Antidiabetic features of AdipoAI, a novel AdipoR agonist.

Adiponectin is an antidiabetic endogenous adipokine that plays a protective role against the unfavorable metabolic sequelae of obesity. Recent evidence suggests a sinister link between hypoadiponectinemia and development of insulin resistance/type 2 diabetes (T2D). Adiponectin's insulin-sensitizing property is mediated through the specific adiponectin receptors R1 and R2, which activate the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) α pathways. AdipoAI is a novel synthetic analogue of endogenous adiponectin with possibly similar pharmacological effects. Thus, there is a need of orally active small molecules that activate Adipoq subunits, and their downstream signaling, which could ameliorate obesity related type 2 diabetes. In the study we aim to investigate the effects of AdipoAI on obesity and T2D. Through in-vitro and in-vivo analyses, we investigated the antidiabetic potentials of AdipoAI and compared it with AdipoRON, another orally active adiponectin receptors agonist. Our results showed that in-vitro treatment of AdipoAI (0-5 µM) increased adiponectin receptor subunits AdipoR1/R2 with increase in AMPK and APPL1 protein expression in C2C12 myotubes. Similarly, in-vivo, oral administration of AdipoAI (25 mg/kg) observed similar effects as that of AdipoRON (50 mg/kg) with improved control of blood glucose and insulin sensitivity in diet-induced obesity (DIO) mice models. Further, AdipoAI significantly reduced epididymal fat content with decrease in inflammatory markers and increase in PPAR-α and AMPK levels and exhibited hepatoprotective effects in liver. Further, AdipoAI and AdipoRON also observed similar results in adipose tissue. Thus, our results suggest that low doses of orally active small molecule agonist of adiponectin AdipoAI can be a promising therapeutic target for obesity and T2D.

Long non-coding RNA APDC plays important regulatory roles in metabolism of bone and adipose tissues.

The long noncoding RNA (lncR) ANRIL in the human genome is an established genetic risk factor for atherosclerosis, periodontitis, diabetes, and cancer. However, the regulatory role of lncR-ANRIL in bone and adipose tissue metabolism remains unclear. To elucidate the function of lncRNA ANRIL in a mouse model, we investigated its ortholog, AK148321 (referred to as lncR-APDC), located on chr4 of the mouse genome, which is hypothesized to have similar biological functions to ANRIL. We initially revealed that lncR-APDC in mouse bone marrow cells (BMSCs) and lncR-ANRIL in human osteoblasts (hFOBs) are both increased during early osteogenesis. Subsequently, we examined the osteogenesis, adipogenesis, osteoclastogenesis function with lncR-APDC deletion/overexpression cell models. In vivo, we compared the phenotypic differences in bone and adipose tissue between APDC-KO and wild-type mice. Our findings demonstrated that lncR-APDC deficiency impaired osteogenesis while promoting adipogenesis and osteoclastogenesis. Conversely, the overexpression of lncR-APDC stimulated osteogenesis, but impaired adipogenesis and osteoclastogenesis. Furthermore, KDM6B was downregulated with lncR-APDC deficiency and upregulated with overexpression. Through binding-site analysis, we identified miR-99a as a potential target of lncR-APDC. The results suggest that lncR-APDC exerts its osteogenic function via miR-99a/KDM6B/Hox pathways. Additionally, osteoclasto-osteogenic imbalance was mediated by lncR-APDC through MAPK/p38 and TLR4/MyD88 activation. These findings highlight the pivotal role of lncR-APDC as a key regulator in bone and fat tissue metabolism. It shows potential therapeutic for addressing imbalances in osteogenesis, adipogenesis, and osteoclastogenesis.

AdipoAI suppresses osteoclastogenesis by activating AdipoR1/APPL1: An in vivo experimental study in diabetes-associated peri-implantitis.

