URB is abundantly expressed in adipose tissue and dysregulated in obesity

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-α, H₂O₂ and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.     

Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish

We have explored the possible mechanisms by which mineralocorticoid (MR) and glucocorticoid (GR) receptors regulate the response to freshwater transfer in the gills of the euryhaline killifish Fundulus heteroclitus. Killifish were implanted with RU486 (GR antagonist) or spironolactone (MR antagonist) at doses of 0.1–1.0 mg g⁻¹, and subsequently transferred from 10‰ brackish water to freshwater. Compared to brackish water sham fish, mRNA expression of CFTR and NKCC1 decreased in the gills of sham fish transferred to freshwater, whereas Na⁺,K⁺–ATPase α_1a mRNA expression and α protein abundance, as well as cell proliferation (detected using BrdU) increased. Spironolactone inhibited the normal increase in cell proliferation and Na⁺,K⁺-ATPase expression after freshwater transfer. RU486 increased plasma cortisol levels and may have slightly inhibited Na⁺,K⁺–ATPase activity, but did not change α _1a expression. RU486 had no effect on cell proliferation in the non-lamellar region of the gills, but increased proliferation in the lamellar region. Neither antagonist inhibited the suppression of CFTR or NKCC1 expression after freshwater transfer. Glucocorticoid receptor expression was reduced in all sham and antagonist treatments compared to untreated controls, but no other consistent differences were observed. The effects of spironolactone suggest that MR is important for regulating ion transport in killifish gills after freshwater transfer.     

Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity,and Inflammation in Fat

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H₂O₂, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.     

Apelin Attenuates Oxidative Stress in Human Adipocytes

It has been recently recognized that the increased oxidative stress (ROS overproduction) in obese condition is a key contributor to the pathogenesis of obesity-associated metabolic diseases. Apelin is an adipocytokine secreted by adipocytes, and known for its anti-obesity and anti-diabetic properties. In obesity, both oxidative stress and plasma level of apelin are increased. However, the regulatory roles of apelin on oxidative stress in adipocytes remain unknown. In the present study, we provide evidence that apelin, through its interaction with apelin receptor (APJ), suppresses production and release of reactive oxygen species (ROS) in adipocytes. This is further supported by the observations that apelin promotes the expression of anti-oxidant enzymes via MAPK kinase/ERK and AMPK pathways, and suppresses the expression of pro-oxidant enzyme via AMPK pathway. We further demonstrate that apelin is able to relieve oxidative stress-induced dysregulations of the expression of anti- and pro-oxidant enzymes, mitochondrial biogenesis and function, as well as release of pro- and anti-inflammatory adipocytokines. This study, for the first time, reveals the antioxidant properties of apelin in adipocytes, and suggests its potential as a novel therapeutic target for metabolic diseases.     

Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.     

11-Beta hydroxysteroid dehydrogenase type 2 expression in white adipose tissue is strongly correlated with adiposity

Glucocorticoid action within the cells is regulated by the levels of glucocorticoid receptor (GR) expression and two enzymes, 11-beta hydroxysteroid dehydrogenase type 1 (11betaHSD1), which converts inactive to active glucocorticoids, and 11-beta hydroxysteroid dehydrogenase type 2 (11betaHSD2), which regulates the access of active glucocorticoids to the receptor by converting cortisol/corticosterone to the glucocorticoid-inactive form cortisone/dehydrocorticosterone. Male Wistar rats developed obesity by being fed a high-fat diet for 56 days, and GR, 11betaHSD1 and 11betaHSD2 gene expression were compared with control-diet fed animals. Gene expression analysis of 11betaHSD1, 11betaHSD2 and GR were performed by RT-PCR in subcutaneous and retroperitoneal adipose tissue. High-fat fed animals overexpressed 11betaHSD2 in subcutaneous but not in retroperitoneal fat. Interestingly, mRNA levels strongly correlated in both tissues with different parameters related to obesity, such as body weight, adiposity and insulin resistance, suggesting that this gene is a reliable marker of adiposity in this rat model of obesity. Thus, 11betaHSD2 is expressed in adipose tissue by both adipocytes and stromal-vascular cells, which suggests that this enzyme may play an important role in preventing fat accumulation in adipose tissue.     

