Cancer Cachexia in STK11/LKB1 -mutated NSCLC is Dependent on Tumor-secreted GDF15.

Cachexia is a wasting syndrome comprised of adipose, muscle, and weight loss observed in cancer patients. Tumor loss-of-function mutations in STK11/LKB1 , a regulator of the energy sensor AMP-activated protein kinase, induce cancer cachexia (CC) in preclinical models and are associated with cancer-related weight loss in NSCLC patients. Here we characterized the relevance of the NSCLC-associated cachexia factor growth differentiation factor 15 (GDF15) in several patient-derived and genetically engineered STK11/LKB1 -mutant NSCLC cachexia lines. Both tumor mRNA expression and serum concentrations of tumor-derived GDF15 were significantly elevated in multiple mice transplanted with patient-derived STK11/LKB1 -mutated NSCLC lines. GDF15 neutralizing antibody administered to mice transplanted with patient- or mouse-derived STK11/LKB1 -mutated NSCLC lines suppressed cachexia-associated adipose loss, muscle atrophy, and changes in body weight. The silencing of GDF15 in multiple human NSCLC lines was also sufficient to eliminate in vivo circulating GDF15 levels and abrogate cachexia induction, suggesting that tumor and not host tissues represent a key source of GDF15 production in these cancer models. Finally, reconstitution of wild-type STK11/LKB1 in a human STK11/LKB1 loss-of-function NSCLC line that normally induces cachexia in vivo correlated with the absence of tumor-secreted GDF15 and rescue from the cachexia phenotype. The current data provide evidence for tumor-secreted GDF15 as a conduit and a therapeutic target through which NSCLCs with STK11/LKB1 loss-of-function mutations promote cachexia-associated wasting.

Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity.

In an effort to identify novel candidate regulators of adipogenesis, gene profiling of differentiating 3T3-L1 preadipocytes was analyzed using a novel algorithm. We report here the characterization of xanthine oxidoreductase (XOR) as a novel regulator of adipogenesis. XOR lies downstream of C/EBPbeta and upstream of PPARgamma, in the cascade of factors that control adipogenesis, and it regulates PPARgamma activity. In vitro, knockdown of XOR inhibits adipogenesis and PPARgamma activity while constitutive overexpression increases activity of the PPARgamma receptor in both adipocytes and preadipocytes. In vivo, XOR -/- mice demonstrate 50% reduction in adipose mass versus wild-type littermates while obese ob/ob mice exhibit increased concentrations of XOR mRNA and urate in the adipose tissue. We propose that XOR is a novel regulator of adipogenesis and of PPARgamma activity and essential for the regulation of fat accretion. Our results identify XOR as a potential therapeutic target for metabolic abnormalities beyond hyperuricemia.

Hypomorphic mutation of PGC-1beta causes mitochondrial dysfunction and liver insulin resistance.

PGC-1beta is a transcriptional coactivator that potently stimulates mitochondrial biogenesis and respiration of cells. Here, we have generated mice lacking exons 3 to 4 of the Pgc-1beta gene (Pgc-1beta(E3,4-/E3,4-) mice). These mice express a mutant protein that has reduced coactivation activity on a subset of transcription factors, including ERRalpha, a major target of PGC-1beta in the induction of mitochondrial gene expression. The mutant mice have reduced expression of OXPHOS genes and mitochondrial dysfunction in liver and skeletal muscle as well as elevated liver triglycerides. Euglycemic-hyperinsulinemic clamp and insulin signaling studies show that PGC-1beta mutant mice have normal skeletal muscle response to insulin but have hepatic insulin resistance. These results demonstrate that PGC-1beta is required for normal expression of OXPHOS genes and mitochondrial function in liver and skeletal muscle. Importantly, these abnormalities do not cause insulin resistance in skeletal muscle but cause substantially reduced insulin action in the liver.

Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3.

Leptin is an adipocyte-derived hormone that regulates energy balance and neuroendocrine function primarily by acting on specific hypothalamic pathways. Resistance to the weight reducing effects of leptin is a feature of most cases of human and rodent obesity, yet the molecular basis of leptin resistance is poorly understood. We have previously identified suppressor of cytokine signaling-3 (Socs3) as a leptin-induced negative regulator of leptin receptor signaling and potential mediator of leptin resistance. However, due to the non-viability of mice with targeted disruption of Socs3 (ref. 6), the importance of Socs3 in leptin action in vivo was unclear. To determine the functional significance of Socs3 in energy balance in vivo we undertook studies in mice with heterozygous Socs3 deficiency (Socs3(+/-)). We report here that Socs3(+/-) mice display greater leptin sensitivity than wild-type control mice: Socs3(+/-) mice show both enhanced weight loss and increased hypothalamic leptin receptor signaling in response to exogenous leptin administration. Furthermore, Socs3(+/-) mice are significantly protected against the development of diet-induced obesity and associated metabolic complications. The level of Socs3 expression is thus a critical determinant of leptin sensitivity and obesity susceptibility in vivo and this molecule is a potential target for therapeutic intervention.

TLR4 links innate immunity and fatty acid-induced insulin resistance.

TLR4 is the receptor for LPS and plays a critical role in innate immunity. Stimulation of TLR4 activates proinflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. Here we show that nutritional fatty acids, whose circulating levels are often increased in obesity, activate TLR4 signaling in adipocytes and macrophages and that the capacity of fatty acids to induce inflammatory signaling in adipose cells or tissue and macrophages is blunted in the absence of TLR4. Moreover, mice lacking TLR4 are substantially protected from the ability of systemic lipid infusion to (a) suppress insulin signaling in muscle and (b) reduce insulin-mediated changes in systemic glucose metabolism. Finally, female C57BL/6 mice lacking TLR4 have increased obesity but are partially protected against high fat diet-induced insulin resistance, possibly due to reduced inflammatory gene expression in liver and fat. Taken together, these data suggest that TLR4 is a molecular link among nutrition, lipids, and inflammation and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.

Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization.

Nicotinamide N-methyltransferase (Nnmt) methylates nicotinamide, a form of vitamin B3, to produce N(1)-methylnicotinamide (MNAM). Nnmt has emerged as a metabolic regulator in adipocytes, but its role in the liver, the tissue with the strongest Nnmt expression, is not known. In spite of its overall high expression, here we find that hepatic expression of Nnmt is highly variable and correlates with multiple metabolic parameters in mice and humans. Further, we find that suppression of hepatic Nnmt expression in vivo alters glucose and cholesterol metabolism and that the metabolic effects of Nnmt in the liver are mediated by its product MNAM. Supplementation of high-fat diet with MNAM decreases serum and liver cholesterol and liver triglycerides levels in mice. Mechanistically, increasing Nnmt expression or MNAM levels stabilizes sirtuin 1 protein, an effect that is required for their metabolic benefits. In summary, we describe here a novel regulatory pathway for vitamin B3 that could provide a new opportunity for metabolic disease therapy.

Melanin-concentrating hormone receptor 1 activates extracellular signal-regulated kinase and synergizes with G(s)-coupled pathways.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a key role in energy homeostasis. Like many neuropeptides, it signals through two G protein-coupled receptors. MCH receptor 1 (MCHR1) is the sole receptor expressed in rodents and couples to G(i) and G(q) proteins. Little is known about the intracellular pathways engaged by MCH and its receptor. Using HEK293 cells stably expressing MCHR1, we demonstrate that MCH, acting through MCHR1, antagonizes the action of forskolin, an adenylate cyclase activator that increases intracellular levels of cAMP. MCH also inhibits cAMP induction by the G(s)-coupled beta-adrenergic receptor. Activation of either the G(i)- or G(s)-dependent pathway typically results in ERK phosphorylation in HEK293 cells. In contrast to opposing actions on cAMP synthesis, simultaneous MCH and forskolin treatment results in synergistic activation of ERK. This synergy proceeds through pertussis toxin-independent pathways and requires several enzymatic activities such as protein kinase A, protein kinase C, phospholipase C, and Src kinase. Finally, we provide evidence that such positive interactions are not limited to cell lines but can also be observed in the brain.

Obesity-related upregulation of monocyte chemotactic factors in adipocytes: involvement of nuclear factor-kappaB and c-Jun NH2-terminal kinase pathways.

OBJECTIVE: We sought to evaluate the entire picture of all monocyte chemotactic factors that potentially contribute to adipose tissue macrophage accumulation in obesity. RESEARCH DESIGN AND METHODS: Expression and regulation of members in the entire chemokine superfamily were evaluated in adipose tissue and isolated adipocytes of obese versus lean mice. Kinetics of adipose tissue macrophage infiltration was characterized by fluorescence-activated cell sorting. The effects of fatty acids on stimulation of chemokine expression in adipocytes and underlying mechanisms were investigated. RESULTS: Six monocyte chemotactic factors were found to be predominantly upregulated in isolated adipocytes versus stromal vascular cells in obese mice for the first time, although most of them were previously reported to be upregulated in whole adipose tissue. In diet-induced obese mice, adipose tissue enlargement, increase of adipocyte number, and elevation of multiple chemokine expression precede the initiation of macrophage infiltration. Free fatty acids (FFAs) are found to be inducers for upregulating these chemokines in 3T3-L1 adipocytes, and this effect can be partially blunted by reducing Toll-like receptor 4 expression. FFAs induce expression of monocyte chemotactic factors in adipocytes via both transcription-dependent and -independent mechanisms. In contrast to the reported role of JNK as the exclusive mediator of FFA-induced monocyte chemoattractant protein-1 (MCP-1) expression in macrophages, we show a novel role of inhibitor of kappaB kinase-beta (IKKbeta) in mediating FFA-induced upregulation of all six chemokines and a role of JNK in FFA-induced upregulation of MCP-1 and MCP-3. CONCLUSIONS: Multiple chemokines derived from adipocytes might contribute to obesity-related WAT macrophage infiltration with FFAs as potential triggers and involvement of both IKKbeta and JNK pathways.

Complex role of the vitamin D receptor and its ligand in adipogenesis in 3T3-L1 cells.

The vitamin D receptor (VDR) and its ligand 1,25-OH2-VD3 (calcitriol) play an essential role in mineral homeostasis in mammals. Interestingly, the VDR is expressed very early in adipogenesis in 3T3-L1 cells, suggesting that the VDR signaling pathway may play a role in adipocyte biology and function. Indeed, it has been known for a number of years that calcitriol is a potent inhibitor of adipogenesis in this model but with no clear mechanism identified. In this study, we have further defined the molecular mechanism by which the unliganded VDR and calcitriol-liganded VDR regulate adipogenesis. In the presence of calcitriol, the VDR blocks adipogenesis by down-regulating both C/EBPbeta mRNA expression and C/EBPbeta nuclear protein levels at a critical stage of differentiation. In addition, calcitriol allows for the up-regulation of the recently described C/EBPbeta corerepressor, ETO, which would further inhibit the action of any remaining C/EBPbeta, whose action is required for adipogenesis. In contrast, in the absence of calcitriol, the unliganded VDR appears necessary for lipid accumulation, since knock-down of the VDR using siRNA both delays and prevents this process. Taken together, these data support the notion that the intracellular concentrations of calcitriol can play an important role in either promoting or inhibiting adipogenesis via the VDR and the transcriptional pathways that it targets. Further examination of this hypothesis in vivo may shed new light on the biology of adipogenesis.

Suppressor of cytokine signaling 3 is a physiological regulator of adipocyte insulin signaling.

Many proinflammatory cytokines and hormones have been demonstrated to be involved in insulin resistance. However, the molecular mechanisms whereby these cytokines and hormones inhibit insulin signaling are not completely understood. We observed that several cytokines and hormones that induce insulin resistance also stimulate SOCS3 expression in 3T3-L1 adipocytes and that SOCS3 mRNA is increased in adipose tissue of obese/diabetic mice. We then hypothesized that SOCS3 may mediate cytokine- and hormone-induced insulin resistance. By using SOCS3-deficient adipocytes differentiated from mouse embryonic fibroblasts, we found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 phosphorylation, IRS-associated phosphatidylinositol 3-kinase activity, and insulin-stimulated glucose uptake. Moreover, lack of SOCS3 substantially limits the inhibitory effects of tumor necrosis factor-alpha to suppress IRS1 and IRS2 tyrosine phosphorylation, phosphatidylinositol 3-kinase activity, and glucose uptake in adipocytes. The ameliorated insulin signaling in SOCS3-deficient adipocytes is mainly due to the suppression of tumor necrosis factor-alpha-induced IRS1 and IRS2 protein degradation. Therefore, our data suggest that endogenous SOCS3 expression is a key determinant of basal insulin signaling and is an important molecular mediator of cytokine-induced insulin resistance in adipocytes. We conclude that SOCS3 plays an important role in mediating insulin resistance and may be an excellent target for therapeutic intervention in insulin resistance and type II diabetes.

Complex effects of rexinoids on ligand dependent activation or inhibition of the xenobiotic receptor, CAR.

BACKGROUND: CAR/RXR heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands. This constitutive activity of murine CAR can be inhibited by the inverse agonist ligand androstanol or increased by the agonist TCPOBOP. RXR agonists activate some RXR heterodimer complexes, which are termed permissive, while other non-permissive complexes are not responsive to such ligands. RESULTS: Direct protein-protein interaction studies demonstrate that the RXR agonist 9-cis-RA increases interaction of CAR/RXR heterodimers with the coactivator SRC-3, but also inhibits the ability of TCPOBOP to increase and androstanol to decrease coactivator binding. CAR transactivation of a response element with a five nucleotide spacer (DR-5) is unaffected by 9-cis-RA or the synthetic RXR agonist LG1069. In agreement with the inhibitory effect observed in vitro, these rexinoids block both the TCPOBOP mediated transactivation of this element and the androstanol dependent inhibition. In contrast, CAR transactivation of other response elements is increased by rexinoids. Stable expression of CAR in a HepG2 derived cell line increases expression of the endogenous CAR target CYP2B6. This expression is further increased by TCPOBOP but decreased by either androstanol or LG1069, and LG1069 blocks the stimulatory effect of TCPOBOP but not the inhibitory effect of androstanol. CONCLUSION: We conclude that CAR/RXR heterodimers are neither strictly permissive nor non-permissive for RXR signaling. Instead, rexinoids have distinct effects in different contexts. These results expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses.

Decreased expression of peroxisome proliferator activated receptor gamma in cftr-/- mice.

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARgamma. We hypothesized that PPARgamma expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARgamma mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild-type and cftr-/- mice by quantitative RT-PCR. PPARgamma expression was decreased twofold in CFTR-regulated tissues (colon, ileum, and lung) from cftr-/- mice compared to wild-type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARgamma in ileum and colon revealed a predominantly nuclear localization in wild-type mucosal epithelial cells while tissues from cftr-/- mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARgamma expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARgamma/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr-/- mice. Treatment of cftr-/- mice with the PPARgamma ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARgamma expression in cftr-/- mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

Regulated production of a peroxisome proliferator-activated receptor-gamma ligand during an early phase of adipocyte differentiation in 3T3-L1 adipocytes.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPARgamma include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPARgamma ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPARgamma ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a beta-galactosidase-based PPARgamma ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated beta-galactosidase activity that signifies activation of PPARgamma via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPARgamma through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPARgamma. The active species appears to be different from other PPARgamma ligands identified previously. Our findings suggest that a novel biologically relevant PPARgamma ligand is transiently produced in 3T3-L1 cells during adipogenesis.