Cold induces brain region-selective cell activity-dependent lipid metabolism.

It has been well documented that cold is an enhancer of lipid metabolism in peripheral tissues, yet its effect on central nervous system lipid dynamics is underexplored. It is well recognized that cold acclimations enhance adipocyte functions, including white adipose tissue (WAT) lipid lipolysis and beiging, and brown adipose tissue (BAT) thermogenesis in mammals. However, it remains unclear whether and how lipid metabolism in the brain is also under the control of cold acclimations. Here, we show that cold exposure predominantly increases the expressions of the lipid lipolysis genes and proteins within the paraventricular nucleus of the hypothalamus (PVH). Mechanistically, we find that cold activates cells within the PVH and pharmacological inactivation of cells blunts cold-induced effects on lipid peroxidation, accumulation of lipid droplets (LDs), and lipolysis in the PVH. Together, these findings suggest that PVH lipid metabolism is cold sensitive and integral to cold-induced broader regulatory responses.

Magel2 knockdown in hypothalamic POMC neurons innervating the medial amygdala reduces susceptibility to diet-induced obesity.

Hyperphagia and obesity profoundly affect the health of children with Prader-Willi syndrome (PWS). The Magel2 gene among the genes in the Prader-Willi syndrome deletion region is expressed in proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC). Knockout of the Magel2 gene disrupts POMC neuronal circuits and functions. Here, we report that loss of the Magel2 gene exclusively in ARCPOMC neurons innervating the medial amygdala (MeA) causes a reduction in body weight in both male and female mice fed with a high-fat diet. This anti-obesity effect is associated with an increased locomotor activity. There are no significant differences in glucose and insulin tolerance in mice without the Magel2 gene in ARCPOMC neurons innervating the MeA. Plasma estrogen levels are higher in female mutant mice than in controls. Blockade of the G protein-coupled estrogen receptor (GPER), but not estrogen receptor-α (ER-α), reduces locomotor activity in female mutant mice. Hence, our study provides evidence that knockdown of the Magel2 gene in ARCPOMC neurons innervating the MeA reduces susceptibility to diet-induced obesity with increased locomotor activity through activation of central GPER.

Overexpressing the hydroxycarboxylic acid receptor 1 in mouse brown adipose tissue restores glucose tolerance and insulin sensitivity in diet-induced obese mice.

Interscapular brown adipose tissue (BAT) plays an important role in controlling glucose homeostasis. Increased glucose entry and glycolysis in BAT result in lactate production and release. The adipose tissue expresses the lactate receptor hydrocarboxylic acid receptor 1 (HCAR1), markedly downregulated in male diet-induced obese (DIO) and ob/ob mice. In this study, we examined the role of HCAR1 in BAT in controlling glucose homeostasis in male DIO mice. We overexpressed HCAR1 in BAT by injecting adeno-associated viruses (AAVs) expressing HCAR1 into the BAT pads of male DIO C57BL/6J mice. Overexpressing HCAR1 in BAT resulted in augmented glucose uptake by BAT in response to treatment with the HCAR1 agonist. HCAR1 overexpression elevated BAT temperature associated with increased thermogenic gene expression in BAT. HCAR1 overexpression prevented body weight gain in male DIO mice. Importantly, mice overexpressing HCAR1 in BAT exhibited improved glucose tolerance and insulin sensitivity. HCAR1 overexpression upregulated the Slc2a4 gene expression and promoted GLUT4 trafficking to the plasma membrane. In addition, mice overexpressing HCAR1 displayed a decrease in hormone-sensitive lipase (HSL) phosphorylation and increased lipogenic enzyme gene expression in BAT. Unlike DIO mice, overexpressing HCAR1 in BAT of mice fed a low-fat diet did not change body weight gain and glucose homeostasis. Taken together, our results support the interpretation that HCAR1 expressed in BAT promotes glucose entry and reduces lipolysis in BAT of male DIO mice. As activation of HCAR1 in BAT restores body weight, glucose tolerance, and insulin sensitivity in male DIO mice, our study suggests that interoceptive lactate detection via HCAR1 in BAT can regulate glucose and lipid substrate utilization and/or availability to promote healthy metabolism.NEW & NOTEWORTHY HCAR1 expressed in BAT can promote glucose entry and reduce lipolysis, resulting in body weight loss and increased insulin sensitivity. Hence, targeting HCAR1 in BAT would provide an alternative way to control body weight and euglycemia in individuals with obesity.

Zingerone Attenuates Pi-induced Vascular Calcification via AMPK-mediated TIMP4 Expression.

OBJECTIVE: Vascular calcification requires the differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. This phenomenon can be enhanced by inflammation and oxidative stress. Zingerone is one of the active ingredients present in the ginger plant that has anti-inflammatory and antioxidant effects. Other functions include anti-obesity, anti-nausea effects. However, the functions of zingerone on vascular calcification has not yet been elucidated. This study investigated the effect of zingerone on vascular calcification and its molecular mechanism. METHODS: Reverse transcription-polymerase chain reaction (PCR), real-time PCR and Western blot analysis was used to measure expression levels of osteogenic marker genes and to investigate whether calcification was regulated by the expression of AMP-activated protein kinase (AMPK) and tissue inhibitor of metalloproteinase 4 (TIMP4). Alizarin red S staining was used to measure calcium deposition. Studies were carried out in VSMCs. RESULTS: Zingerone induced the expression of 2 markers of VSMCs differentiation (α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α)) and decreased the expression of core-binding factor α-1 (CBFA1). Additionally, zingerone decreased inorganic phosphate (Pi)-induced expression of distal-less homeobox 5 and CBFA1. AMPK phosphorylation and TIMP4 expression were increased by zingerone. Importantly, zingerone protected VSMCs from calcification, and this protective effect was confirmed by increased TIMP4 via overexpression of AMPK, and inhibition of TIMP4 by Compound C. Zingerone upregulated AMPK/TIMP4 expression and recovered Pi-induced inhibition of TIMP4. CONCLUSIONS: Taken together, our results show that zingerone inhibits Pi-induced vascular calcification by regulating the AMPK/TIMP4 signaling cascade in VSMCs. These results suggest that the natural product zingerone could be useful for treating vascular and metabolic diseases.

Vitexin enhances osteoblast differentiation through phosphorylation of Smad and expression of Runx2 at in vitro and ex vivo.

Vitexin (apigenin-8-C-d-glucopyranoside) is a flavonoid isolated from natural sources. It has been employed as an anti-oxidant, anti-inflammatory, and anti-cancer agent, and is used as a traditional Chinese medicine to treat a variety of illnesses. The present study investigated the effect of vitexin on osteoblast differentiation of C3H10T1/2 mesenchymal stem cells, MC3T3-E1 preosteoblast, mouse calvarial primary cells, and primary bone marrow stem cells (BMSCs). RT-PCR and quantitative PCR demonstrated that vitexin increased mRNA expression of the osteogenic genes distal-less homeobox 5 (Dlx5) and Runxt-related transcription factor 2 (Runx2). Vitexin also increased the Dlx5 and Runx2 protein levels, Smad1/5/9 phosphorylation, and alkaline phosphatase (ALP) activity. In addition, vitexin increased Runx2-luciferase activity. Moreover, knockdown of Runx2 attenuated the increase in ALP activity induced by vitexin. These results demonstrate that vitexin enhances osteoblast differentiation via Runx2.

Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop with p-AMPK.

Fat mass and obesity-associated (FTO) gene helps to regulate energy homeostasis in mammals by controlling energy expenditure. In addition, FTO functions in the regulation of obesity and adipogenic differentiation; however, a role in osteogenic differentiation is unknown. This study investigated the effects of FTO on osteogenic differentiation of C3H10T1/2 cells and the underlying mechanism. Expression of osteogenic and endoplasmic reticulum (ER) stress markers were characterized by reverse-transcriptase polymerase chain reaction and western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity. BMP2 treatment increased mRNA expression of osteogenic genes and FTO. Overexpression of FTO increased expression of the osteogenic genes distal-less homeobox5 (Dlx5) and runt-related transcription factor 2 (Runx2). Activation of adenosine monophosphate-activated protein kinase (AMPK) increased FTO expression, and there was a positive feedback loop between FTO and p-AMPK. p-AMPK and FTO induced mild ER stress; however, tunicamycin-induced severe ER stress suppressed FTO expression and AMPK activation. In summary, FTO induces osteogenic differentiation of C3H10T1/2 cells upon BMP2 treatment by inducing mild ER stress via a positive feedback loop with p-AMPK. FTO expression and AMPK activation induce mild ER stress. By contrast, severe ER stress inhibits osteogenic differentiation by suppressing FTO expression and AMPK activation.

Alpha-pinene promotes osteoblast differentiation and attenuates TNFα-induced inhibition of differentiation in MC3T3-E1 pre-osteoblasts.

Alpha-pinene (α-pinene) is an organic compound, found in the oils of many species of coniferous trees, especially pine. α-Pinene reportedly has antioxidant and anti-inflammatory activities; however, its effects on osteoblasts are unknown. This study investigated the effects of α-pinene on osteoblast differentiation and tumour necrosis factor-alpha (TNFα)-induced inhibition of osteogenesis. Culture in control or osteogenic medium containing α-pinene increased osteogenic marker expression. Alkaline phosphatase staining and alizarin red S staining confirmed that α-pinene enhanced osteoblast differentiation. Also, α-pinene attenuated TNFα-induced inhibition of Smad1/5/9 phosphorylation and extracellular matrix mineralization. Taken together, our findings suggest that α-pinene enhances osteoblast differentiation and mineralization in MC3T3-E1 pre-osteoblasts.

Estradiol-induced RORα expression positively regulates osteoblast differentiation.

The retinoic acid receptor-related orphan receptor alpha (RORα) is a member of the nuclear hormone receptor superfamily. Several studies show that estradiol is related to RORα expression. However, the link between estradiol and RORα in osteoblast differentiation remains unknown. Here, we showed that estradiol induces RORα expression in C3H10T1/2 and MC3T3-E1 cells. RORα overexpression increased the expression of osteogenic genes including bone morphogenetic protein 2 (BMP2), distal-less homeobox 5, inhibitor of DNA binding, runt-related transcription factor 2 (Runx2), and osteocalcin. In addition, RORα increased phosphorylation of smad1/5/9. Furthermore, RORα knockdown suppressed estradiol-induced BMP2 and Runx2 protein level. Also, we confirmed that estradiol-induced ALP staining and matrix mineralization was attenuated in RORα knockdown. Summarily, these results suggest that estradiol-induced RORα promotes osteoblast differentiation.

OVO homologue-like 1 promotes osteoblast differentiation through BMP2 expression.

OVO homologue-like 1 (OVOL1) encodes a C2H2 zinc finger protein and is an evolutionarily conserved gene in mammals. The OVOL1 expression is required for development. However, the function of OVOL1 in bone metabolism remains unreported. Here, we show for the first time the role of OVOL1 in osteoblast differentiation. To determine the role of OVOL1 in osteogenic differentiation, we analyzed OVOL1 expression in the preosteoblastic cell line. OVOL1 messenger RNA expression was induced during osteoblast differentiation. In addition, OVOL1 overexpression enhanced the expression of osteogenic genes including bone morphogenetic protein 2 (BMP2), the inhibitor of DNA binding 1 (Id1), distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), osteocalcin (OC), and alkaline phosphatase (ALP). Moreover, mineralization of the extracellular matrix was increased by OVOL1 overexpression in MC3T3-E1 cells. Furthermore, knockdown of the OVOL1 experiment demonstrated that OVOL1 is required for osteoblast differentiation. Collectively, these results suggest that OVOL1 function as an important regulator of osteoblast differentiation by inducing BMP2 expression in MC3T3-E1 cells.

Bmal1 induces osteoblast differentiation via regulation of BMP2 expression in MC3T3-E1 cells.

AIMS: Mammalian circadian rhythms regulate many metabolic processes. Recent studies suggest that brain and muscle Arnt-like 1 (BMAL1), an important component of mammalian circadian rhythm, is associated with insulin signaling. Several studies have shown that insulin is associated with bone metabolism; however, the relationship between BMAL1 and osteoblasts remains unclear. MAIN METHODS: Expression of osteogenic markers and Bmal1 in MC3T3-E1 cells was measured by RT-PCR and Western blotting. Alizarin red S staining was performed to assess matrix mineralization in MC3T3-E1 cells. KEY FINDINGS: mRNA levels of osteogenic genes and Bmal1 were up-regulated in MC3T3-E1 cells upon insulin treatment. In addition, Bmal1 overexpression increased the expression of osteogenic genes including inhibitor of DNA binding (Id1), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC). Interestingly, expression of Bone morphogenetic protein-2 (BMP2), an important upstream factor of Id1, Runx2, and OC, was markedly increased by Bmal1. Finally, we confirmed that insulin-induced BMP2 expression was attenuated in Bmal1 knockout (KO) cells. PCR analysis and alizarin red S staining showed that insulin-mediated increases gene expression and calcium deposition were reduced in Bmal1 KO cells compared to wild-type cells. SIGNIFICANCE: Taken together, these results demonstrate that Bmal1 promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells.