Neuronal Androgen Receptor Regulates Insulin Sensitivity via Suppression of Hypothalamic NF-κB–Mediated PTP1B Expression

Clinical investigations highlight the increased incidence of metabolic syndrome in prostate cancer (PCa) patients receiving androgen deprivation therapy (ADT). Studies using global androgen receptor (AR) knockout mice demonstrate that AR deficiency results in the development of insulin resistance in males. However, mechanisms by which AR in individual organs coordinately regulates insulin sensitivity remain unexplored. Here we tested the hypothesis that functional AR in the brain contributes to whole-body insulin sensitivity regulation and to the metabolic abnormalities developed in AR-deficient male mice. The mouse model selectively lacking AR in the central nervous system and AR-expressing GT1-7 neuronal cells were established and used to delineate molecular mechanisms in insulin signaling modulated by AR. Neuronal AR deficiency leads to reduced insulin sensitivity in middle-aged mice. Neuronal AR regulates hypothalamic insulin signaling by repressing nuclear factor-κB (NF-κB)–mediated induction of protein-tyrosine phosphatase 1B (PTP1B). Hypothalamic insulin resistance leads to hepatic insulin resistance, lipid accumulation, and visceral obesity. The functional deficiency of AR in the hypothalamus leads to male mice being more susceptible to the effects of high-fat diet consumption on PTP1B expression and NF-κB activation. These findings suggest that in men with PCa undergoing ADT, reduction of AR function in the brain may contribute to insulin resistance and visceral obesity. Pharmacotherapies targeting neuronal AR and NF-κB may be developed to combat the metabolic syndrome in men receiving ADT and in elderly men with age-associated hypogonadism.Prostate cancer (PCa), one of the most frequently diagnosed malignancies in men in the Western world, represents 25% of cancers among men (1). Androgen deprivation therapy (ADT) is the fundamental management for men with locally confined, advanced, and metastatic PCa to suppress the functions of androgen/androgen receptor (AR) signaling using AR antagonists in conjunction with bilateral orchiectomy. Although ADT is the frontline and effective treatment for PCa, the resulting profound hypogonadism has adverse effects associated with metabolic syndrome and cardiovascular-related mortality (2–4). The accumulation of visceral adiposity during a short-term ADT period is associated with increasing insulin levels, which may be an initiating event leading to metabolic dysregulation (5). Men receiving long-term ADT treatment develop significant insulin resistance, hyperglycemia, and cardiovascular mortality compared with the non-ADT and control groups (2,6,7). These studies highlight the increased risk of metabolic syndrome, cardiovascular disease, and type 2 diabetes in men with PCa receiving ADT.Consistent with the relationship of decreased AR function with metabolic syndrome are previous studies demonstrating that genetic inactivation and global loss of AR (AR knockout [ARKO]) lead to the development of excess adiposity associated with insulin resistance and altered glucose homeostasis (8). As testosterone replacement cannot reverse the metabolic abnormalities and insulin resistance observed in ARKO male mice, this suggests that AR is critical in mediating the effects of androgens to regulate glucose and lipid homeostasis in males. Moreover, male mice with hepatic-specific AR deletion more rapidly develop hepatic steatosis and insulin resistance induced by high-fat diet (HFD) feeding and age (9). These findings provide strong evidence that functional deficiency of AR leads to insulin resistance in male mice.Compelling evidence is mounting that the brain is an insulin target organ that plays a key role in glucose homeostasis and energy balance. Central insulin resistance is suggested to participate critically in the pathophysiology of obesity, type 2 diabetes, and related metabolic disorders (10–12). Differential sensitivity to exogenous insulin in the male and female central nervous system has been observed in animals and humans (13–15). Male rats decrease their food intake and body weight when receiving intracerebroventricular insulin administration, whereas female rats remain largely unaffected (13). Analogous studies have been reported for humans using an intranasal route of insulin delivery, showing that men, but not women, decrease body weight and body fat after 8 weeks of intranasal insulin (14). Moreover, a single dose of insulin reduces food intake in men, but not in women (15).Although the development of insulin resistance in different tissues may be temporally and mechanistically distinct, there are complicated interorgan communications among the various sites of insulin action. For example, defective hypothalamic insulin signaling is able to promote hepatic insulin resistance in brain-specific insulin receptor (IR) knockout mice (10). Restoration of liver insulin signaling in the whole body of the IR knockout mice fails to normalize insulin action to suppress hepatic glucose production, further supporting the importance of the hypothalamus in insulin signaling (16).We hypothesized that functional AR in the brain may contribute to insulin sensitivity in the male brain and to the metabolic abnormalities in ARKO male mice, which may implicate the insulin resistance developed in men with PCa undergoing ADT. We used neuronal-specific AR knockout (NARKO) mice to directly determine the function of brain AR in insulin sensitivity. Our results demonstrate that neuronal AR deficiency results in dysregulation of central insulin action and abnormalities in whole-body glucose homeostasis. Loss of neuronal AR leads to increased activation of hypothalamic nuclear factor-κB (NF-κB), which induces the expression of protein-tyrosine phosphatase 1B (PTP1B), which interferes with hypothalamic insulin signaling. Impaired hypothalamic insulin signaling contributes to increased hepatic glucose production, systemic insulin resistance, and excessive fat deposition, further supporting the critical role of functional brain AR in restraining the development of obesity.     

Strong cytoplasmic expression of NF-κB/p65 correlates with a good prognosis in patients with triple-negative breast cancer

Purpose

Recent studies have indicated that constitutive NF-κB activity could be involved in the proliferation of triple-negative breast cancer.

Methods

The NF-κB/p65 expression and the effects of a NF-κB inhibitor, (?)-DHMEQ, were examined in triple-negative MDA-MB-231 breast cancer cells. Women with triple-negative breast cancer treated with neoadjuvant chemotherapy between 2002 and 2012 were retrospectively analyzed for their expression of NF-κB/p65, Bcl2 and Ki67 by immunohistochemistry in pre- and post-treatment specimens. The factors predicting the response to neoadjuvant chemotherapy and the prognosis were analyzed.

Results

NF-κB/p65 was predominantly expressed in the cytoplasm of MDA-MB-231 cells. Of 34 triple-negative breast cancer patients, positive staining for NF-κB/p65 expression was detected in the nuclei of a few cells in seven tumors before neoadjuvant chemotherapy, while the expression of NF-κB/p65 in the cytoplasm was detected in almost all tumor cells of 33 tumors. The expression levels of NF-κB/p65 were not associated with the response to neoadjuvant chemotherapy, although the cytoplasmic NF-κB/p65 staining intensity was significantly decreased in the post-treatment tumor samples compared with the pretreatment samples. All patients whose tumors showed strong cytoplasmic NF-κB/p65 expression before neoadjuvant chemotherapy are currently disease free.

Conclusion

Our results suggest that strong cytoplasmic NF-κB/p65 expression could be a prognostic marker for patients with triple-negative breast cancer.

     

Therapeutic effect of p65 AS-ODN, a subset of NF-κB, on pancreatic cancer

核因子-κB(NF-κB)是细胞分裂和生存的关键调节因子,参与肿瘤发生、增殖、转移等多方面的调控环节,在胰腺癌的发生、发展过程中发挥着重要作用.     

Therapeutic effect of p65 AS-ODN, a subset of NF-κB, on pancreatic cancer

核因子-κB(NF-κB)是细胞分裂和生存的关键调节因子,参与肿瘤发生、增殖、转移等多方面的调控环节,在胰腺癌的发生、发展过程中发挥着重要作用.     

Effects of knockout receptor activator of NF-κB (RANK) in podocytes on proteinuria in mice

Objective To investigate the role of RANK in injured podocyte and its effects on proteinuria. Methods PCR analysis was carried out to identify the genotypes of podocyte-specific RANK knockout mice. Six (6-8 weeks old) female RANK-/- mice were chosen as KO group. Six (6-8 weeks old) littermates and six (6-8 weeks old) C57/B6j mice were chosen as WT group and C57 group respectively. Each group was injected with LPS to create podocyte-injured proteinuria model. 24h urine was collected before and after injection. Urinary albumin, urine creatinine and UACR were detected by automatic biochemical analyzer. The mice were sacrificed after 48 h. Cortical kidney tissue samples were stained by PAS and collected for transmission electron microscopy to detect the foot process effacement. The number of podocytes was examined at different time points by immunohistochemistry .The expression of intergrin-β1, integrin-β3 and uPAR was detected by immunofluorescence and westernblot. Results (1) Comparing KO group, WT group and C57 group, it was found that there was no difference in weight, morphology, function of kidney and ACR. (2) After LPS injection, proteinuria and ACR were increased in three groups, but less significantly in KO group (23.70±9.90), compared to those in WT group (107.56±22.32) and C57 group (132.13±14.26) (P＜0.001). (3) In three groups, the number of podocytes was decreased after LPS injection and the decrease in the KO group was the slightest. (4) Compared to WT and C57 group, podocyte foot process effacement was less obvious in KO group after LPS injection in transmission electron microscopy. (5) Immunofluorescence results showed that after LPS injection, integrin-β1 was decreased in three groups, but most significantly in KO group. Integrin-β3 was increased in three groups, but less obvious in KO group. (6) Westernblot results showed consistency with immunofluorescence. The expression of uPAR protein was increased in three groups, with the increase in KO group being the slightest. Conclusions Podocyte-specific knockout of RANK reduces LPS-induced proteinuria, suggesting that RANK might be involved in the development of proteinuria in podocyt injury. Its mechanism is probably related with integrin-β1, integrin-β3 and uPAR.     

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism

NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2-3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from p50/p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.     

Effect of Nephrotoxins on Tubulointerstitial Injury and NF-κB Activation in Adriamycin Nephropathy

In a previous study we found that an episode of acute subclinical nephrotoxicity with gentamicin (G) (but not that induced by another proximal tubular cell nephrotoxin: ferric nitrilotriacetate, FeNTA), paradoxically reduced the progression of renal function and injury in uninephrectomized rats with nephrotic glomerular disease due to Adriamycin nephropathy (AN). Here, we hypothesized that subclinical exposure to G reduces early renal cortical tubulointerstitial inflammation and NF-κB activation in AN. To test this hypothesis, male Wistar rats with established AN received either G (10, 40, or 80 mg/kg by daily s.c.i. for 3 days), FeNTA (1.25, 5, or 10 mg/kg by a single i.p.i.), or vehicle (n = 8 per group), 13 to 15 days after disease induction. Although G and FeNTA caused acute tubular necrosis in a dose-dependant manner (day 17), only the highest doses (10 mg/kg and 80 mg/kg) produced an acute elevation in the serum creatinine. On day 33, chronic tubulointerstitial inflammation (tubular atrophy, interstitial ED-1 + /CD8 + cell accumulation) and NF-κB activation were exacerbated only in the groups that caused functional nephrotoxicity. These data suggest that: 1) the protective effect of subclinical G nephrotoxicity in chronic AN does not involve early changes in interstitial inflammation or NF-κB activation; and 2) a single episode of G exposure must be accompanied by clinically apparent nephrotoxicity in order to accelerate progression in a nonuremic model of chronic glomerular disease.     

Receptor activator of NF-κB (RANK)-mediated induction of metastatic spread and association with poor prognosis in renal cell carcinoma

Background

Inhibition of the receptor activator of NF-κB ligand (RANKL) has become a standard of care supportive treatment to prevent skeletal related events in cancer patients. Moreover, RANKL inhibition has been implicated with better survival outcome in lung cancer, while RANKL expression induces tumor progression and metastatic spread in vivo in breast cancer. Whether RANK/RANKL may have an impact on the pathogenesis of clear cell renal cell carcinoma (ccRCC) is currently unknown.

NF-κB and chemokine-cytokine expression in renal tubulointerstitium in experimental hyperoxaluria. Role of the renin-angiotensin system

Recent evidence indicates that the renin-angiotensin system (RAS) seems to play a considerable role in the development of tubulointerstitial (TI) lesions caused by hyperoxaluria (Hox). The purpose of the present study was to evaluate the specific mechanism by which Hox involving RAS induces chemokine and cytokine expression and, therefore, renal TI damage in the ethylene-glycol (ETG) induced hyperoxaluric rat model. Sprague-Dawley rats, separated into five groups, received: G1 regular water, and G2, G3, G4 and G5 1% ETG (a precursor for oxalates) in their drinking water for 4 weeks. An angiotensin converting enzyme inhibitor, benazepril (BZ) 10 mg/kg/day, angiotensin II receptor antagonists, subtype 1 (AT1) losartan (LOS) 40 mg/kg/day and subtype 2 (AT2) PD 123,319 (PD) 10 mg/kg/day, were administered daily to G3, G4 and G5, respectively. At the end of the study, the inflammatory response to Hox was evaluated using anti-NF-B (p50), anti-IL-6, anti-MCP-1; anti-RANTES and anti-ED1 (monocytes/macrophages) in each group. In spite of the same urine oxalate levels, rats belonging to the hyperoxaluric groups treated with either BZ or LOS showed significantly (P<0.01) less TI lesions together with a lower immunoexpression of inflammatory mediators when compared with untreated hyperoxaluric animals. NF-B (p50) was increased in tubular cells in the ETG group (43.6±8.7 positive cells/mm²) and was significantly (P<0.01) reduced by LOS (11.2±4 positive cells/mm²) and even more by BZ (6.1±2.4 positive cells/mm²). There was a significant (P<0.01) correlation between NF-B (p50) positive cells and ED1 cells in the ETG group (r=0.88) and in the ETG+LOS group (r=0.92). LOS showed better control on IL-6 and MCP-1 with respect to untreated rats, while BZ showed the best control on RANTES and ED1 cells in comparison with untreated animals. Renal function was significantly (P<0.01) better preserved in BZ and LOS treated groups compared to both untreated animals and rats with PD, as indicated by creatinine clearance values. These results suggest that Hox stimulates the NF-B cascade and, therefore, induces the overexpression of inflammatory mediators like IL-6, MCP-1, and RANTES. This pathway seems to be mediated not only by AT1 but also by AT2 receptors of angiotensin II.     

EGFR Tyrosine Kinase Inhibitor (PD153035) Improves Glucose Tolerance and Insulin Action in High-Fat Diet�CFed Mice

OBJECTIVE

In obesity, an increased macrophage infiltration in adipose tissue occurs, contributing to low-grade inflammation and insulin resistance. Epidermal growth factor receptor (EGFR) mediates both chemotaxis and proliferation in monocytes and macrophages. However, the role of EGFR inhibitors in this subclinical inflammation has not yet been investigated. We investigated, herein, in vivo efficacy and associated molecular mechanisms by which PD153035, an EGFR tyrosine kinase inhibitor, improved diabetes control and insulin action.

RESEARCH DESIGN AND METHODS

The effect of PD153035 was investigated on insulin sensitivity, insulin signaling, and c-Jun NH₂-terminal kinase (JNK) and nuclear factor (NF)-κB activity in tissues of high-fat diet (HFD)-fed mice and also on infiltration and the activation state of adipose tissue macrophages (ATMs) in these mice.

RESULTS

PD153035 treatment for 1 day decreased the protein expression of inducible nitric oxide synthase, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in the stroma vascular fraction, suggesting that this drug reduces the M1 proinflammatory state in ATMs, as an initial effect, in turn reducing the circulating levels of TNF-α and IL-6, and initiating an improvement in insulin signaling and sensitivity. After 14 days of drug administration, there was a marked improvement in glucose tolerance; a reduction in insulin resistance; a reduction in macrophage infiltration in adipose tissue and in TNF-α, IL-6, and free fatty acids; accompanied by an improvement in insulin signaling in liver, muscle, and adipose tissue; and also a decrease in insulin receptor substrate-1 Ser³⁰⁷ phosphorylation in JNK and inhibitor of NF-κB kinase (IKKβ) activation in these tissues.

CONCLUSIONS

Treatment with PD153035 improves glucose tolerance, insulin sensitivity, and signaling and reduces subclinical inflammation in HFD-fed mice.Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are used in the clinic to treat malignancies (1). It has recently been observed that a modest number of patients, suffering from both malignancies and type 2 diabetes, were successfully treated not only for their malignancies but also for diabetes when given some tyrosine kinase inhibitors (2–5). However, the molecular mechanisms that account for the effect of these drugs on insulin action and glucose metabolism are unknown.Insulin stimulates a signaling network composed of a number of molecules, initiating the activation of insulin receptor tyrosine kinase and phosphorylation of insulin receptor substrates, including insulin receptor substrate (IRS)-1 and IRS-2 (6–8). Following tyrosine phosphorylation, IRS-1/IRS-2 bind and activate the enzyme phosphatidylinositol 3-kinase (PI3-K). The activation of PI3-K increases serine phosphorylation of Akt, which is responsible for most of the metabolic actions of insulin, such as glucose transport, lipogenesis, and glycogen synthesis (7,8).In the most prevalent forms of insulin resistance, diet-induced obesity, and type 2 diabetes, there is a downregulation in this signaling pathway in insulin-sensitive tissues, parallel to a state of chronic low-grade inflammation (6). Several serine/threonine kinases are activated by inflammatory or stressful stimuli and contribute to inhibition of insulin signaling, including c-Jun NH₂-terminal kinase (JNK) (9–13) and inhibitor of nuclear factor (NF)-κB kinase (IKKβ) (12,14). In obesity, an increased macrophage infiltration in adipose tissue occurs, contributing to this low-grade inflammation (15–17), which has an important role in the increased tissue production of proinflammatory molecules and acute-phase proteins associated with obesity (13,14). EGFR has been described in monocytes and in macrophages and mediates both chemotaxis and proliferation in macrophages (18–20). However, the role of EGFR inhibitors on this subclinical inflammation of obesity was not yet investigated.PD153035 has been shown to possess highly potent and selectively inhibitory activity against EGFR tyrosine kinase and rapidly suppresses autophosphorylation of EGFR at low nanomolar concentrations in fibroblasts and human epidermoid carcinoma cells, as well as selectively blocking EGF-mediated cellular processes, including mitogenesis and early gene expression (21–23). In addition, PD153035 has been shown to reduce JNK and IKK/IκB/NF-κB pathways (24,25). Moreover, EGFR and other tyrosine kinase inhibitors have also been shown to inhibit the growth of monocyte/macrophages, suggesting possible mechanisms to improve insulin action (26–29).Herein, we investigated the in vivo efficacy and associated molecular mechanisms by which PD153035, an EGFR tyrosine kinase inhibitor, improved diabetes control and insulin action. We studied the effect of acute (1 day) or chronic (14 days) administration of PD153035 on insulin sensitivity, insulin signaling, and JNK and NF-κB activity in liver, muscle, and adipose tissue of high-fat diet (HFD)-fed mice and also on the infiltration and activation state of adipose tissue macrophages (ATMs) in these mice.     

Determination of NF-κB and RANKL levels in peripheral blood osteoclast precursor cells in chronic kidney disease patients

International Urology and Nephrology - Chronic kidney disease (CKD) is a progressive condition characterized by irreversible loss of functional nephron mass due to variety of causes; an inevitable...     

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-κB pathways

Summary  Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-κB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. Introduction  A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-κB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. Methods  The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-κB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-κB activity. Results  Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-κB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. Conclusions  Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-κB activation. R.-S. Yang, and W.-M. Fu contributed equally to this study.