The fractalkine/CX3CR1 system regulates ß cell function and insulin secretion.

Here, we demonstrate that the fractalkine (FKN)/CX3CR1 system represents a regulatory mechanism for pancreatic islet ß cell function and insulin secretion. CX3CR1 knockout (KO) mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca(2+) and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated ß cell state, whereas treatment of wild-type (WT) islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and high-fat diet/obesity, suggesting that decreased FKN/CX3CR1 signaling could be a mechanism underlying ß cell dysfunction in type 2 diabetes.

GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.

Omega-3 fatty acids (omega-3 FAs), DHA and EPA, exert anti-inflammatory effects, but the mechanisms are poorly understood. Here, we show that the G protein-coupled receptor 120 (GPR120) functions as an omega-3 FA receptor/sensor. Stimulation of GPR120 with omega-3 FAs or a chemical agonist causes broad anti-inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages. All of these effects are abrogated by GPR120 knockdown. Since chronic macrophage-mediated tissue inflammation is a key mechanism for insulin resistance in obesity, we fed obese WT and GPR120 knockout mice a high-fat diet with or without omega-3 FA supplementation. The omega-3 FA treatment inhibited inflammation and enhanced systemic insulin sensitivity in WT mice, but was without effect in GPR120 knockout mice. In conclusion, GPR120 is a functional omega-3 FA receptor/sensor and mediates potent insulin sensitizing and antidiabetic effects in vivo by repressing macrophage-induced tissue inflammation.

High-mobility group box 2 protein is essential for the early phase of adipogenesis.

Understanding of the mechanism of adipogenesis is essential for the control of obesity, which predisposes toward numerous health problems. High-mobility group box protein 2 (HMGB2) is a non-histone chromosomal protein that facilitates DNA replication, transcription, recombination, and repair. Here, we studied the role of HMGB2 in adipogenic differentiation. The expression of HMGB2 was measured at the mRNA and protein levels in cultured 3T3-L1 pre-adipocyte cells and during the process of adipogenic differentiation induced bya cocktail of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone. This increased in the early phase and decreased in the late phase of differentiation. However, 3T3-L1 pre-adipocyte cells did not differentiate into adipocytes after the knockdown of HMGB2 expression by small interfering RNA (siRNA). Similarly, mesenchymal stem cells (MSCs) isolated from Hmgb2-/- mice did not express peroxisome proliferator-activated receptor gamma (PPARγ) in response to the adipocyte differentiation cocktail and did not differentiate. Wnt/ß-catenin signaling is a negative regulator of adipogenic differentiation. We found that ß-catenin expression was downregulated during 3T3-L1 adipogenic differentiation, as expected, but not when endogenous HMBG2 expression was knocked down using siRNA. These results indicate that HMGB2 plays an essential role in the early phase of the differentiation of pre-adipocytes and MSCs, and probably interacts with other regulators, such as PPARγ and Wnt/ß-catenin signaling.

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K+ channel activity.

AIMS/HYPOTHESIS: The TGF-ß superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-ß family signalling pathway is required for essential islet functions in the adult pancreas. METHODS: To identify distinct roles for the downstream components of the canonical TGF-ß signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-ß signals, in pancreatic beta cells (i.e. Smad2ß knockout [KO] mice). The activity of ATP-sensitive K(+) channels (KATP channels) was recorded in mutant beta cells using patch-clamp techniques. RESULTS: The Smad2ßKO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in beta cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of KATP channels was decreased in mutant beta cells. CONCLUSIONS/INTERPRETATION: These results suggest that in the adult pancreas, TGF-ß signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of KATP channel activity.

Neuronal Sirt1 deficiency increases insulin sensitivity in both brain and peripheral tissues.

Sirt1 is a NAD(+)-dependent class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. To assess this idea, we generated Sirt1 neuron-specific knockout (SINKO) mice. On both standard chow and HFD, SINKO mice were more insulin sensitive than Sirt1(f/f) mice. Thus, SINKO mice had lower fasting insulin levels, improved glucose tolerance and insulin tolerance, and enhanced systemic insulin sensitivity during hyperinsulinemic euglycemic clamp studies. Hypothalamic insulin sensitivity of SINKO mice was also increased over controls, as assessed by hypothalamic activation of PI3K, phosphorylation of Akt and FoxO1 following systemic insulin injection. Intracerebroventricular injection of insulin led to a greater systemic effect to improve glucose tolerance and insulin sensitivity in SINKO mice compared with controls. In line with the in vivo results, insulin-induced AKT and FoxO1 phosphorylation were potentiated by inhibition of Sirt1 in a cultured hypothalamic cell line. Mechanistically, this effect was traced to a reduced effect of Sirt1 to directly deacetylate and repress IRS-1 function. The enhanced central insulin signaling in SINKO mice was accompanied by increased insulin receptor signal transduction in liver, muscle, and adipose tissue. In summary, we conclude that neuronal Sirt1 negatively regulates hypothalamic insulin signaling, leading to systemic insulin resistance. Interventions that reduce neuronal Sirt1 activity have the potential to improve systemic insulin action and limit weight gain on an obesigenic diet.

FoxO1 regulates Tlr4 inflammatory pathway signalling in macrophages.

The macrophage-mediated inflammatory response is a key etiologic component of obesity-related tissue inflammation and insulin resistance. The transcriptional factor FoxO1 is a key regulator of cell metabolism, cell cycle and cell death. Its activity is tightly regulated by the phosphoinositide-3-kinase-AKT (PI3K-Akt) pathway, which leads to phosphorylation, cytoplasmic retention and inactivation of FoxO1. Here, we show that FoxO1 promotes inflammation by enhancing Tlr4-mediated signalling in mature macrophages. By means of chromatin immunoprecipitation (ChIP) combined with massively parallel sequencing (ChIP-Seq), we show that FoxO1 binds to multiple enhancer-like elements within the Tlr4 gene itself, as well as to sites in a number of Tlr4 signalling pathway genes. While FoxO1 potentiates Tlr4 signalling, activation of the latter induces AKT and subsequently inactivates FoxO1, establishing a self-limiting mechanism of inflammation. Given the central role of macrophage Tlr4 in transducing extrinsic proinflammatory signals, the novel functions for FoxO1 in macrophages as a transcriptional regulator of the Tlr4 gene and its inflammatory pathway, highlights FoxO1 as a key molecular adaptor integrating inflammatory responses in the context of obesity and insulin resistance.

Activation of activin type IB receptor signals in pancreatic ß cells leads to defective insulin secretion through the attenuation of ATP-sensitive K+ channel activity.

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic ß cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic ß cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic ß cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic ß cells. The resultant mice (pancreatic ß cell-specific ActRIB transgenic (Tg) mice; ActRIBCAßTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K(+) channels (KATP channels) was decreased in mutant ß cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic ß cells, and that activin signaling involves modulation of KATP channel activity.

GPR105 ablation prevents inflammation and improves insulin sensitivity in mice with diet-induced obesity.

GPR105, a G protein-coupled receptor for UDP-glucose, is highly expressed in several human tissues and participates in the innate immune response. Because inflammation has been implicated as a key initial trigger for type 2 diabetes, we hypothesized that GPR105 (official gene name: P2RY14) might play a role in the initiation of inflammation and insulin resistance in obesity. To this end, we investigated glucose metabolism in GPR105 knockout (KO) and wild-type (WT) mice fed a high-fat diet (HFD). We also examined whether GPR105 regulates macrophage recruitment to liver or adipose tissues by in vivo monocyte tracking and in vitro chemotaxis experiments, followed by transplantation of bone marrow from either KO or WT donors to WT recipients. Our data show that genetic deletion of GPR105 confers protection against HFD-induced insulin resistance, with reduced macrophage infiltration and inflammation in liver, and increased insulin-stimulated Akt phosphorylation in liver, muscle, and adipose tissue. By tracking monocytes from either KO or WT donors, we found that fewer KO monocytes were recruited to the liver of WT recipients. Furthermore, we observed that uridine 5-diphosphoglucose enhanced the in vitro migration of bone marrow-derived macrophages from WT but not KO mice, and that plasma uridine 5-diphosphoglucose levels were significantly higher in obese versus lean mice. Finally, we confirmed that insulin sensitivity improved in HFD mice with a myeloid cell-specific deletion of GPR105. These studies indicate that GPR105 ablation mitigates HFD-induced insulin resistance by inhibiting macrophage recruitment and tissue inflammation. Hence GPR105 provides a novel link between innate immunity and metabolism.

