Effects of gp130 cytokines on adipocytes

Members of the gp130 cytokine family are known for their pleiotropic roles in various cell types. Our work has focused on their actions in adipocytes. CNTF administration has been shown to ameliorate most complications associated with obesity and type 2 diabetes through an unknown mechanism. In this study CNTF is shown to activate JAK/STAT and MAPK signaling, both in vitro and in vivo. In 3T3-L1 adipocytes, chronic CNTF also regulates the expression of SREBP-1, FAS, IRS-1 and GLUT4. Acute CNTF administration enhances the activation of IRS-1 and Akt by insulin. Our data also demonstrate that the expression of CNTF-specific receptor, CNTFRalpha, is decreased during adipocyte differentiation, but that this protein is expressed in several other tissues, and the level of CNTFRalpha glycosylation varies between different tissues. Most importantly, the expression of CNTFRalpha is dramatically increased in the fat pads of obese and diabetic animals. Even though CT-1 is mostly known for its actions in cardiomyocytes, the work presented demonstrates that, in fat cells, CT-1 can potently induce JAK/STAT and MAPK signaling. Neither CT-1 nor CNTF affected adipocyte differentiation, but chronic CT-1 did induce a decrease in FAS and IRS-1 protein expression. Both CNTF and CT-1 also induced a transient increase in SOCS-3 and a transient decrease in PPARgamma mRNA levels, in a MAPK-independent manner. Our studies also demonstrate that several gp130 cytokines, namely CT-1, LIF and OSM, have the ability to block signaling by other gp130 cytokines, but not GH. These cytokines can also dramatically reduce the half-life of LIFR protein in adipocytes, and this change in LIFR stability correlates with the ability of CT-1, LIF and OSM, to block subsequent gp130 cytokine signaling. The loss of LIFR protein can be prevented by the lysosome inhibitors leupeptin and chloroquine, but not by proteasome inhibitors. In summary, these studies demonstrate than gp130 cytokines have a wide spectrum of effects on both cultured and native adipocytes, and that their actions may be important in various aspects of adipocyte physiology

Analysis of circadian pattern reveals tissue-specific alternative transcription in leptin signaling pathway

*Background*
It has been previously reported that most mammalian genes display a circadian oscillation in their baseline expression. Consequently, the phase and amplitude of each component of a signal transduction cascade has downstream consequences. 

*Results*
We report our analysis of alternative transcripts in the leptin signaling pathway which is responsible for the systemic regulation of macronutrient storage and energy balance. We focused on the circadian expression pattern of a critical component of the leptin signaling system, suppressor of cytokine signaling 3 (SOCS3). On an Affymetrix GeneChip 430A2 microarray, this gene is represented by three probe sets targeting different regions within the 3’ end of the last exon. We demonstrate that in murine brown adipose tissue two downstream 3’ probe sets experience circadian baseline oscillation in counter-phase to the upstream probe set. Such differences in expression patterns are a telltale sign of alternative splicing within the last exon of SOCS3. In contrast, all three probe sets oscillated in a common phase in murine liver and white adipose tissue. This suggests that the regulation of SOCS3 expression in brown fat is tissue specific. Another component of the signaling pathway, Janus kinase (JAK), is directly regulated by SOCS and has alternative transcript probe sets oscillating in counter-phase in a white adipose tissue specific manner.
 
*Conclusion*
We hypothesize that differential oscillation of alternative transcripts may provide a mechanism to maintain steady levels of expression in spite of circadian baseline variation

The clock genes Period 2 and Cryptochrome 2 differentially balance bone formation

Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters

Oncostatin m is produced in adipose tissue and is regulated in conditions of obesity and type 2 diabetes

CONTEXT: Adipose tissue is a highly active endocrine organ that secretes many factors that affect other tissues and whole-body metabolism. Adipocytes are responsive to several glycoprotein 130 (gp130) cytokines, some of which have been targeted as potential antiobesity therapeutics. OBJECTIVE: Oncostatin M (OSM) is a gp130 family member known to inhibit adipocyte differentiation in vitro, but its effects on other adipocyte properties are not characterized. The expression of OSM in white adipose tissue (WAT) has not been evaluated in the context of obesity. Thus, our objective was to examine the expression of adipose tissue OSM in obese animals and humans. DESIGN: OSM expression was examined in adipose tissues from mice with diet-induced and genetic obesity and in obese humans as well as in fractionated adipose tissue from mice. Murine adipocytes were used to examine OSM receptor expression and the effects of OSM on adipocytes, including the secretion of factors such as plasminogen activator inhibitor 1 and IL-6, which are implicated in metabolic diseases. RESULTS: OSM expression is increased in rodent and human obesity/type 2 diabetes mellitus. In humans, OSM levels correlate with body weight and insulin and are inversely correlated with glucose disposal rate as measured by hyperinsulinemic-euglycemic clamp. OSM is not produced from the adipocytes in WAT but derives from cells in the stromovascular fraction, including F4/80(+) macrophages. The specific receptor of OSM, OSM receptor-β, is expressed in adipocytes and adipose tissue and increased in both rodent models of obesity examined. OSM acts on adipocytes to induce the expression and secretion of plasminogen activator inhibitor 1 and IL-6. CONCLUSIONS: These data indicate that WAT macrophages are a source of OSM and that OSM levels are significantly induced in murine and human obesity/type 2 diabetes mellitus. These studies suggest that OSM produced from immune cells in WAT acts in a paracrine manner on adipocytes to promote a proinflammatory phenotype in adipose tissue

Digital Signal Processing Reveals Circadian Baseline Oscillation in Majority of Mammalian Genes

In mammals, circadian periodicity has been described for gene expression in the hypothalamus and multiple peripheral tissues. It is accepted that 10%–15% of all genes oscillate in a daily rhythm, regulated by an intrinsic molecular clock. Statistical analyses of periodicity are limited by the small size of datasets and high levels of stochastic noise. Here, we propose a new approach applying digital signal processing algorithms separately to each group of genes oscillating in the same phase. Combined with the statistical tests for periodicity, this method identifies circadian baseline oscillation in almost 100% of all expressed genes. Consequently, circadian oscillation in gene expression should be evaluated in any study related to biological pathways. Changes in gene expression caused by mutations or regulation of environmental factors (such as photic stimuli or feeding) should be considered in the context of changes in the amplitude and phase of genetic oscillations

Fine-Tuning Reception in the Bone: PPARγ and Company

PPARγ plays a central role in the formation of fat. Regulation of PPARγ activity depends on numerous factors ranging from dietary ligands to nuclear hormone coactivators and corepressors to oxygen-sensing mechanisms. In addition, the interplay of PPARγ with other nuclear hormone receptors has implications for the balance between adipogenesis and osteogenesis in mesenchymal stem cells of the bone marrow stroma. This review will explore a range of factors influencing PPARγ activity and how these interactions may affect osteogenesis

Permutation test for periodicity in short time series data

Abstract Background Periodic processes, such as the circadian rhythm, are important factors modulating and coordinating transcription of genes governing key metabolic pathways. Theoretically, even small fluctuations in the orchestration of circadian gene expression patterns among different tissues may result in functional asynchrony at the organism level and may contribute to a wide range of pathologic disorders. Identification of circadian expression pattern in time series data is important, but equally challenging. Microarray technology allows estimation of relative expression of thousands of genes at each time point. However, this estimation often lacks precision and microarray experiments are prohibitively expensive, limiting the number of data points in a time series expression profile. The data produced in these experiments carries a high degree of stochastic variation, obscuring the periodic pattern and a limited number of replicates, typically covering not more than two complete periods of oscillation. Results To address this issue, we have developed a simple, but effective, computational technique for the identification of a periodic pattern in relatively short time series, typical for microarray studies of circadian expression. This test is based on a random permutation of time points in order to estimate non-randomness of a periodogram. The Permutated time, or Pt-test, is able to detect oscillations within a given period in expression profiles dominated by a high degree of stochastic fluctuations or oscillations of different irrelevant frequencies. We have conducted a comprehensive study of circadian expression on a large data set produced at PBRC, representing three different peripheral murine tissues. We have also re-analyzed a number of similar time series data sets produced and published independently by other research groups over the past few years. Conclusion The Permutated time test (Pt-test) is demonstrated to be effective for detection of periodicity in short time series typical for high-density microarray experiments. The software is a set of C++ programs available from the authors on the open source basis.</p