AIM: Diabetics experience severe peri-implant inflammatory bone damage. We aimed to provide powerful evidence supporting the novel adiponectin receptor agonist AdipoAI in treating diabetes-associated peri-implantitis. MATERIALS AND METHODS: Twenty-four ZDF-Leprfa/Crl rats were randomly allocated to three groups (N = 8). After feeding with a high-fat diet to establish diabetic rats, experimental peri-implantitis was induced by implanting titanium rods (1.5 mm diameter and 20 mm length) contaminated with Staphylococcus aureus into the femurs. Radiographic evaluation, microCT, histological analyses and qRT-PCR were used to detect inflammatory infiltration and bone destruction. In vitro, the inhibition by AdipoAI of osteoclastogenesis, including the number and function of osteoclasts, was investigated by TRAP staining, immunofluorescence, qRT-PCR and Western blotting. Immunofluorescence, qRT-PCR and Western blotting were also utilized to explore AdipoR1, APPL1, NF-κB and Wnt5a-Ror2 signalling molecules in this process. One-way ANOVA with Tukey's post hoc test was used to compare the data. RESULTS: AdipoAI reduced inflammation and bone destruction caused by peri-implantitis in diabetic rats, which were manifested by a reduction in F4/80-positive macrophage infiltration by 72%, the number of osteoclasts by 58% and the levels of cytokines (p < .05) in disease group. In vitro, 1 µM AdipoAI decreased the number of osteoclasts to 51%, inhibited F-actin ring formation and reduced the levels of related markers (p < .05). Mechanistically, AdipoAI activated AdipoR1/APPL1 and conversely suppressed the phosphorylation of IκB-α, nuclear translocation of P65 and the Wnt5a-Ror2 signalling pathway (p < .05). CONCLUSIONS: AdipoAI suppressed osteoclastogenesis in diabetes-associated peri-implantitis by inhibiting the NF-κB and Wnt5a-Ror2 pathways via the AdipoR1/APPL1 axis.

A novel adiponectin receptor agonist (AdipoAI) ameliorates type 2 diabetes-associated periodontitis by enhancing autophagy in osteoclasts.

BACKGROUND AND OBJECTIVE: Type 2 diabetes (T2D)-associated periodontitis is severe and refractory in many cases. Considered an inflammatory disease, T2D predisposes to periodontitis by increasing whole-body inflammation. One mechanism of increased inflammation is thatT2D is mediated by loss of production or function of the anti-inflammatory hormone adiponectin. In our previous report, AdipoRon, an adiponectin receptor agonist, and AdipoAI, a newly discovered, more specific agonist, attenuated T2D-associated inflammation by inhibiting osteoclastogenesis and LPS-induced endotoxemia. Autophagy plays an important role during osteoclast differentiation and function. The impact of AdipoAI on osteoclast function and autophagy involved in osteoclastogenesis is not known. Here, we compare AdipoRon and AdipoAI potency, side effects and mechanism of action in T2D-associated periodontitis. METHODS: The RAW 264.7 cell line was used for in vitro studies. We analyzed the potential cytotoxicity of AdipoAI using the CCK-8 assay. The anti-osteoclastogenic potential of AdipoAI was studied by real-time qPCR and tartrate-resistant acid phosphatase staining. The actions of AdipoAI involved in autophagy were tested by real-time qPCR, western blot and immunofluorescence staining. In the diet-induced obesity model of T2D, we investigated the impact of AdipoAI on fasting blood glucose, alveolar bone loss, and gingival inflammation in mice with experimental periodontitis. RESULTS: AdipoRon inhibited osteoclastogenesis and AdipoAI inhibited osteoclastogenesis at lower doses than AdipoRon without any cytotoxicity. In DIO mice with experimental periodontitis, AdipoAI reduced mouse body weight in 14 days, reducing fasting glucose levels, alveolar bone destruction, osteoclast number along the alveolar bone surface, and decreased the expression of pro-inflammatory factors in periodontal tissues. AdipoAI and AdipoRon also enhanced LC3A/B expression when cultured with RANKL.3-Methyladenine, a known autophagy inhibitor, decreased LC3A/B expression and reversed the inhibition of osteoclastogenesis during AdipoAI treatment. CONCLUSIONS: Our results demonstrate that AdipoAI ameliorates the severity of T2D-associated periodontitis by enhancing autophagy in osteoclasts at lower doses than AdipoRon without demonstrable side effects. Thus, AdipoAI has pharmaceutical potential for treating diabetes-associated periodontal disease.

Irisin deficiency disturbs bone metabolism.

Balancing the process of bone formation and resorption is important in the maintenance of healthy bone. Therefore, the discovery of novel factors that can regulate bone metabolism remains needed. Irisin is a newly identified hormone-like peptide. Recent studies have reported the involvement of irisin in many physiological and pathological conditions with bone mineral density changes, including osteopenia and osteoporotic fractures. In this study, we generated the first line of Osx-Cre:FNDC5/irisin KO mice, in which FNDC5/irisin was specifically deleted in the osteoblast lineage. Gene and protein expressions of irisin were remarkably decreased in bones but no significant differences in other tissues were observed in knockout mice. FNDC5/irisin deficient mice showed a lower bone density and significantly delayed bone development and mineralization from early-stage to adulthood. Our phenotypical analysis exhibited decreased osteoblast-related gene expression and increased osteoclast-related gene expression in bone tissues, and reduced adipose tissue browning due to bone-born irisin deletion. By harvesting and culturing MSCs from the knockout mice, we found that osteoblastogenesis was inhibited and osteoclastogenesis was increased. By using irisin stimulated wildtype primary cells as a gain-of-function model, we further revealed the effects and mechanisms of irisin on promoting osteogenesis and inhibiting osteoclastogenesis in vitro. In addition, positive effects of exercise, including bone strength enhancement and body weight loss were remarkably weakened due to irisin deficiency. Interestingly, these changes can be rescued by supplemental administration of recombinant irisin during exercise. Our study indicates that irisin plays an important role in bone metabolism and the crosstalk between bone and adipose tissue. Irisin represents a potential molecule for the prevention and treatment of bone metabolic diseases.

AdipoRon promotes diabetic fracture repair through endochondral ossification-based bone repair by enhancing survival and differentiation of chondrocytes.

Diabetic bone defects may exhibit impaired endochondral ossification (ECO) leading to delayed bone repair. AdipoRon, a receptor agonist of adiponectin polymers, can ameliorate diabetes and related complications, as well as overcome the disadvantages of the unstable structure of artificial adiponectin polymers. Here, the effects of AdipoRon on the survival and differentiation of chondrocytes in a diabetic environment were explored focusing on related mechanisms in gene and protein levels. In vivo, AdipoRon was applied to diet-induced-obesity (DIO) mice, a model of obesity and type 2 diabetes, with femoral fracture. Sequential histological evaluations and micro-CT were examined for further verification. We found that AdipoRon could ameliorate cell viability, apoptosis, and reactive oxygen species (ROS) production and promote mRNA expression of chondrogenic markers and cartilaginous matrix production of ATDC5 cells in high glucose medium via activating ERK1/2 pathway. Additionally, DIO mice with intragastric AdipoRon administration had more neocartilage and accelerated new bone formation. These data suggest that AdipoRon could stimulate bone regeneration via ECO in diabetes.

Potential roles of miR-335-5p on pathogenesis of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a prevalent oral disease responsible for tooth loss. MicroRNAs have been proven crucial in bone disorders over the past decades. Promotive effect on osteogenic activities by microRNA-335-5p (miR-335-5p) has been well demonstrated, but its role involved in the pathogenesis of periodontitis remains elusive. In this study, we established experimental periodontitis (EP) on transgenic mice overexpressing miR-335-5p (335-Tg) to investigate the novel effects of miR-335-5p on periodontal inflammation and bone loss. METHODS: Experimental periodontitis was established via ligation. The expression of inflammatory and osteoclastic genes was examined by quantitative real-time PCR (qPCR). Morphology of alveolar bone was analyzed by microcomputed tomography (µCT). Hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and Toll-like receptor 4 (TLR4) immunohistochemistry (IHC) staining were conducted for histological analysis. RESULTS: The expression of miR-335-5p decreased significantly in the periodontal tissues of EP. Compared to the WT-EP group, µCT analysis showed less bone loss in the 335-Tg-EP group accompanying with a decreased number of TRAP-positive osteoclasts. H&E and IHC staining exhibited attenuated inflammation and TLR4 expression in the 335-Tg-EP group. Furthermore, reduced expressions of IL-1ß, IL-6, TNF-α, and TLR4 were also detected in the 335-Tg-EP group. Overexpression of miR-335-5p in vivo weakened the periodontal bone destruction and inflammation compared with the WT-EP group. CONCLUSIONS: Our data exhibit novel roles of miR-335-5p in preventing bone loss and inflammation in experimental periodontitis.

Central adiponectin induces trabecular bone mass partly through epigenetic downregulation of cannabinoid receptor CB1.

Central adiponectin (APN) in either the globular (gAPN) or full-length forms decreases sympathetic tone and increases trabecular bone mass in mice through the hypothalamus. It is known that cannabinoid type-1 (CB1) receptors are expressed in the hypothalamic ventromedial nucleus and participate in energy metabolism by controlling sympathetic activity. However, whether central APN could influence endocannabinoid signaling through CB1 receptor to regulate bone metabolism has not been characterized. Here we demonstrate that gAPN downregulated CB1 expression in embryonic mouse hypothalamus N1 cells in vitro. gAPN intracerebroventricular (icv) infusions also decreased hypothalamic CB1 expression and bone formation parameters in APN-knockout (APN-KO) and wild-type mice. Most importantly, mice pretreated with icv infusions with the CB1 receptor agonist arachidonyl-2'-chloroethylamine or antagonist rimonabant attenuated or enhanced respectively central APN induction of bone formation. We then investigated whether epigenetic signaling mechanisms were involved in the downregulation of hypothalamic CB1 expression by gAPN. We found gAPN enhanced expression levels of various histone deacetylases (HDACs), especially HDAC5. Furthermore, chromatin immunoprecipitation assays revealed HDAC5 bound to the transcriptional start site transcription start site 2 region of the CB1 promoter. In summary, our study identified a possible novel central APN-HDAC5-CB1 signaling mechanism that promotes peripheral bone formation through epigenetic regulation of hypothalamic CB1 expression.

MicroRNA-99a is a novel regulator of KDM6B-mediated osteogenic differentiation of BMSCs.

Skeletal tissue originates from mesenchymal stem cells (MSCs) with differentiation potential into the osteoblast lineage regulated by essential transcriptional and post-transcriptional mechanisms. Recently, miRNAs and histone modifications have been identified as novel key regulators of osteogenic differentiation of MSCs. Here, we identified miR-99a and its target lysine (K)-specific demethylase 6B (KDM6B) gene as novel modulators of osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Microarray profiling and further validation by quantitative real-time RT-PCR revealed that miR-99a was up-regulated during osteoblastic differentiation of BMSCs, and decreased in differentiated osteoblasts. Transfection of miR-99a mimics inhibited osteoblastic commitment and differentiation of BMSCs, whereas inhibition of miR-99a by inhibitors enhances these processes. KDM6B was determined as one of important targets of miR-99a, which was further confirmed by luciferase assay of 3'-UTR of KDM6B. Moreover, HOX gene level decreased after transfection of miR-99a mimics in BMSCs, which indicated that KDM6B is a bona fide target of miR-99a. Furthermore, in a model of in vivo bone regeneration, osteoblast-specific gain- and loss-of-function experiments performed using cranial bone defects revealed that miR-99a mimics-transfected BMSCs reduced bone formation, and conversely, miR-99a inhibitors-transfected BMSCs increased in vivo bone formation. Tissue-specific inhibition of miR-99a may be a potential novel therapeutic approach for enhancing BMSCs-based bone formation and regeneration.

Runx2/DICER/miRNA Pathway in Regulating Osteogenesis.

DICER is the central enzyme that cleaves precursor microRNAs (miRNAs) into 21-25 nucleotide duplex in cell lineage differentiation, identity, and survival. In the current study, we characterized the specific bone metabolism genes and corresponding miRNAs and found that DICER and Runt-related transcription factor 2 (Runx2) expressions increased simultaneously during osteogenic differentiation. Luciferase assay showed that Runx2 significantly increased the expression levels of DICER luciferase promoter reporter. Our analysis also revealed weaker DICER expression in embryos of Runx2 knock out mice (Runx2 -/-) compared with that of Runx2 +/- and Runx2 +/+ mice. We further established the calvarial bone critical-size defect (CSD) mouse model. The bone marrow stromal cells (BMSCs) transfected with siRNA targeting DICER were combined with silk scaffolds and transplanted into calvarial bone CSDs. Five weeks post-surgery, micro-CT analysis revealed impaired bone formation, and repairing in calvarial defects with the siRNA targeting DICER group. In conclusion, our results suggest that DICER is specifically regulated by osteogenic master gene Runx2 that binds to the DICER promoter. Consequently, DICER cleaves precursors of miR-335-5p and miR-17-92 cluster to form mature miRNAs, which target and decrease the Dickkopf-related protein 1 (DKK1), and proapoptotic factor BIM levels, respectively, leading to an enhanced Wnt/ß-catenin signaling pathway. These intriguing results reveal a central mechanism underlying lineage-specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. Our study, also suggests a potential application of modulating DICER expression for bone tissue repair and regeneration. J. Cell. Physiol. 232: 182-191, 2017. © 2016 Wiley Periodicals, Inc.

Epigenetic Modulation in Periodontitis: Interaction of Adiponectin and JMJD3-IRF4 Axis in Macrophages.

Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down-regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down-regulated in obesity, functions to switch macrophage polarization from M1 to M2, thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up-regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up-regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain-function experiments of APN in vivo with APN(-/-) mice with experimental periodontitis and in vitro with macrophage isolated from APN(-/-) mice. For the first time, we found that APN can help to reduce periodontitis-related bone loss, modulate JMJD3 and IRF4 expression, and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti-inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage-centered epigenetic therapeutic strategies.

Adiponectin regulates bone marrow mesenchymal stem cell niche through a unique signal transduction pathway: an approach for treating bone disease in diabetes.

Adiponectin (APN) is an adipocyte-secreted adipokine that exerts well-characterized antidiabetic properties. Patients with type 2 diabetes (T2D) are characterized by reduced APN levels in circulation and impaired stem cell and progenitor cell mobilization from the bone marrow for tissue repair and remodeling. In this study, we found that APN regulates the mobilization and recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) to participate in tissue repair and regeneration. APN facilitated BMSCs migrating from the bone marrow into the circulation to regenerate bone by regulating stromal cell-derived factor (SDF)-1 in a mouse bone defect model. More importantly, we found that systemic APN infusion ameliorated diabetic mobilopathy of BMSCs, lowered glucose concentration, and promoted bone regeneration in diet-induced obesity mice. In vitro studies allowed us to identify Smad1/5/8 as a novel signaling mediator of APN receptor (AdipoR)-1 in BMSCs and osteoblasts. APN stimulation of MC3T3-E1 osteoblastic cells led to Smad1/5/8 phosphorylation and nuclear localization and increased SDF-1 mRNA expression. Although APN-mediated phosphorylation of Smad1/5/8 occurred independently from adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1, it correlated with the disassembly of protein kinase casein kinase 2 and AdipoR1 in immunoprecipitation experiments. Taken together, this study identified APN as a regulator of BMSCs migration in response to bone injury. Therefore, our findings suggest APN signaling could be a potential therapeutic target to improve bone regeneration and homeostasis, especially in obese and T2D patients.

Central adiponectin administration reveals new regulatory mechanisms of bone metabolism in mice.

Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.

Induction of B7-H1 expression by human cytomegalovirus in extravillous cytotrophoblast cells and role of MAPK pathway.

OBJECTIVE: This paper is aimed at to evaluate B7-H1 expression as induced by human cytomegalovirus (HCMV) in extravillous cytotrophoblast cell line HPT-8 and possible underlying mechanism. METHOD: Real time PCR and flow cytometry were used to determine B7-H1 mRNA and protein before and after HCMV infection in HPT-8 cells. Western blot analysis was used to determine the level of MAPK phosphorylation in HPT-8 cell lines infected with HCMV. RESULTS: 100TCID50 was found to be the most effective dose, capable of stimulating B7-H1 mRNA and protein expression in HPT-8 cells. When empty control group was considered to have a B7-H1 mRNA value of 1, B7-H1 mRNA was 4.32 in 100TCID50 group. In flow cytometry study, mean fluorescence intensity (MFI) of 100TCID50 group was 16.14, while empty control group was 1.34. Both mRNA and protein expression were found to be significantly increased (P<0.05) in 100TCID50 group compared to empty control group. The result of Western blot analysis showed increase in B7-H1 expression caused by the extracellular signaling that was related to ERK activation and the ERK inhibitor U0126 was found to reverse this increase. CONCLUSION: HCMV upregulates B7-H1 expression in human extravillous cytotrophoblast cell line HPT-8, which is related to MAPK activation. Our result would be helpful in finding better therapies against intrauterine HCMV infection.

Establishment of a novel cellular model for Alzheimer's disease in vitro studies.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss, cognitive impairment, and behavioral and psychological symptoms of dementia. The limited efficacy of drugs for the treatment of neurodegenerative diseases reflects their complex etiology and pathogenesis. A novel in vitro model may help to bridge the gap between existing preclinical animal models and human clinical trials, thus identifying promising therapeutic targets that can be explored in upcoming clinical trials. By assisting in the identification of the mechanism of action and potential dangers, in vitro testing can also shorten the time and expense of translation. AIM: As a result of these factors, our objective is to develop a powerful and informative cellular model of AD within a short period of time. Through triggering the MAPK and NF-κß signaling pathways with the aid of small chemical compounds (PAF C-16 and BetA), respectively, in mouse microglial (SIM-A9) and neuroblast Neuro-2a (N2a) cell lines. RESULTS: PAF C-16, initiated an activation effect at a concentration of 3.12 nM to 25 nM in the SIM-A9 and N2a cell lines after 72 h. BetA, activated the NF-κß pathway with a concentration of 12.5 nM to 25 nM in the SIM-A9 and N2a cell lines after 72 h. The combination of the activator chemicals provided suitable activation for MEK1/2-ERK and NF-κß in more than three subcultures. Activators significantly initiate APP and MAPT gene expression, as well as the expression of proteins APP, ß. Amyloid, tau, and p-tau. The activation of the targeted pathways leads to significant morphological changes. CONCLUSION: We can infer that the MEK1/2-ERK and NF-κß pathways, respectively, are directly activated by the PAF C-16 and BetA chemicals. The activation of MEK1/2-ERK pathway results in the activation of the APP gene, which in turn activates the ß. Amyloid protein, which in turn results in plaque. Furthermore, NF-κß activation results in the activation of the MAPT gene, which leads to Tau and p-Tau protein activation, which ultimately results in tangles. This can be put into practice in just three days, with a high level of activity and stability that is passed down to the next three generations (subculture), with significant morphological changes. In microglial and neuroblast cell lines, we were successful in creating a novel AD-cell model.

Adiponectin mRNA Conjugated with Lipid Nanoparticles Specifically Targets the Pathogenesis of Type 2 Diabetes.

Type 2 diabetes (T2D) is a widespread health condition both in the United States and around the world, with insulin resistance playing a critical role in its development. Effective treatment strategies are essential for managing T2D and mitigating associated risks. Adiponectin (APN), secreted by adipocytes, exhibits an inverse correlation with obesity-related adiposity, and its levels are negatively associated with insulin resistance and body mass index. This study aimed to enhance endogenous APN levels in a diet-induced obese (DIO) mouse model using lipid nanoparticles (LNP) as safe delivery agents for APN mRNA conjugates. The results indicate that APN-mRNA-LNP administration successfully induced APN synthesis in various tissues, including muscle, liver, kidney, pancreas, and adipose cells. This induction was associated with several positive outcomes, such as preventing diet-induced body weight gain, improving hyperglycemia by promoting Glut-4 expression, alleviating diabetic nephropathy symptoms by blocking the EGFR pathway, and reducing pro-inflammatory cytokine production. In addition, the treatment demonstrated enhanced insulin sensitivity by activating DGKd and inhibiting PKCε. This resulted in reactivation of insulin receptors in insulin target tissues and stimulation of insulin secretion from pancreatic beta cells. The findings of the present study highlight the potential of APN-mRNA-LNP-based nucleic acid therapy as a treatment for type 2 diabetes, offering a comprehensive approach to addressing its complexities.

AdipoRon accelerates bone repair of calvarial defect in diet-induced obesity mice.

Objectives: To investigate the role of AdipoRon in bone wound healing of calvaria critical-sized defects (CSD) in diet-induced obesity (DIO) mice. Materials and methods: After establishing the calvaria CSD in normal-chow (NC), DIO and Adiponectin knockout (APNKO) mice, AdipoRon or vehicle was orally gavaged for 3 weeks. The bone defects were analyzed by micro-CT and H&E staining. The expression of osteogenesis-related factor in the defect area, and the chemotactic gradient of SDF-1 between bone marrow and bone defect area were further analyzed. Results: AdipoRon downregulated body weight and alleviated fasting blood glucose level of DIO mice after treatment with AdipoRon in 14 and 21 days. Newly formed bone was significantly increased in the defect area of DIO and APNKO mice after treatment with AdipoRon compared with vehicle treatment. No significant difference was shown in NC mice. Furthermore, compared with NC mice, a significant decrease of BV/TV%, Tb.N value and formed bone percentage were shown in DIO and APNKO mice. The treatment with AdipoRon could reverse of decreased value and increase the newly formed bone in those mice. AdipoRon promoted col-1α expression in wound sites in DIO and APNKO mice. AdipoRon nearly quadrupled the chemotactic gradient of SDF-1 by decreasing SDF-1 expression in bone marrow and increasing it in the bone defect area in APNKO and DIO treated mice. Conclusion: AdipoRon alleviates the obesity status in DIO mice with calvarial defect and increase new bone formation in calvarial defects in DIO and APNKO mice by modulating chemotactic gradient of SDF-1.

The adiponectin receptor agonist AdipoAI attenuates periodontitis in diabetic rats by inhibiting gingival fibroblast-induced macrophage migration.

BACKGROUND AND PURPOSE: Low-grade inflammation, a common feature of both diabetes and periodontitis, partly accounts for the complexity and refractoriness of diabetes-associated periodontitis. Adiponectin (APN), the most abundant adipokine in human blood, has been widely reported to have anti-inflammatory functions. Herein, we investigated the ability of an APN receptor agonist, AdipoAI, to alleviate diabetes-associated periodontitis. Furthermore, we revealed the possible mechanism underlying its anti-inflammatory effects. EXPERIMENTAL APPROACH: The maxillary first molar of Zucker diabetic fatty (ZDF) rats was ligated to construct a diabetes-associated periodontitis model, and rats were administered AdipoAI by gavage. We examined diabetes-related indexes, pathological changes in insulin target organs, alveolar bone resorption and systemic and local inflammation. In vitro, transwell assays were used to evaluate monocyte/macrophage migration induced by human gingival fibroblasts (hGFs) with/without AdipoAI treatment. Additionally, we examined chemokine expression levels in hGFs and hGF-induced monocyte/macrophage migration upon siRNA knockdown of Adiponectin receptor expression. Expression of Adipo1/Adipo2 receptors and inflammation-related signalling pathways were examined by IHC and WB, followed by confirmation with an NF-κB P65 inhibitor (BAY 11-7082). KEY RESULTS: AdipoAI lowered fasting blood glucose and serum insulin in ZDF rats and alleviated inflammation in insulin target tissues. Locally, AdipoAI reduced alveolar bone absorption and gingival inflammation. Mechanistically, AdipoAI inhibited hGF-induced monocyte/macrophage migration by reducing CCL2 secretion. In hGFs, AdipoAI attenuated LPS-induced activation of NF-κB P65 and CCL2 expression, which was dependent on the Adipo receptor 1. CONCLUSION AND IMPLICATIONS: AdipoAI, with its ability to alleviate inflammatory damage in tissues, is a candidate for diabetes-associated periodontitis treatment.