Perilipin 5, a lipid droplet-binding protein, protects heart from oxidative burden by sequestering fatty acid from excessive oxidation.

Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart.

Activin stimulates CYP19A gene expression in human ovarian granulosa cell-like KGN cells via the Smad2 signaling pathway.

Activin, a transforming growth factor ß family member, has a wide range of physiological roles during embryonic development and organogenesis. In the ovary, activin, secreted from ovarian granulosa cells, not only acts on the pituitary gland to regulate the gonadotropin secretion from the pituitary gland in an endocrine manner but also acts on granulosa cells in a paracrine/autocrine manner to regulate folliculogenesis. Previously, we showed that activin signals through activin type IB receptor (ActRIB) and up-regulates follicle-stimulating hormone receptor expression and P450 aromatase activity in human ovarian granulose cell-like KGN cells. In the current study, we demonstrate the direct involvement of Smad2 as a downstream signal mediator of ActRIB in the transcriptional regulation of the P450 aromatase gene (CYP19A) in KGN cells. Upon activin stimulation, Smad2 activation and an increase in P450 aromatase messenger RNA (mRNA) were observed in KGN cells. Interestingly, Smad2 phosphorylation correlated well with the increase in P450 aromatase mRNA. Reciprocally, knockdown of Smad2 mRNA in KGN cells led to a decrease in the P450 aromatase mRNA expression, suggesting that Smad2 regulates CYP19A gene expression. Further analysis of CYP19A promoter activity revealed that the 5' upstream region between -2069 and -1271bp is required for the activation by Smad2. Finally, we provide compelling evidence that Smad2 shows follicular stage-specific expression, which is high in granulosa cells of preantral or early antral follicles in mice. Our results suggest that activin signaling through the ActRIB-Smad2 pathway plays a pivotal role in CYP19A expression and thus in follicular development.

Pancreatic beta-cell failure in obese mice with human-like CMP-Neu5Ac hydroxylase deficiency.

Type 2 diabetes is highly prevalent in human populations, particularly in obese individuals, and is characterized by progressive pancreatic ß-cell dysfunction and insulin resistance. Most mammals, including Old World primates, express two major kinds of sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), typically found at the distal ends of glycoconjugate chains at the cell surface. Humans are uniquely unable to produce endogenous Neu5Gc due to an inactivating mutation in the CMP-Neu5Ac hydroxylase (CMAH) gene. The CMAH enzyme catalyzes the generation of CMP-Neu5Gc by the transfer of a single oxygen atom to the acyl group of CMP-Neu5Ac. Here, we show that mice bearing a human-like deletion of the Cmah gene exhibit fasting hyperglycemia and glucose intolerance following a high-fat diet. This phenotype is caused not by worsened insulin resistance but by compromised pancreatic ß-cell function associated with a 65% decrease in islet size and area and 50% decrease in islet number. Obese Cmah-null mice also show an ∼40% reduction in response to insulin secretagogues in vivo. These findings show that human evolution-like changes in sialic acid composition impair pancreatic ß-cell function and exacerbate glucose intolerance in mice. This may lend insight into the pathogenesis of type 2 diabetes in obese humans.

Author Correction: HMGB2 is a novel adipogenic factor that regulates ectopic fat infiltration in skeletal muscles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular and biophysical properties of voltage-gated Na+ channels in murine vas deferens.

