Biology subjects

Czernik, P. J.

Publications and source records attributed to Czernik, P. J..

6 recordsLinked to original sources

bioRxiv2026

Osteocytes secrete adiponectin and display adipocyte-like phenotype under control of PPARG nuclear receptor

Osteocytes and adipocytes represent cells with disparate functions. Osteocytes regulate bone metabolism (remodeling) and bone homeostasis, while adipocytes regulate energy metabolism and energy storage. Here, we demonstrate that osteocyte phenotype consists of adipocytic features which are under control of peroxisome proliferator-activated receptor gamma (PPARG), a master regulator of adipocyte differentiation and function. Using a mouse model with osteocyte-specific deletion of PPARG (OT{gamma}KO) and osteocyte cellular model of MLO-Y4 cells edited with CRISPR/Cas9 for PPARG deficiency, we are demonstrating that under PPARG control osteocytes produce and secrete adiponectin (ADIPOQ), and they are equipped in adipocyte-specific mechanisms for lipid-storage and their metabolism. Under PPARG, osteocytes accumulate lipid droplets which correlate with their capability to cover up to 20% of energy requirements from fatty acids metabolism. Although osteocytes like osteoblasts mainly express perilipin 2 (Plin2), however similarly to adipocytes, lipid droplets accumulation is associated with expression of perilipin 1 (Plin1) under PPARG control. Similarly, lipids accumulation and metabolism involve adipocyte-specific activities including fatty acids binding protein 4 (Fabp4), hormone-specific lipase (Hsl) and adipocyte-specific triglyceride lipase (Atgl), which expression are under PPARG control. These studies provide a new understanding of osteocyte biology which include adipocyte-like endocrine and lipid metabolism features probably reflecting an adaptation to their unique localization and a need for a maintenance of functional fitness in these conditions. They deepen our comprehension of the crossroads of osteocyte and adipocyte function and underscore the therapeutic potential of targeting common molecular pathways in both cell types for managing metabolic disorders and skeletal diseases.

cell biology↗

bioRxiv2024

Critical-Size Defect Tibialis Anterior (TA) Muscle Regeneration using Ex-Vivo Mice Hindlimbs Culturing under Dynamic Mechanical Loading

In this study, we introduced an innovative computer-controlled ex vivo mice hindlimb culturing platform operating under dynamic loading, coupled with injectable cell-laden nanofibrous matrix (PNCOL), to investigate tissue response and therapeutic outcomes in critical size defect tibialis anterior (TA) muscle regeneration. The combination of mechanical stimulation and cell therapy offers a distinctive opportunity to delve into the regenerative rehabilitation field and create sustainable solutions in musculoskeletal (MSK) tissue regeneration. The application of mechanical loading on the whole mice hindlimbs increased total bone area and marrow area suggesting an increase in periosteal bone formation and resorption on the endosteal surface. Viability assessments confirmed the sustained culturing of the samples throughout the study. Then, the effect of mechanical loading and PNCOL injection on muscle regeneration at the TA defect site was evaluated. Histological analyses revealed enhanced muscle regeneration in PNCOL-treated hindlimbs. Structural analysis of the defect area through scanning electron microscopy (SEM) showed regeneration of ECM fibers at the defect site in PNCOL-treated groups. An analysis of cytokine levels in conditioned media at the end experiment showed changes in the number of proteins with the role in wound healing, muscle regeneration WNT, and IGF-1 signaling suggesting an anabolic effect of mechanical stimulation on muscle and bone. Similarly, gene expression analysis showed a significant upregulation of PAX7, Mrf4, MYF5, and TGF{beta}1 mRNA levels, indicating enhanced muscle regeneration after coupled mechanical loading and PNCOL treatments. Lastly, immunostaining showed an increase in tissue regeneration and anti-inflammatory response (CD206) in PNCOL-treated groups. Overall, the ex vivo hindlimb organ culturing platform- maintained tissue functions under mechanical loading, while PNCOL treatment promoted muscle tissue regeneration and reduced inflammation. These findings demonstrated the potential of multidimensional approaches for enhancing therapeutic outcomes in MSK disorders. In addition, this study aligns with the growing emphasis on minimizing the number of animals used in research and developing a robust sense of responsible animal experimentation through introducing dynamic ex-vivo muscle organ culturing platform.

bioengineering↗

bioRxiv2024

IGF-1 and insulin receptors in LepRb neurons jointly regulate body growth, bone mass, reproduction, and metabolism

Leptin receptor (LepRb)-expressing neurons are known to link body growth and reproduction, but whether these functions are mediated via insulin-like growth factor 1 receptor (IGF1R) signaling is unknown. IGF-1 and insulin can bind to each others receptors, permitting IGF-1 signaling in the absence of IGF1R. Therefore, we created mice lacking IGF1R exclusively in LepRb neurons (IGF1RLepRb mice) and simultaneously lacking IGF1R and insulin receptor (IR) in LepRb neurons (IGF1R/IRLepRb mice) and then characterized their body growth, bone morphology, reproductive and metabolic functions. We found that IGF1R and IR in LepRb neurons were required for normal timing of pubertal onset, while IGF1R in LepRb neurons played a predominant role in regulating adult fertility and exerted protective effects against reproductive aging. Accompanying these reproductive deficits, IGF1RLepRb mice and IGF1R/IRLepRb mice had transient growth retardation. Notably, IGF1R in LepRb neurons was indispensable for normal trabecular and cortical bone mass accrual in both sexes. These findings suggest that IGF1R in LepRb neurons is involved in the interaction among body growth, bone development, and reproduction. Though only mild changes in body weight were detected, simultaneous deletion of IGF1R and IR in LepRb neurons caused dramatically increased fat mass composition, decreased lean mass composition, lower energy expenditure, and locomotor activity in both sexes. Male IGF1R/IRLepRb mice exhibited impaired insulin sensitivity. These findings suggest that IGF1R and IR in LepRb neurons jointly regulated body composition, energy balance, and glucose homeostasis. Taken together, our studies identified the sex-dependent complex roles of IGF1R and IR in LepRb neurons in regulating body growth, reproduction, and metabolism.

neuroscience↗

bioRxiv2024

PPARG in osteocytes controls cell bioenergetics and systemic energy metabolism independently of sclerostin levels in circulation

ObjectiveThe skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels. MethodsIn vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp1CrePpar{gamma}flfl male and female mice ({gamma}OTKO) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Ppar{gamma}. As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress. ResultsCirculating sclerostin levels of {gamma}OTKO male and female mice were not different from control mice. Male {gamma}OTKO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from {gamma}OTKO males suggested profound changes in cellular metabolism, fuel transport and usage, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, shifts in fuel utilization, and accumulation of reactive oxygen species (ROS). ConclusionsPPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism. HighlightsO_LIOsteocytes function as a body energostat via their bioenergetics C_LIO_LIPPARG protein acts as a "molecular break" of osteocyte mitochondrial activity C_LIO_LIPPARG deficiency activates TCA cycle, oxidative stress and ROS accumulation C_LIO_LIPPARG controls osteocyte insulin signaling and fuel utilization C_LI

physiology↗

bioRxiv2020

PPARG in osteocytes is essential for sclerostin expression, bone mass, marrow adiposity and TZD-induced bone loss

PPARG role in regulation of osteocyte function is largely unknown. We report that PPARG is essential for sclerostin production, a recently approved target to treat osteoporosis. There is an excellent correlation in osteocytes between Sost/sclerostin and PPARG at the transcript and protein levels, and increased bone mass in mice with osteocyte-specific deletion of PPARG ({gamma}OTKO) correlated with increased WNT signaling and bone forming activity of endosteal osteoblasts and decreased marrow fat. The 8 kb sequence upstream of Sost gene transcription start site possesses multiple PPARG binding elements (PPREs) with at least two of them binding PPARG with dynamics reflecting its activation and the levels of Sost transcript and sclerostin protein expression. Older {gamma}OTKO female mice are largely protected from TZD-induced bone loss providing proof of concept that PPARG in osteocytes can be pharmacologically targeted. Our study opens the possibility to consider repurposing PPARG as a target for treatment of osteoporosis.

physiology↗

bioRxiv2020

Reconstitution of the host holobiont acutely increases bone growth of the gnotobiotic rat

In recent years there has been growing evidence regarding the effect of microbiota on the skeletal growth and homeostasis. Here we present, for the first time, accelerated longitudinal and radial bone growth in young (7-week-old) germ-free male rats after short-term exposure to a newly established gut microbiota. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in tibia diaphysis. In addition, the number of small-in-size adipocytes increased, while the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats. The observed changes in bone status were paralleled with a positive shift in microbiota composition towards short chain fatty acids (SCFA)-producing microbes, which reflected a dramatic increase in cecal concentration of SCFA, specifically butyrate. Further, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels, implicating an involvement of the somatotropic axis. Increased serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase and resembled reversal of dysbiosis in adolescence, which is marked by rapid skeletal expansion. These findings may help in developing microbiota-based therapeutics to combat bone related disorders resulting from hormonal defects and/or malnutrition in children and adolescence.

physiology↗