Diurnal rhythms of bone turnover markers in three ethnic groups

Context: Ethnic groups differ in fragility fracture risk and bone metabolism. Differences in diurnal rhythms (DRs) of bone turnover and PTH may play a role.  Objective: We investigated the DRs of plasma bone turnover markers (BTMs), PTH, and 1,25(OH)2D in three groups with pronounced differences in bone metabolism and plasma PTH.  Participants: Healthy Gambian, Chinese, and white British adults (ages 60–75 years; 30 per country).  Interventions: Observational study with sample collection every 4 hours for 24 hours.  Main Outcomes: Levels of plasma C-terminal telopeptide of type I collagen, procollagen type-1 N-propeptide, N-mid osteocalcin, bone alkaline phosphatase, PTH, and 1,25-dihydroxyvitamin D were measured. DRs were analyzed with random-effects Fourier regression and cross-correlation and regression analyses to assess associations between DRs and fasting and 24-hour means of BTMs and PTH.  Results: Concentrations of BTMs, PTH, and 1,25-dihydroxyvitamin D were higher in Gambians compared to other groups (P < .05). The DRs were significant for all variables and groups (P < .03) and were unimodal, with a nocturnal peak and a daytime nadir for BTMs, whereas PTH had two peaks. The DRs of BTMs and PTH were significantly cross-correlated for all groups (P < .05). There was a significant positive association between C-terminal telopeptide of type I collagen and PTH in the British and Gambian groups (P = .03), but not the Chinese group.  Conclusions: Despite ethnic differences in plasma BTMs and PTH, DRs were similar. This indicates that alteration of rhythmicity and loss of coupling of bone resorption and formation associated with an elevated PTH in other studies may not uniformly occur across different populations and needs to be considered in the interpretation of PTH as a risk factor of increased bone loss

The regulation and activation of ciliary neurotrophic factor signaling proteins in adipocytes

Ciliary neurotrophic factor (CNTF) is primarily known for its roles as a lesion factor released by the ruptured glial cells that prevent neuronal degeneration. However, CNTF has also been shown to cause weight loss in a variety of rodent models of obesity/type II diabetes, whereas a modified form also causes weight loss in humans. CNTF administration can correct or improve hyperinsulinemia, hyperphagia, and hyperlipidemia associated with these models of obesity. In order to investigate the effects of CNTF on fat cells, we examined the expression of CNTF receptor complex proteins (LIFR, gp130, and CNTFRα) during adipocyte differentiation and the effects of CNTF on STAT, Akt, and MAPK activation. We also examined the ability of CNTF to regulate the expression of adipocyte transcription factors and other adipogenic proteins. Our studies clearly demonstrate that the expression of two of the three CNTF receptor complex components, CNTFRα and LIFR, decreases during adipocyte differentiation. In contrast, gp130 expression is relatively unaffected by differentiation. In addition, preadipocytes are more sensitive to CNTF treatment than adipocytes, as judged by both STAT 3 and Akt activation. Despite decreased levels of CNTFRα expression in fully differentiated 3T3-L1 adipocytes, CNTF treatment of these cells resulted in a time-dependent activation of STAT 3. Chronic treatment of adipocytes resulted in a substantial decrease in fatty-acid synthase and a notable decline in SREBP-1 levels but had no effect on the expression of peroxisome proliferator-activated receptor γ, acrp30, adipocyte-expressed STAT proteins, or C/EBPa. However, CNTF resulted in a significant increase in IRS-1 expression. CNTFRα receptor expression was substantially induced in the fat pads of four rodent models of obesity/type II diabetes as compared with lean littermates. Moreover, we demonstrated that CNTF can activate STAT 3 in adipose tissue and skeletal muscle in vivo. In summary, CNTF affects adipocyte gene expression, and the specific receptor for this cytokine is induced in rodent models of obesity/type II diabetes

Cross-talk among gp130 cytokines in adipocytes

The interleukin-6 (IL-6) family of cytokines is a family of structurally and functionally related proteins, including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These proteins are also known as gp130 cytokines because they all share gp130 as a common transducer protein within their functional receptor complexes. Several of these cytokines (LIF, OSM, CNTF, and CT-1) also utilize the LIF receptor (LIFR) as a component of their receptor complex. We have shown that all of these cytokines are capable of activating both the JAK/STAT and p42/44 mitogen-activated protein kinase signaling pathways in 3T3-L1 adipocytes. By performing a variety of preincubation studies and examining the ability of these cytokines to activate STATs, ERKs, and induce transcription of SOCS-3 mRNA, we have also examined the ability of gp130 cytokines to modulate the action of their family members. Our results indicate that a subset of gp130 cytokines, in particular CT-1, LIF, and OSM, has the ability to impair subsequent signaling activity initiated by gp130 cytokines. However, IL-6 and CNTF do not exhibit this cross-talk ability. Moreover, our results indicate that the cross-talk among gp130 cytokines is mediated by the ability of these cytokines to induce ligand-dependent degradation of the LIFR, in a proteasome-independent manner, which coincides with decreased levels of LIFR at the plasma membrane. In summary, our results demonstrate that an inhibitory cross-talk among specific gp130 cytokines in 3T3-L1 adipocytes occurs as a result of specific degradation of LIFR via a lysosome-mediated pathway. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc