Weijin Zang

Osteocalcin reverses endoplasmic reticulum stress and improves impaired insulin sensitivity secondary to diet-induced obesity through nuclear factor-κB signaling pathway.

Osteocalcin, a synthetic osteoblast-specific protein, has recently emerged as an important regulator of energy metabolism, but the underlying mechanisms are not fully understood. In the present study, mice fed a high-fat diet and receiving osteocalcin showed reduced body weight gain, less fat pad gain, and improved insulin sensitivity as well as increased energy expenditure compared with mice fed a high-fat diet and receiving vehicle. Meanwhile, increased endoplasmic reticulum (ER) stress, defective insulin signaling, and mitochondrial dysfunction induced by obesity were also effectively alleviated by treatment with osteocalcin. Consistent with these findings, the addition of osteocalcin to the culture medium of 3T3-L1 adipocytes, Fao liver cells, and L6 muscle cells markedly reduced ER stress and restored insulin sensitivity. These effects were nullified by blockade of nuclear factor-κB (NF-κB) or phosphatidylinositol 3-kinase but not by U0126, a mitogen-activated protein kinase inhibitor, indicating the causative role of phosphatidylinositol 3-kinase/NF-κB in action of osteocalcin. In addition, the reversal effects of osteocalcin in cells deficient in X-box-binding protein-1, a transcription factor that modulates ER stress response, further confirmed its protective role against ER stress and insulin resistance. Our findings suggest that osteocalcin attenuates ER stress and rescues impaired insulin sensitivity in insulin resistance via the NF-κB signaling pathway, which may offer novel opportunities for treatment of obesity and diabetes.

Dehydroepiandrosterone-induced proliferation of prostatic epithelial cell is mediated by NFKB via PI3K/AKT signaling pathway.

Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. In this study, rat ventral prostatic epithelial cells were used to determine whether DHEA-modulated proliferation and prostate-specific antigen (PSA listed as KLKB1 in the MGI Database) production were mediated via the androgen receptor (AR) and its potential mechanism. We demonstrated that proliferation of prostatic epithelial cells and increase of PSA expression induced by DHEA were neutralized by Casodex or Ar siRNA, two specific AR blockers. DHEA stimulated Nfkb DNA binding activity, with this effect being blunted by Casodex or Ar siRNA. Moreover, the inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT nullified the effects of DHEA on NFKB activation. These findings suggested that DHEA stimulated normal prostatic epithelial cell proliferation, and AR is involved in DHEA-induced PSA expression in normal prostatic epithelial cells. This stimulation effect induced by DHEA is mediated by the activation of NFKB via PI3K/AKT pathway.

Intermittent injections of osteocalcin reverse autophagic dysfunction and endoplasmic reticulum stress resulting from diet-induced obesity in the vascular tissue via the NFκB-p65-dependent mechanism

The osteoblast-specific secreted molecule osteocalcin behaves as a hormone-regulating glucose and lipid metabolism, but the role of osteocalcin in cardiovascular disease (CVD) is not fully understood. In the present study, we investigated the effect of osteocalcin on autophagy and endoplasmic reticulum (ER) stress secondary to diet-induced obesity in the vascular tissue of mice and in vascular cell models and clarified the intracellular events responsible for osteocalcin-mediated effects. The evidences showed that intermittent injections of osteocalcin in mice fed the high-fat diet were associated with a reduced body weight gain, decreased blood glucose and improved insulin sensitivity compared with mice fed the high-fat diet receiving vehicle. Simultaneously, the administration of osteocalcin not only attenuated autophagy and ER stress but also rescued impaired insulin signaling in vascular tissues of mice fed a high-fat diet. Consistent with these results in vivo, the addition of osteocalcin reversed autophagy and ER stress and restored defective insulin sensitivity in vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) in the presence of tunicamycin or in knockout XBP-1 (a transcription factor which mediates ER stress response) cells or in Atg7−/− cells. The protective effects of osteocalcin were nullified by suppression of Akt, mammalian target of rapamycin (mTOR) or nuclear factor kappa B (NFκB), suggesting that osteocalcin inhibits autophagy, ER stress and improves insulin signaling in the vascular tissue and cells under insulin resistance in a NFκB-dependent manner, which may be a promising therapeutic strategies of cardiovascular dysfunction secondary to obesity.

Effects of GH Secretagogues on Contractility and Ca2+ Homeostasis of Isolated Adult Rat Ventricular Myocytes

Ghrelin and its synthetic analogue hexarelin are specific ligands of GH secretagogue receptor (GHS-R) and induce a variety of cardiovascular protective and cardiac positive inotropic effects. The signaling system underlying immediate effects of both GHSs in cardiomyocytes remains undefined. In the present study, we investigated the immediate effects of GHSs on isolated ventricular myocyte shortening, intracellular Ca(2+) ([Ca(2+)](i)) transients, and the L-type Ca(2+) current (I(Ca,L)). Putative intracellular signalling cascades were studied with specific receptor and signalling blockers. In fresh isolated adult Wistar rat ventricular myocytes, GHSs produced a positive inotropic effect in a concentration-dependent manner and increased the amplitude of [Ca(2+)](i) transients and the I(Ca,L). The positive inotropic response was abolished by the GHS-R1a antagonist [D-Lys(3)]-GH-releasing peptide-6 (10 microm). GHS-induced increase in the I(Ca,L) was abolished by [D-Lys(3)]-GH-releasing peptide-6 and protein kinase C inhibitor, chelerythrine chloride (5 microm), but not by protein kinase A inhibitor, KT 5720 (10 microm). We conclude that hexarelin and ghrelin increase the I(Ca,L), through GHS-R1a receptor and protein kinase C signalling cascade, which contribute to its direct positive inotropic effect on cardiomyocytes.

Circulating PGRN Is Significantly Associated With Systemic Insulin Sensitivity and Autophagic Activity in Metabolic Syndrome

Progranulin (PGRN) is a secreted protein that has recently emerged as an important regulatory adipokine of glucose metabolism and insulin sensitivity. We report here that serum PGRN concentrations were significantly higher in patients with metabolic syndrome (MS) than in subjects without MS and correlated positively with body mass index, waist circumference, fasting insulin, fasting plasma glucose, glycated hemoglobin A1c, triglyceride, and homeostasis model assessment of insulin resistance, and were inversely related to high-density lipoprotein cholesterol and homeostasis model assessment of β cell function. Subgroup analysis in 32 subjects showed that elevated expression levels of PGRN were positively correlated with increased autophagy markers LC3 and Atg7 proteins in omental adipose tissue of subjects with MS. Consistent with these findings, the enhanced PGRN levels were also observed in multiple insulin-resistant cellular models, whereas PGRN-deficient adipocytes were more susceptible to insulin action and refractory to tunicamycin-induced autophagic disorders. PGRN remarkably attenuated insulin sensitivity, increased autophagic activity, and triggered endoplasmic reticulum (ER) stress in cultured human adipocytes, whereas these effects were nullified by reduction of ER stress with phenylbutyric acid chemical chaperone treatment. In addition, PGRN-induced ER stress and impaired insulin sensitivity were improved in TNFR1(-/-) cells, indicating a causative role of TNF receptor in the action of PGRN. Collectively, our findings suggest that circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in adipose tissue and support the notion that PGRN functions as a potential link between chronic inflammation and insulin resistance.

Serum Levels of Progranulin Are Closely Associated with Microvascular Complication in Type 2 Diabetes

Objective. Progranulin (PGRN) was recently introduced as a novel marker of chronic inflammatory response in obesity and type 2 diabetes capable of directly affecting the insulin signaling pathway. This study aimed to investigate the correlation between PGRN and type 2 diabetics with microvascular complications. Methods. PGRN serum levels and glucose metabolism related substance were measured in 84 type 2 diabetic patients with or without microangiopathies and 12 health persons. Further analyses of serum PGRN in different stages of diabetic microangiopathies were conducted. Results. Serum levels of PGRN were markedly higher in type 2 diabetic patients with microangiopathies. PGRN serum levels increased with the progress of diabetic microangiopathies with significantly highest values detectable in clinical diabetic nephropathy (CDN) and proliferative diabetic retinopathy (PDR) groups. Serum PGRN concentrations in all individuals positively and markedly correlated with systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), triglyceride (TG), urinary albumin excretion rate (UAER), blood urea nitrogen (BUN), creatinine (CRE), white blood cell (WBC), disease duration, IL-6, and TNF-α, while correlating negatively and significantly with eGFR. Multiple linear regression analysis showed that only UAER and CRE were independently associated with serum PGRN. Conclusion. PGRN might be considered as a marker for diabetic microangiopathy and its severity.

Growth Hormone Secretagogues Reduce Transient Outward K Current via Phospholipase C/Protein Kinase C Signaling Pathway in Rat Ventricular Myocytes

Endogenous ghrelin and its synthetic counterpart hexarelin are peptide GH secretagogues (GHS) that exert a positive ionotropic effect in the cardiovascular system. The mechanism by which GHS modulate cardiac electrophysiology properties to alter myocyte contraction is poorly understood. In the present study, we examined whether GHS regulates the transient outward potassium current (I(to)) as well as the putative intracellular signaling cascade responsible for such regulation. GHS and experimental agents were applied locally onto freshly isolated adult Sprague-Dawley rat ventricular myocytes and action potential morphology and I(to) was recorded using nystatin-perforated whole-cell patch-clamp recording technique. Under current clamp, ghrelin and hexarelin (10 nm) significantly prolonged action potential duration. Under voltage clamp, hexarelin and ghrelin inhibited I(to) in a concentration-dependent manner. This inhibition was abolished in the presence of the GHS receptor (GHS-R) antagonist [D-Lys(3)]GH-releasing peptide-6 (10 microm) and GHS-R1a-specific antagonist BIM28163 (1 microm). GHS-induced I(to) inhibition was totally reversed by the phospholipase C inhibitor U73122 (5 microm) and protein kinase C inhibitors GO6983 (1 microm) and calphostin C (0.1 microm) but not by the cAMP antagonist Rp-cAMP (100 microm) or the PKA inhibitor H89 (1 microm). We conclude that hexarelin and ghrelin activate phospholipase C and protein kinase C signaling cascade through the stimulation of the GHS-R, resulting in a decrease in the I(to) current and subsequent prolongation of action potential duration.

The reciprocal interaction between autophagic dysfunction and ER stress in adipose insulin resistance

Autophagy, a predominantly cytoprotective process, is an important regulator in diabetic metabolism and endoplasmic reticulum (ER) stress responses. However, the interaction and biological significance between autophagic imbalance and ER stress involved in insulin resistance remain not fully elucidated. In the present study, when compared with normal glucose tolerance (NGT) subjects, enhanced ER stress and pronounced protein and mRNA levels of the autophagic genes such as Atg7, LC3A, and LC3B were evident in adipose tissue of patients with type 2 diabetes. An increased number of autophagosomes and elevated autophagy flux in adipose explants incubated with lysomoal inhibitor were also observed in type 2 diabetes. In addition, adipocytes differentiation was significantly repressed by exogenous ER stress and defective autophagy in vitro. Tunicamycin-induced ER stress in adipocytes can trigger autophagic response and insulin insensitivity that was partially attributed to the upregulation of IRE1-JNK pathway, whereas autophagy deficiency resulted in ER stress and impaired insulin signaling, further supporting the crucial roles of autophagy in ER stress and insulin resistance. Moreover, disturbance of autophagy and insulin sensitivity induced by tunicamycin can be effectively corrected by the addition of osteocalcin in an NFκB-dependent manner in vitro. In conclusion, our results demonstrated a reciprocal functional interaction among autophagy, ER stress, and insulin signaling in adipose tissue of type 2 diabetes and adipocytes, supporting an adaptive role of autophagy-dependent mechanism in response to ER stress-induced insulin resistance in type 2 diabetes.

DHEA suppresses longitudinal bone growth by acting directly at growth plate through estrogen receptors.

Dehydroepiandrosterone (DHEA) is produced by the adrenal cortex and is the most abundant steroid in humans. Although in some physiological and pathological conditions the increased secretion of DHEA and its sulfated form is associated with accelerated growth rate and skeletal maturation, it is unclear whether DHEA can affect longitudinal bone growth and skeletal maturation by acting directly at the growth plate. In our study, DHEA suppressed metatarsal growth, growth plate chondrocyte proliferation, and hypertrophy/differentiation. In addition, DHEA increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, DHEA reduced chondrocyte proliferation and induced apoptosis. The DHEA-induced inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was nullified by culturing metatarsals with DHEA in the presence of ICI 182,780, an inhibitor of estrogen receptor, but not in the presence of Casodex, an inhibitor of androgen receptor. Lastly, nuclear factor-κB DNA binding activity was inhibited by the addition of DHEA in the medium of cultured chondrocyte. Our findings indicate that DHEA suppressed bone growth by acting directly at growth plate through estrogen receptor. Such growth inhibition is mediated by decreased chondrocyte proliferation and hypertrophy/differentiation and by increased chondrocyte apoptosis.

Proepithelin Stimulates Growth Plate Chondrogenesis via Nuclear Factor-κB-p65-dependent Mechanisms

Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.