non-invasive-skeletal-muscle-quantification-small-animals-using-micro

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

Universidade Federal do Rio de Janeiro

Previous studies have proposed a role for neuromedin B (NB), a bombesin-like peptide, in the control of body weight homeostasis. However, the nature of this role is unclear. The actions of NB are mediated preferentially by NB-preferring receptors (NBRs). Here we examined the consequences of targeted deletion of NBRs in female mice on body weight homeostasis in mice fed a normolipid diet (ND) or a high-fat diet (HFD) for 13 weeks. Body weight and food ingestion of neuromedin B receptor knockout (NBR-KO) mice fed a normolipid diet showed no difference in relation to wild-type (WT). However, the high-fat diet induced an 8.9- and 4.8-fold increase in body weight of WT and NBR-KO, respectively, compared to their controls maintained with a normolipid diet, even though the mice ingested the same amount of calories, regardless of genotype. Comparing mice fed the high-fat diet, NBR-KO mice accumulated approximately 45% less fat depot mass than WT, exhibited a lower percentage of fat in their carcasses (19.2 vs. 31.3%), and their adipocytes were less hypertrophied. Serum leptin and leptin mRNA in inguinal and perigonadal fat were lower in HFD NBR-KO than HFD WT, and serum adiponectin was similar among HFD groups and unaltered in comparison to ND-fed mice. HFD-fed WT mice developed glucose intolerance but not the HFD-fed NBR-KO mice, although they had similar glycaemia and insulinaemia. NBR-KO and WT mice on the normolipid diet showed no differences in any parameters, except for a trend to lower insulin levels. Therefore, disruption of the neuromedin B receptor pathway did not change body weight homeostasis in female mice fed a normolipid diet; however, it did result in partial resistance to diet-induced obesity.

Female mice target deleted for the neuromedin B receptor have partial resistance to diet-induced obesity.

Thyroid hormones (TH) are crucial for development, growth, differentiation, metabolism and thermogenesis. Skeletal muscle (SM) contractile function, myogenesis and bioenergetic metabolism are influenced by TH. These effects depend on the presence of the TH transporters MCT8 and MCT10 in the plasma membrane, the expression of TH receptors (THRA or THRB) and hormone availability, which is determined either by the activation of thyroxine (T) into triiodothyronine (T) by type 2 iodothyronine deiodinases (D2) or by the inactivation of T into reverse T by deiodinases type 3 (D3). SM relaxation and contraction rates depend on T regulation of myosin expression and energy supplied by substrate oxidation in the mitochondria. The balance between D2 and D3 expression determines TH intracellular levels and thus influences the proliferation and differentiation of satellite cells, indicating an important role of TH in muscle repair and myogenesis. During critical illness, changes in TH levels and in THR and deiodinase expression negatively affect SM function and repair. This review will discuss the influence of TH action on SM contraction, bioenergetics metabolism, myogenesis and repair in health and illness conditions.

Role of thyroid hormone in skeletal muscle physiology.

Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.

Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice.

The mdx mouse model of Duchenne Muscular Dystrophy (DMD) presents sarcolemma instability and develops a mild multi-stage dystrophinopathy characterized by intense myonecrosis with inflammatory infiltrate at 4-weeks; muscular regeneration at 12-weeks and persistent fibrosis onwards. Mdx diaphragm muscle has a more severe phenotype with structural and functional deterioration that closely resembles the diaphragm impairment responsible for DMD human patients' morbidity. Herein, we compared calcium deposits, activity of calcium-related proteases, and expression of muscle-specific proteins in mdx diaphragm at 4-weeks and 12-weeks. We found increased calcium deposits mainly at 12-weeks, concomitant with high activity of calpains and matrix metalloprotease-9, but decreased expression of Myh4 (Myhc IIb) and Atp2a1 (SERCA1), and high expression of the myogenic regulatory factors Myod1 and Myog. Our results suggest that increased calcium deposits and persistent activity of calcium dependent proteases throughout the disease are involved in the degeneration and regeneration processes in the mdx diaphragm.

Persistent mdx diaphragm alterations are accompanied by increased expression and activity of calcium and muscle-specific proteins.

Infection alters the expression of transporters that mediate the placental exchange of xenobiotics, lipids and cytokines. We hypothesized that lipopolysaccharide (LPS) modifies the expression of placental transport systems and lipid homeostasis. LPS (150 μg/kg; i.p.) treatments were administered for 4 h or 24 h, animals were euthanized at gestational days (GD) 15.5 or 18.5, and maternal blood, fetuses and placentae were collected. Increased rates of fetal demise were observed at GD15.5 following LPS treatment, whereas at GD18.5, high rates of early labour occurred and were associated with distinct proinflammatory responses. Lipopolysaccharide did not alter ATP-binding cassette (ABC) transporter mRNA expression but decreased fatty acid binding protein associated with plasma membrane () at GD15.5 (LPS-4 h) and increased fatty acid translocase () mRNA at GD18.5 (LPS-4 h). At the protein level, breast cancer-related protein (Bcrp) and ABC sub-family G member 1 (Abcg1) levels were decreased in the placental labyrinth zone (Lz) at GD15.5, whereas P-glycoprotein (P-gp) and Bcrp Lz-immunostaining was decreased at GD18.5. In the placental junctional zone (Jz), P-gp, Bcrp and Abcg1 levels were higher at GD18.5. Specific maternal plasma and placental changes in triacylglycerol, free fatty acid, cholesterol, cholesterol ester and monoacylglycerol levels were detected in a gestational age-dependent manner. In conclusion, LPS-increased risk of fetal death and early labour were associated with altered placental ABC and lipid transporter expression and deranged maternal plasma and placental lipid homeostasis. These changes may potentially modify fetal xenobiotic exposure and placental lipid exchange in cases of bacterial infection.

Effect of Sublethal Prenatal Endotoxaemia on Murine Placental Transport Systems and Lipid Homeostasis.

Assisted reproductive technologies (ART) may increase risk for abnormal placental development, preterm delivery and low birthweight. We investigated placental morphology, transporter expression and paired maternal/umbilical fasting blood nutrient levels in human term pregnancies conceived naturally ( = 10) or by intracytoplasmic sperm injection (ICSI;  = 11). Maternal and umbilical vein blood from singleton term (>37 weeks) C-section pregnancies were assessed for levels of free amino acids, glucose, free fatty acids (FFA), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low-density lipoprotein (VLDL) and triglycerides. We quantified placental expression of GLUT1 (glucose), SNAT2 (amino acids), P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) (drug) transporters, and placental morphology and pathology. Following ICSI, placental SNAT2 protein expression was downregulated and umbilical cord blood levels of citrulline were increased, while FFA levels were decreased at term ( < 0.05). Placental proliferation and apoptotic rates were increased in ICSI placentae ( < 0.05). No changes in maternal blood nutrient levels, placental GLUT1, P-gp and BCRP expression, or placental histopathology were observed. In term pregnancies, ICSI impairs placental SNAT2 transporter expression and cell turnover, and alters umbilical vein levels of specific nutrients without changing placental morphology. These may represent mechanisms through which ICSI impacts pregnancy outcomes and programs disease risk trajectories in offspring across the life course.

Altered Umbilical Cord Blood Nutrient Levels, Placental Cell Turnover and Transporter Expression in Human Term Pregnancies Conceived by Intracytoplasmic Sperm Injection (ICSI).

Perinatal maternal obesity and excessive fructose consumption have been associated with liver metabolic diseases. The study investigates whether moderate maternal high-fat diet affects the liver mitochondria responses to fructose intake in adult offspring.

Maternal Isocaloric High-Fat Diet Induces Liver Mitochondria Maladaptations and Homeostatic Disturbances Intensifying Mitochondria Damage in Response to Fructose Intake in Adult Male Rat Offspring.

Thyroid hormone (TH) signaling controls muscle progenitor cells differentiation. However, inflammation can alter muscle TH signaling by modulating the expression of TH transporters (, receptors (), and deiodinase enzymes ( and ). Thus, a proinflammatory environment could affect myogenesis. The role of a low-grade inflammatory milieu in TH signaling during myogenesis needs further investigation. Herein, we aimed to study the impact of the bacterial lipopolysaccharide (LPS)-induced inflammatory stimulus on the TH signaling during myogenesis. C2C12 myoblasts differentiation was induced without (CTR) or with 10 ng/mL LPS presence. The myoblasts under LPS stimulus release the proinflammatory cytokines (IL-6 and IL-1β) and chemokines (CCL2 and CXCL-1). LPS decreases  expression by 28% during the initial myogenesis, thus reducing the myogenic stimulus. At the same time, LPS reduced the expression of  by 41% but doubled the D2 enzymatic activity. The late differentiation was not affected by inflammatory milieu, which only increased the  gene expression by 38%. LPS altered the intracellular metabolism of TH and reduced the initial myogenic stimulus. However, it did not affect late differentiation. Increased intracellular TH activation may be the compensatory pathway involved in the recovery of myogenic differentiation under a low-grade inflammatory milieu.

Low Inflammatory Stimulus Increases D2 Activity and Modulates Thyroid Hormone Metabolism during Myogenesis In Vitro.

Hashimoto thyroiditis is an autoimmune disease characterized by hypothyroidism and a high level of anti-thyroid autoantibodies. It has shown to negatively impact female fertility; however, the mechanisms are unclear. Ovarian follicular fluid appears to be the key to understanding how Hashimoto thyroiditis affecst fertility. Thus, we aimed to evaluated the metabolic profile of follicular fluid and antithyroid autoantibody levels in the context of Hashimoto thyroiditis. We collected follicular fluid from 61 patients, namely 38 women with thyroid autoantibody positivity and 23 women as negative controls, undergoing in vitro fertilization treatment. Follicular fluid samples were analyzed using metabolomics, and thyroid autoantibodies were measured. Fifteen metabolites with higher concentrations in the follicular fluid samples from Hashimoto thyroiditis were identified, comprising five possible affected pathways: the glycerophospholipid, arachidonic acid, linoleic acid, alpha-linolenic acid, and sphingolipid metabolism pathways. These pathways are known to regulate ovarian functions. In addition, antithyroglobulin antibody concentrations in both serum and follicular fluid were more than tenfold higher in women with Hashimoto thyroiditis than in controls. Our data showed that the metabolic profile of follicular fluid is altered in women with Hashimoto thyroiditis, suggesting a potential mechanistic explanation for the association of this disease with female infertility.

Article	Year
Female mice target deleted for the neuromedin B receptor have partial resistance to diet-induced obesity. The Journal of physiology\| PubMed ID: 20211980	2010
Role of thyroid hormone in skeletal muscle physiology. The Journal of endocrinology\| PubMed ID: 29051191	2018
Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice. Scientific reports\| PubMed ID: 30760802	2019
Persistent mdx diaphragm alterations are accompanied by increased expression and activity of calcium and muscle-specific proteins. Histology and histopathology\| PubMed ID: 33825181	2021
Effect of Sublethal Prenatal Endotoxaemia on Murine Placental Transport Systems and Lipid Homeostasis. Frontiers in microbiology\| PubMed ID: 34394055	2021
Altered Umbilical Cord Blood Nutrient Levels, Placental Cell Turnover and Transporter Expression in Human Term Pregnancies Conceived by Intracytoplasmic Sperm Injection (ICSI). Nutrients\| PubMed ID: 34444747	2021
Maternal Isocaloric High-Fat Diet Induces Liver Mitochondria Maladaptations and Homeostatic Disturbances Intensifying Mitochondria Damage in Response to Fructose Intake in Adult Male Rat Off... Molecular nutrition & food research\| PubMed ID: 35175665	2022
Low Inflammatory Stimulus Increases D2 Activity and Modulates Thyroid Hormone Metabolism during Myogenesis In Vitro. Metabolites\| PubMed ID: 35629920	2022
Metabolomic analysis of follicular fluid from women with Hashimoto thyroiditis. Scientific reports\| PubMed ID: 37532758	2023

Flavia F. Bloise

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