<section xmlns="" id="Sec1" class="Section1 RenderAsSection1" style="box-sizing: border-box; border-top-width: 1px; border-top-style: solid; border-top-color: rgb(212, 212, 212); padding-top: 36px; margin-top: 36px; color: rgb(51, 51, 51); font-family: 'Open Sans', sans-serif; font-size: medium; line-height: 25px; margin-bottom: 5px !important;">Myostatin initially designated as growth differentiation factor 8 (GDF-8), is a distinctive member of the TGF-β super-family, retaining many of the characteristic features found in this family [1]. The functional importance of myostatin is inferred by the high level of sequence homology and conservation seen across a number of species. Synthesized as a precursor protein, myostatin is proteolytically cleaved twice to release the biologically mature form of myostatin, with dimerisation of the mature protein creating the active form of myostatin [1]. The binding of the myostatin dimer to its receptor (ActRIIB) initiates the Smad mediated signaling pathway, which results in the transcription and expression of genes needed to mediate the negative regulation of muscle development. Myostatin function is controlled by a number of inhibitors, the most potent being follistatin like-3 (FSTL-3) [2].
Myostatin was initially identified as a negative regulator of muscle development where its inactivation in mice resulted in offspring with a two to three-fold increase in muscle mass [3]. Subsequently, myostatin was identified to affect glucose uptake in the human placenta [4]. Recently, altered placenta myostatin concentrations were identified in developmentally programmed rat model that supports a role as a mediator of this phenomenon [5]. Myostatin has also been shown to be crucial in the modulation of glucose homeostasis and adipogenesis [6, 7]. The aim of this study was to utilise a model of trans-generational maternal under-nutrition to investigate potential sexual dimorphic changes in the expression of myostatin and FSTL-3 in rat skeletal muscle.
</section><section xmlns="" id="Sec2" class="Section1 RenderAsSection1" style="box-sizing: border-box; border-top-width: 1px; border-top-style: solid; border-top-color: rgb(212, 212, 212); padding-top: 36px; margin-top: 36px; color: rgb(51, 51, 51); font-family: 'Open Sans', sans-serif; font-size: medium; line-height: 25px; margin-bottom: 5px !important;">Materials and methods
The animal model depicted (Figure 1) was based upon an established model of under-nutrition [8–10]. Briefly, virgin female Wistar rats (F0) reared on an ad libitum (AD) standard chow diet (2018 Teklad Global Rodent Diet; Bicester, UK) were mated at D120 ± 5 of age with males also fed an AD standard chow diet. Following confirmation of mating, the females were individually housed and received either an AD (AD group) or 30% of the AD chow diet (UN group) throughout gestation. During lactation dams of both groups were on an AD diet. Litter size was standardized on D1 to 10 pups per litter to standardize nutrition until weaning. Female offspring from these pregnancies (F1) were weaned and fed chow AD until D120 ± 5 of age before being mated with AD males and fed either AD or UN during gestation. Resulting in three groups ADAD, ADUN and UNAD; UNUN animals were not bred for ethical reasons.<figure xmlns="" class="Figure" id="Fig1" style="box-sizing: border-box; margin: 24px 16px; padding: 16px; border: 1px solid rgb(231, 231, 231);">
<figcaption class="Caption" lang="en" style="box-sizing: border-box; font-size: 0.875rem; line-height: 1.5em; margin-top: 16px;">Figure 1The experimental design employed in this study. Gestational diets are represented by AD = ad libitum and UN = 30% of the ad libitum diet. Hatched boxes are males and in black are the females. The relative difference in body size between F2 generation males and females is represented diagrammatically.
</figcaption></figure>
F1 females litter size was standardized on D1 to 10 pups, 5 males and 5 females where possible. All these F2 pups were nursed by their own dams that were fed AD until weaning (D22). At weaning half the offspring from each of the three groups were fed either a chow or high fat (HF, 60% kcal as fat; D12492 Research Diets Inc, NJ, USA) diet ad libitum until being euthanized at D140. The body weights of F2 animals were recorded from birth until D140. On D130, body measurements and composition were determined in F2 animals (n = 10 per sex per diet per group) by DEXA using a LUNAR Prodigy Scanner (GE Medical Systems, WI, USA). On D140 after an overnight fast, the animals were euthanised via decapitation under sodium pentobarbital anesthesia (60 mg kg, I.P.). Blood was collected, centrifuged for 15 mins at 1500 ×g at 4°C and stored at -20°C for further analysis. Skeletal muscle samples were collected from the left hind leg quadriceps, snap frozen and stored at -80°C. All animal procedures undertaken were approved by the University of Auckland Animal Ethics Committee.
Plasma glucose was measured along with quality controls using commercial kits (Roche Diagnostics Corp.; IN, USA) on an auto-analyzer (Roche/Hitachi 9002 Analyzer; Hitachi, Tokyo, Japan). Plasma insulin was determined by a rat insulin ELISA (Mercodia; Uppsala, Sweden) following the manufacturers' instructions. The insulin assay sensitivity was 0.07 μg/l and the intra- and inter-assay coefficients of variation (CVs) were 3.4% and 6.3%.
<section id="Sec3" class="Section2 RenderAsSection2" style="box-sizing: border-box;">Protein extraction and Western blotting
Proteins were extracted from male and female D140 muscle samples and Western blot analysis performed [11, 12]. Antibodies from Santa Cruz Biotechnology (CA, USA) were used for the detection of myostatin (1:3000 dilution; Goat Polyclonal GDF-8 (F-13), Sc-34781) and FSTL-3 (1:5000 dilution; Goat Polyclonal FLRG (N-18), Sc-21302) in conjunction with a secondary Polyclonal Rabbit Anti IgG/HRP antibody (1:10000 dilution). The relative optical densities were normalized to sheep muscle expression of myostatin and FSTL-3 protein.
</section><section id="Sec4" class="Section2 RenderAsSection2" style="box-sizing: border-box;">Statistical analysis
All analyses were run using a general linear mixed model followed by Tukey's post hoc comparison performed on SAS software version 9.1 (SAS institute, Cavy, NC). Differences in bodyweight and composition, plasma metabolite concentrations and the relative expression of myostatin and FSTL-3 in skeletal muscle were analysed with group, sex and diet included in the model, and all interactions between the main effects were analysed. All data presented are as means ± SEM.
</section>
</section><section xmlns="" id="Sec5" class="Section1 RenderAsSection1" style="box-sizing: border-box; border-top-width: 1px; border-top-style: solid; border-top-color: rgb(212, 212, 212); padding-top: 36px; margin-top: 36px; color: rgb(51, 51, 51); font-family: 'Open Sans', sans-serif; font-size: medium; line-height: 25px; margin-bottom: 5px !important;">Results
Comparisons of D130 body composition are shown in Table 1 and Table 2. Male offspring were heavier (P < 0.0001) than females (Table 1). Males and females were heavier (P < 0.05) on an HF diet compared to their chow fed contemporaries; the ADAD group was the heaviest within HF fed animals (P < 0.01). A trend toward a higher percentage of body fat in the males compared to females was observed with significance reached in the ADUN chow and HF fed offspring (P < 0.05). HF fed offspring had an elevated body fat percentage but lower lean body percentage compared to chow-fed offspring. Interestingly, both UNAD HF fed males and females had a lower (P < 0.01) body fat percentage compared to ADAD contemporaries (Table 1). Differences in plasma glucose and insulin concentrations were also evident between sexes and groups (Table 2). Most strikingly an elevated glucose concentration was seen in UNAD HF females (not males) compared to all other groups.
</section>
Myostatin was initially identified as a negative regulator of muscle development where its inactivation in mice resulted in offspring with a two to three-fold increase in muscle mass [3]. Subsequently, myostatin was identified to affect glucose uptake in the human placenta [4]. Recently, altered placenta myostatin concentrations were identified in developmentally programmed rat model that supports a role as a mediator of this phenomenon [5]. Myostatin has also been shown to be crucial in the modulation of glucose homeostasis and adipogenesis [6, 7]. The aim of this study was to utilise a model of trans-generational maternal under-nutrition to investigate potential sexual dimorphic changes in the expression of myostatin and FSTL-3 in rat skeletal muscle.
</section><section xmlns="" id="Sec2" class="Section1 RenderAsSection1" style="box-sizing: border-box; border-top-width: 1px; border-top-style: solid; border-top-color: rgb(212, 212, 212); padding-top: 36px; margin-top: 36px; color: rgb(51, 51, 51); font-family: 'Open Sans', sans-serif; font-size: medium; line-height: 25px; margin-bottom: 5px !important;">Materials and methods
The animal model depicted (Figure 1) was based upon an established model of under-nutrition [8–10]. Briefly, virgin female Wistar rats (F0) reared on an ad libitum (AD) standard chow diet (2018 Teklad Global Rodent Diet; Bicester, UK) were mated at D120 ± 5 of age with males also fed an AD standard chow diet. Following confirmation of mating, the females were individually housed and received either an AD (AD group) or 30% of the AD chow diet (UN group) throughout gestation. During lactation dams of both groups were on an AD diet. Litter size was standardized on D1 to 10 pups per litter to standardize nutrition until weaning. Female offspring from these pregnancies (F1) were weaned and fed chow AD until D120 ± 5 of age before being mated with AD males and fed either AD or UN during gestation. Resulting in three groups ADAD, ADUN and UNAD; UNUN animals were not bred for ethical reasons.<figure xmlns="" class="Figure" id="Fig1" style="box-sizing: border-box; margin: 24px 16px; padding: 16px; border: 1px solid rgb(231, 231, 231);">
<figcaption class="Caption" lang="en" style="box-sizing: border-box; font-size: 0.875rem; line-height: 1.5em; margin-top: 16px;">Figure 1The experimental design employed in this study. Gestational diets are represented by AD = ad libitum and UN = 30% of the ad libitum diet. Hatched boxes are males and in black are the females. The relative difference in body size between F2 generation males and females is represented diagrammatically.
</figcaption></figure>
F1 females litter size was standardized on D1 to 10 pups, 5 males and 5 females where possible. All these F2 pups were nursed by their own dams that were fed AD until weaning (D22). At weaning half the offspring from each of the three groups were fed either a chow or high fat (HF, 60% kcal as fat; D12492 Research Diets Inc, NJ, USA) diet ad libitum until being euthanized at D140. The body weights of F2 animals were recorded from birth until D140. On D130, body measurements and composition were determined in F2 animals (n = 10 per sex per diet per group) by DEXA using a LUNAR Prodigy Scanner (GE Medical Systems, WI, USA). On D140 after an overnight fast, the animals were euthanised via decapitation under sodium pentobarbital anesthesia (60 mg kg, I.P.). Blood was collected, centrifuged for 15 mins at 1500 ×g at 4°C and stored at -20°C for further analysis. Skeletal muscle samples were collected from the left hind leg quadriceps, snap frozen and stored at -80°C. All animal procedures undertaken were approved by the University of Auckland Animal Ethics Committee.
Plasma glucose was measured along with quality controls using commercial kits (Roche Diagnostics Corp.; IN, USA) on an auto-analyzer (Roche/Hitachi 9002 Analyzer; Hitachi, Tokyo, Japan). Plasma insulin was determined by a rat insulin ELISA (Mercodia; Uppsala, Sweden) following the manufacturers' instructions. The insulin assay sensitivity was 0.07 μg/l and the intra- and inter-assay coefficients of variation (CVs) were 3.4% and 6.3%.
<section id="Sec3" class="Section2 RenderAsSection2" style="box-sizing: border-box;">Protein extraction and Western blotting
Proteins were extracted from male and female D140 muscle samples and Western blot analysis performed [11, 12]. Antibodies from Santa Cruz Biotechnology (CA, USA) were used for the detection of myostatin (1:3000 dilution; Goat Polyclonal GDF-8 (F-13), Sc-34781) and FSTL-3 (1:5000 dilution; Goat Polyclonal FLRG (N-18), Sc-21302) in conjunction with a secondary Polyclonal Rabbit Anti IgG/HRP antibody (1:10000 dilution). The relative optical densities were normalized to sheep muscle expression of myostatin and FSTL-3 protein.
</section><section id="Sec4" class="Section2 RenderAsSection2" style="box-sizing: border-box;">Statistical analysis
All analyses were run using a general linear mixed model followed by Tukey's post hoc comparison performed on SAS software version 9.1 (SAS institute, Cavy, NC). Differences in bodyweight and composition, plasma metabolite concentrations and the relative expression of myostatin and FSTL-3 in skeletal muscle were analysed with group, sex and diet included in the model, and all interactions between the main effects were analysed. All data presented are as means ± SEM.
</section>
</section><section xmlns="" id="Sec5" class="Section1 RenderAsSection1" style="box-sizing: border-box; border-top-width: 1px; border-top-style: solid; border-top-color: rgb(212, 212, 212); padding-top: 36px; margin-top: 36px; color: rgb(51, 51, 51); font-family: 'Open Sans', sans-serif; font-size: medium; line-height: 25px; margin-bottom: 5px !important;">Results
Comparisons of D130 body composition are shown in Table 1 and Table 2. Male offspring were heavier (P < 0.0001) than females (Table 1). Males and females were heavier (P < 0.05) on an HF diet compared to their chow fed contemporaries; the ADAD group was the heaviest within HF fed animals (P < 0.01). A trend toward a higher percentage of body fat in the males compared to females was observed with significance reached in the ADUN chow and HF fed offspring (P < 0.05). HF fed offspring had an elevated body fat percentage but lower lean body percentage compared to chow-fed offspring. Interestingly, both UNAD HF fed males and females had a lower (P < 0.01) body fat percentage compared to ADAD contemporaries (Table 1). Differences in plasma glucose and insulin concentrations were also evident between sexes and groups (Table 2). Most strikingly an elevated glucose concentration was seen in UNAD HF females (not males) compared to all other groups.
</section>
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