diabetes (db/ db) genotype-mutants groups are represented (x12.5) as indi-cators of length and width index parameters measured rela-tive to the severity of diabetes-obesity syndrome aberrations for body weight and systemic endocrine/metabolic (Table 2) indices Figure 1 Radiological comparisons between the lumbar vertebral segments (LV 1-5) for control, obese (ob/ob) and diabetes (db/ db) genotype-mutants groups are represented (x12.5) as indicators of length and width index parameters measured relative to the severity of diabetes-obesity syndrome aberrations for body weight and systemic endocrine/metabolic (Table 2) indices.
human syndrome complications [8,31,33,36,37,39]. The radiological and morphometric indications of pronounced VC and LV 1-5 length indices, contrasted with diminished LV 1-5 width parameters, in these experimental models are suggestive causes of the recognized increases of vertebral fracture, osteoporosis and torsion stress-induced dislocation susceptibilities in humans experiencing chronic diabetes or obesity-related systemic metabolic, endocrine or nutritional compromise [8,[32][33][34][35]37,38]. The pronounced, progressive suppression of LV 1-5 width maturation, but pronounced individual LV and total VC regional elongation, indices occurred under the systemic influences of hyperglycemic, hyperinsulinemic and hyperlipidemic endocrine/metabolic stimulation. These data indicate that in these novel experimental models of dysregulated metabolic syndromes, variances in VC and LV growth expressions may be evaluated which are recognized to be shared with chronic human syndrome complications [8,37,39]. The restoration of normal LV 1-5 width indices in db/db mutants following E2-HRx indicates that correction of the glucometabolic disturbances with characterize the hypogonadal mutant model [28][29][30] re-establishes osteomaturation indices under persistent missense mutation (Table 1) expression influences [15,16]. These results indicate that the therapeutic re-establishment of a homeostatic systemic glucometabolic environment in db/db mutants supports the expression of normalized LV 1-5 width osteomaturation and VC stabilization, and counter-regulates the hyperglycemia-promoted susceptibilities to osteoporosis and VC destabilization resulting from chronic diabetes syndrome influences [1][2][3][4][5]8,18].

Conclusion
In summary, the current studies define the variable influences of db/db and ob/ob genotype mutations on VC and LV 1-5 osteomaturation indices, and the structural compromise in LV width indices promoted by these dysregulated expression syndromes. Of particular interest were the structural elongation of VC and LV 1-5 osteomaturation parameters, and LV 1-5 width restrictions, in db/db and ob/ob mutants associated with the hypercaloric metabolic state that characterized both mutation syndromes.
Although altered vertebral width expression occurred under such aberrant metabolic syndrome conditions, the therapeutic normalization of systemic glucose concentrations by E2-HRx stimulated LV osteodevelopment (width indices) comparable to control parameters. The chronic influences of these metabolic syndromes on progressive VC and LV osteodevelopment are regarded as intrinsic components of expressed skeletal compromise associated with altered bone cellular proliferation rates, density, cytochemical composition and diminished skeletal strength exhibited by both humans and experimental models exhibiting similar dysregulated metabolic syndromes [16][17][18]25,27]. The results of these studies suggest that these genetic models may be suitable for the further evaluation of novel manipulative or therapeutic treatments for osteomoderating events which are expressed in similar dysregulated metabolic-based human syndromes.