A novel lncRNA-mediated epigenetic regulatory mechanism in periodontitis.

Periodontitis is a highly prevalent chronic inflammatory disease with an exaggerated host immune response, resulting in periodontal tissue destruction and potential tooth loss. The long non-coding RNA, LncR-ANRIL, located on human chromosome 9p21, is recognized as a genetic risk factor for various conditions, including atherosclerosis, periodontitis, diabetes, and cancer. LncR-APDC is an ortholog of ANRIL located on mouse genome chr4. This study aims to comprehend the regulatory role of lncR-APDC in periodontitis progression. Our experimental findings, obtained from lncR-APDC gene knockout (KO) mice with induced experimental periodontitis (EP), revealed exacerbated bone loss and disrupted pro-inflammatory cytokine regulation. Downregulation of osteogenic differentiation occurred in bone marrow stem cells harvested from lncR-APDC-KO mice. Furthermore, single-cell RNA sequencing of periodontitis gingival tissue revealed alterations in the proportion and function of immune cells, including T and B cells, macrophages, and neutrophils, due to lncR-APDC silencing. Our findings also unveiled a previously unidentified epithelial cell subset that is distinctively presenting in the lncR-APDC-KO group. This epithelial subset, characterized by the positive expression of Krt8 and Krt18, engages in interactions with immune cells through a variety of ligand-receptor pairs. The expression of Tff2, now recognized for its role in chronic inflammatory conditions, exhibited a notable increase across various tissue and cell types in lncR-APDC deficient mice. Additionally, our investigation revealed the potential for a direct binding interaction between lncR-APDC and Tff2. Intra-gingival administration of AAV9-lncR-APDC was shown to have therapeutic effects in the EP model. In conclusion, our results suggest that lncR-APDC plays a critical role in the progression of periodontal disease and holds therapeutic potential for periodontitis. Furthermore, the presence of the distinctive epithelial subpopulation and significantly elevated Tff2 levels in the lncR-APDC-silenced EP model offer new perspectives on the epigenetic regulation of periodontitis pathogenesis.

Adiponectin inhibits osteoclastogenesis and bone resorption via APPL1-mediated suppression of Akt1.

Adiponectin is an adipokine playing an important role in regulating energy homeostasis and insulin sensitivity. However, the effect of adiponectin on bone metabolism shows contradictory results according to different research studies. In this study femurs were isolated from genetically double-labeled mBSP9.0Luc/ß-ACT-EGFP transgenic mice and were transplanted into adiponectin knock-out mice or wild type mice to investigate the effect of temporary exposure to adiponectin deficiency on bone growth and metabolism. We found that the growth of bone explants in adiponectin knock-out mice was significantly retarded. Histological analysis, microcomputed tomography analysis, and tartrate-resistant acid phosphatase staining revealed reduced trabecular bone volume, decreased cortical bone, and increased osteoclast number in bone explants in adiponectin knock-out mice. We then found that adiponectin inhibits RANKL-induced osteoclastogenesis from RAW264.7 cells and down-regulates RANKL-enhanced expressions of osteoclastogenic regulators including NFAT2, TRAF6, cathepsin K, and tartrate-resistant acid phosphatase. Adiponectin also increases osteoclast apoptosis and decreases survival/proliferation of osteoclast precursor cells. Using siRNA specifically targeting APPL1, the first identified adaptor protein of adiponectin signaling, we found that the inhibitory effect of adiponectin on osteoclasts was induced by APPL1-mediated down-regulation of Akt1 activity. In addition, overexpression of Akt1 successfully reversed adiponectin-induced inhibition in RANKL-stimulated osteoclast differentiation. In conclusion, adiponectin is important in maintaining the balance of energy metabolism, inflammatory responses, and bone formation.