The biological and molecular properties of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents (I(Na)) in murine vas deferens myocytes were investigated using patch-clamp techniques and molecular biological analyses. In whole-cell configuration, a fast, transient inward current was evoked in the presence of Cd(2+), and was abolished by TTX (K(d) = 11.2 nM), mibefradil (K(d) = 3.3 microM), and external replacement of Na(+) with monovalent cations (TEA(+), Tris(+), and NMDG(+)). The fast transient inward current was enhanced by veratridine, an activator of voltage-gated Na(+) channels, suggesting that the fast transient inward current was a TTX-sensitive I(Na). The values for half-maximal (V(half)) inactivation and activation of I(Na) were -46.3 mV and -26.0 mV, respectively. RT-PCR analysis revealed the expression of Scn1a, 2a, and 8a transcripts. The Scn8a transcript and the alpha-subunit protein of Na(V)1.6 were detected in smooth muscle layers. Using Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, I(Na) were not detected in dispersed smooth muscle cells from the vas deferens, while TTX-sensitive I(Na) were recorded in their wild-type (Na(V)1.6(+/+)) littermates. This study demonstrates that the molecular identity of the voltage-gated Na(+) channels responsible for the TTX-sensitive I(Na) in murine vas deferens myocytes is primarily Na(V)1.6.

Adipose tissue-derived and bone marrow-derived mesenchymal cells develop into different lineage of steroidogenic cells by forced expression of steroidogenic factor 1.

Steroidogenic factor 1 (SF-1)/adrenal 4 binding protein is an essential nuclear receptor for steroidogenesis, as well as for adrenal and gonadal gland development. We have previously clarified that adenovirus-mediated forced expression of SF-1 can transform long-term cultured mouse bone marrow mesenchymal cells (BMCs) into ACTH-responsive steroidogenic cells. In the present study, we extended this work to adipose tissue-derived mesenchymal cells (AMCs) and compared its steroidogenic capacity with those of BMCs prepared from the identical mouse. Several cell surface markers, including potential mesenchymal cell markers, were identical in both cell types, and, as expected, forced expression of SF-1 caused AMCs to be transformed into ACTH-responsive steroidogenic cells. However, more elaborate studies revealed that the steroidogenic property of AMCs was rather different from that of BMCs, especially in steroidogenic lineage. In response to increased SF-1 expression and/or treatment with retinoic acid, AMCs were much more prone to produce adrenal steroid, corticosterone rather than gonadal steroid, testosterone, whereas the contrary was evident in BMCs. Such marked differences in steroidogenic profiles between AMCs and BMCs were also evident by the changes of steroidogenic enzymes. These novel results suggest a promising utility of AMCs for autologous cell regeneration therapy for patients with steroid insufficiency and also a necessity for appropriate tissue selection in preparing mesenchymal stem cells according to the aim. The different steroidogenic potency of AMCs or BMCs might provide a good model for the clarification of the mechanism of tissue- or cell-specific adrenal and gonadal steroidogenic cell differentiation.

Methylation of a conserved intronic CpG island of mouse SF-1 is associated with cell-specific expression of SF-1 in a culture system but not with tissue-specific expression.

The mechanism for the steroidogenic tissue or cell-specific expression of SF-1 has not been well clarified. We examined whether the methylation status of a large CpG island in the first intron of mouse SF-1 gene is associated with the expression level of SF-1 in cultured cells and in tissues. The island consists of three small islands (ICI-1, ICI-2, and ICI-3). In cultured adrenocortical Y-1 cells and in Leydig tumor cells, I-10, that both express high levels of SF-1, the upstream region of ICI-2, ICI-2-1, was clearly hypomethylated compared to cultured mouse bone marrow cells that do not express SF-1. However, this methylation status was not clearly associated with the tissue-specific expression of SF-1, in either adult or during development. These results suggest that methylation of ICI-2-1of SF-1 may partly determine the level of SF-1 expression at the cellular level, but may not be essential at the tissue level.

HMGB2 is a novel adipogenic factor that regulates ectopic fat infiltration in skeletal muscles.

Although various surgical procedures have been developed for chronic rotator cuff tear repair, the re-tear rate remains high with severe fat infiltration. However, little is known about the molecular regulation of this process. Mesenchymal stem cells (MSCs) in the intra-muscular space are origin of ectopic fat cells in skeletal muscle. We have previously shown that high-mobility group box 2 (HMGB2), which is a nuclear protein commonly associated with mesenchymal differentiation, is involved in the early articular cartilage degeneration. In this study, we addressed the role of HMGB2 in adipogenesis of MSCs and fat infiltration into skeletal muscles. HMGB2 was highly expressed in undifferentiated MSCs and co-localized with platelet-derived growth factor receptor α (PDGFRA) known as an MSC-specific marker, while their expressions were decreased during adipocytic differentiation. Under the deficiency of HMGB2, the expressions of adipogenesis-related molecules were reduced, and adipogenic differentiation is substantially impaired in MSCs. Moreover, HMGB2+ cells were generated in the muscle belly of rat supraspinatus muscles after rotator cuff transection, and some of these cells expressed PDGFRA in intra-muscular spaces. Thus, our findings suggest that the enhance expression of HMGB2 induces the adipogenesis of MSCs and the fat infiltration into skeletal muscles through the cascade of HMGB2-PDGFRA.

Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans.

BACKGROUND: Atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP. METHODS: We compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1's role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1-dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII. RESULTS: Atrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this "orphan" receptor. CONCLUSION: The current findings are consistent with atrazine's endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans.

Marked augmentation of PLGA nanoparticle-induced metabolically beneficial impact of γ-oryzanol on fuel dyshomeostasis in genetically obese-diabetic ob/ob mice.

Our previous works demonstrated that brown rice-specific bioactive substance, γ-oryzanol acts as a chaperone, attenuates exaggerated endoplasmic reticulum (ER) stress in brain hypothalamus and pancreatic islets, thereby ameliorating metabolic derangement in high fat diet (HFD)-induced obese diabetic mice. However, extremely low absorption efficiency from intestine of γ-oryzanol is a tough obstacle for the clinical application. Therefore, in this study, to overcome extremely low bioavailability of γ-oryzanol with super-high lipophilicity, we encapsulated γ-oryzanol in polymer poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Nano-Orz), and evaluated its metabolically beneficial impact in genetically obese-diabetic ob/ob mice, the best-known severest diabetic model in mice. To our surprise, Nano-Orz markedly ameliorated fuel metabolism with an unexpected magnitude (∼1000-fold lower dose) compared with regular γ-oryzanol. Furthermore, such a conspicuous impact was achievable by its administration once every 2 weeks. Besides the excellent impact on dysfunction of hypothalamus and pancreatic islets, Nano-Orz markedly decreased ER stress and inflammation in liver and adipose tissue. Collectively, nanotechnology-based developments of functional foods oriented toward γ-oryzanol shed light on the novel approach for the treatment of a variety of metabolic diseases in humans.

Activation of peroxisome proliferator-activated receptor-gamma and retinoid X receptor inhibits aromatase transcription via nuclear factor-kappaB.

Our previous studies demonstrated that a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, troglitazone (TGZ),and/or a retinoid X receptor (RXR) ligand, LG100268 (LG), decreased the aromatase activity in both cultured human ovarian granulosa cells and human granulosa-like tumor KGN cells. In the present study, we further found that a combined treatment of TGZ+LG decreased aromatase promoter II (ArPII) activity in both ovarian KGN cells and fibroblast NIH-3T3 cells in a PPARgamma-dependent manner. Furthermore, the inhibition of both aromatase activity and the transcription of ArPII by TGZ+LG was completely eliminated when nuclear factor-kappaB (NF-kappaB) signaling was blocked by specific inhibitors, suggesting NF-kappaB, which is endogenously expressed in both fibroblast and granulosa cells, might be a mediator of this inhibition. Interestingly, activation of NF-kappaB by either forced expression of the p65 subunit or NF-kappaB-inducing kinase up-regulated ArPII activity. Positive regulation of aromatase by endogenous NF-kappaB was also suggested by the fact that NF-kappaB-specific inhibitors suppress basal activity of the aromatase gene. A concomitant formation of high-order complex between NF-kappaB p65 and ArPII was also observed by chromatin immunoprecipitation assay. Although activation of PPARgamma and RXR affected endogenous expression levels of neither inhibitory kappaBalpha nor p65, it impaired the interaction between NF-kappaB and ArPII and the p65 based transcription as well. Altogether, these results indicate that activation of a nuclear receptor system, constituted by PPARgamma and RXR, down-regulates aromatase expression through the suppression of NF-kappaB-dependent aromatase activation and thus provide a new insight in the mechanism of regulation of the aromatase gene.

Characterization of distinct subpopulations of hepatic macrophages in HFD/obese mice.

The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance.