Novel polychrome staining distinguishing osteochondral tissue and bone cells in decalcified paraffin sections.

The bone is a dynamic and metabolically active organ in which growth and resorption of the osteochondral matrix is orchestrated by osteoblasts and osteoclasts. For decalcified paraffin-embedded specimens, decalcifying agents alter the staining intensity, and excess decalcification interferes with bone staining. Robust bone staining methods independent of the decalcification conditions and animal species are lacking. In this study, we have developed a novel polychrome staining method, named JFRL staining, which stains the components of osteochondral tissue in different colors. With this staining we could visualize the hyaline cartilage as blue by alcian blue, osteoid as red by picrosirius red, and mineralized bone as green by picro-light green SF or picro-naphthol green B and easily distinguished osteoblasts, osteocytes, and osteoclasts. In mineralized bone, this staining revealed the obvious lamellar structures and woven bone. Notably, this staining was independent of the decalcification conditions and experimental animal species examined. To verify the usefulness of JFRL staining, we observed cotton rat tail which has shorter length and shows a false autotomy. The caudal vertebrae were normally developed via endochondral ossification without a fracture plane. At 6 months of age, the number of chondrocytes declined and the hypertrophic zone was absent at the epiphyseal plate, which might reflect the shorter tail. In conclusion, JFRL staining is the first method to simultaneously distinguish osteochondral matrix and bone cells in one section regardless of decalcifying conditions. This robust staining will provide new information for a wide number of biomedical fields, including bone development, physiology, and pathology.

Corrigendum: Citrate synthase insufficiency leads to specific metabolic adaptations in the heart and skeletal muscles upon low-carbohydrate diet feeding in mice.

[This corrects the article DOI: 10.3389/fnut.2022.925908.].

Citrate Synthase Insufficiency Leads to Specific Metabolic Adaptations in the Heart and Skeletal Muscles Upon Low-Carbohydrate Diet Feeding in Mice.

A decrease in TCA cycle activity may lead to impaired nutrition metabolism and cellular energy shortage. Herein, we aimed to characterize the detailed metabolic changes that compensate for energy shortages in energy-consuming organs (heart and skeletal muscles) in mice with knockout of citrate synthase (CS), an important enzyme in the TCA cycle. CS hetero knockout (CS +/-) mice and wild-type mice were fed a low-carbohydrate ketogenic diet (LCKD) or high-fat, high-carbohydrate diet (HFHCD) to induce metabolic changes. Body weight, blood serum parameters, metabolic gene expression, and adenosine triphosphate (ATP) levels were measured in the heart and skeletal muscles. Glycogen content, anabolic and catabolic biomarkers, and morphological changes were also assessed in the skeletal muscles. After diet feeding, there were no differences observed in the body weight and blood serum parameters between wild-type and CS +/- mice. The cardiac expression of genes related to the utilization of fatty acids, monocarboxylates, and branched amino acids increased in LCKD-fed CS +/- mice. In contrast, no significant differences in gene expression were observed in the muscles of LCKD-fed mice or the heart and muscles of HFHCD-fed mice. ATP levels decreased only in the skeletal muscles of LCKD-fed CS +/- mice. Additionally, the decrease in glycogen content, suppression of p70 S6 kinase, and presence of type I fiber atrophy were observed in the muscles of LCKD-fed CS +/- mice. These results suggest that the energy-consuming organs with CS insufficiency may undergo tissue-specific adaption to compensate for energy shortages when the carbohydrate supply is limited.

Gallic acid regulates adipocyte hypertrophy and suppresses inflammatory gene expression induced by the paracrine interaction between adipocytes and macrophages in vitro and in vivo.

Obesity-induced chronic inflammation in adipose tissue plays a critical role in the development of insulin resistance and various lifestyle-related diseases. Although gallic acid (GA) is known to exert protective effects on obesity-related complications, its function in adipose tissue inflammation has not been elucidated. Recently, we reported that GA exerts protective effects against inflammation. To test our hypothesis that the anti-inflammatory effect of GA partially contributes to the improvement of metabolic diseases, we examined the effect of GA on inflammation caused by adipocyte-macrophage crosstalk in obesity. We showed that GA enhanced adipocyte differentiation in 3 T3-L1 adipocytes. Consistent with the enhancement of adipogenesis, GA decreased the gene expression of monocyte chemoattractant protein-1 and increased that of adiponectin and the upstream mediator peroxisome proliferator-activated receptor gamma. GA also reduced inflammatory mediator expression induced by the co-culture of 3 T3-L1 adipocytes with RAW 264 macrophages. Diet-induced obese mice treated with GA showed decreased serum cholesterol levels and adipocyte size, and improved insulin sensitivity without changes in body weight. Moreover, GA-treated mice had decreased expression of interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, F4/80, and sterol regulatory element binding transcription factor-1 in their adipose tissue. These results indicate that GA suppresses adipocyte hypertrophy and inflammation caused by the interaction between adipocytes and macrophages, thereby improving metabolic disorders such as insulin resistance and dyslipidemia.

Slc:Hartley guinea pigs frequently possess duplication of the caudal vena cava.

The formation of the caudal vena cava is a complex process involving development, regression, and anastomosis. In mammals, the normal caudal vena cava runs to the right side of the abdominal aorta, while duplication of the caudal vena cava has been identified as a congenital abnormality in both companion animals and humans. The present study demonstrates that Slc:Hartley guinea pigs frequently possess asymptomatic duplicated caudal vena cava. The prevalence was 30% and 24% for males and females, respectively, with no sex-related differences. In accordance with Saad et al. (2012)'s criteria, duplicated caudal vena cava were classified into two distinct variations. The dominant variation was a complete duplication without iliac anastomosis where the left caudal vena cava continued from the left common iliac vein and joined the left renal vein; the left renal vein ran to the right to join the right caudal vena cava. The alternative variation was an incomplete duplication where the left caudal vena cava joined the right infrarenal caudal vena cava at a more cranial point than in normal cases; the renal segment was unchanged. Iliac anastomosis was not found in any cases. Duplicated caudal vena cava neither affected the body weight nor the kidney weight. In conclusion, Slc:Hartley guinea pigs frequently possess asymptomatic duplicated caudal vena cava in the absence of iliac anastomosis and appear to be a novel and useful animal model for duplicated caudal vena cava in animals and humans.

Short-term and long-term ketogenic diet therapy and the addition of exercise have differential impacts on metabolic gene expression in the mouse energy-consuming organs heart and skeletal muscle.

Although a ketogenic diet (KD) is used to treat various metabolic diseases, the organ-specific metabolic changes that occur in response to a KD remain unclear. Therefore, we tested the hypothesis that duration of KD consumption and regular exercise in addition to KD consumption affect metabolic fuel selection at gene levels in heart and skeletal muscle. Six-week-old male C57BL/6J mice were divided into 2 groups, one fed a standard diet and the other fed a KD, and maintained for either 4 weeks (short term) or 12 weeks (long term). The long-term group was further divided into 2 subgroups, and mice in 1 of the 2 groups had an exercise load 5 days a week. Body weight decreased significantly in the KD groups during the first few weeks only. Plasma ketone levels rose and muscle glycogen levels declined significantly in the KD groups, but these changes were less severe in the KD plus exercise group. KD consumption decreased the expression of genes related to glucose utilization in heart and skeletal muscle; however, this decrease did not occur with KD consumption plus exercise. Long-term but not short-term KD consumption increased the expression of genes related to lipid utilization, regardless of exercise. In the KD groups, the expression of genes related to ketolysis was suppressed, and that of genes related to ketogenesis was increased. These results indicate that KD exposure and pairing a KD with exercise have differential impacts on energy substrate selection at gene expression levels in energy-consuming organs, the heart and skeletal muscle.