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15 ± 0.008 and the basal pHirr was 0.195 ± 0.012 pH units/min (number of tubules/number of tubular areas = 16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10^?12 M) increases the pHirr to approximately 59% of control value, and ALDO (10^?6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10^?6 M) or BAPTA (5 × 10^?5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²⁺]_i was 104 ± 3 nM (15), and ALDO (10^?12 or 10^?6 M) increased the basal [Ca²⁺]_i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²⁺]_i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²⁺]_i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.     

Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin

AimsThe polymethoxyflavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these flavonoids on the hypertrophy of mature adipocytes.Main methodsAll experiments were performed on the murine preadipocyte cell line 3T3-L1. We studied the formation of intracellular lipid droplets in adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyflavonoids on adipocyte differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA.Key findingsWe demonstrated that the combined treatment of differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 adipocytes. Both flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 adipocytes. Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 adipocytes, nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation.SignificanceOur results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature adipocytes.     

Food restriction improves glucose and lipid metabolism through Sirt1 expression: a study using a new rat model with obesity and severe hypertension

AimsTo determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome.Main methodsMale, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPARα, γ and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle.Key findingsFR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPARα and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUT4 expression in skeletal muscle.SignificanceThese results indicate that FR corrects adipokine dysfunction by activating PPARγ via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPARα in the liver.     

Effect of peroxisome proliferator-activated receptor-alpha ligands in the interaction between adipocytes and macrophages in obese adipose tissue

Objective: This study was designed to examine the effect of peroxisome proliferator‐activated receptor‐α (PPAR‐α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR‐α ligands (Wy‐14,643 and fenofibrate) were added to 3T3‐L1 adipocytes, RAW264 macrophages, or co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression and secretion were examined. PPAR‐α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR‐α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR‐α‐deficient mice. Results: In the co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages, PPAR‐α ligands reduced MCP‐1 and TNF‐α mRNA expression and secretion in vitro relative to vehicle‐treated group. The anti‐inflammatory effect of Wy‐14,643 was observed in adipocytes treated with macrophage‐conditioned media or mouse recombinant TNF‐α and in macrophages treated with adipocyte‐conditioned media or palmitate. Systemic administration of PPAR‐α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy‐14,643 also exerted an anti‐inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR‐α‐deficient mice. Discussion: This study provides evidence for the anti‐inflammatory effect of PPAR‐α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity‐related metabolic syndrome.     

Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.     

The metabolic syndrome and adipocytokines

Visceral fat accumulation has been shown to play crucial roles in the development of cardiovascular disease as well as the development of obesity-related disorders such as diabetes mellitus, hyperlipidemia and hypertension and the so-called metabolic syndrome. Given these clinical findings, adipocytes functions have been intensively investigated in the past 10 years, and have been revealed to act as endocrine cells that have been termed adipocytokines, which secrete various bioactive substances. Among adipocytokines, tumor necrosis factor-alpha, plasminogen activator inhibitor type 1 and heparin binding epidermal growth factor-like growth factor are produced in adipocytes as well as other organs, and may contribute to the development of vascular diseases. Visfatin has been identified as a visceral-fat-specific protein that might be involved in the development of obesity-related diseases, such as diabetes mellitus and cardiovascular disease. On the contrary to these adipocytokines, adiponectin, an adipose-tissue-specific, collagen-like protein, has been noted as an important antiatherogenic and antidiabetic protein, or as an anti-inflammatory protein. The functions of adipocytokine secretion might be regulated dynamically by nutritional state. Visceral fat accumulation causes dysregulation of adipocyte functions, including oversecretion of tumor necrosis factor-alpha, plasminogen activator inhibitor type 1 and heparin binding epidermal growth factor-like growth and hyposecretion of adiponectin, which results in the development of a variety of metabolic and circulatory diseases. In this review, the importance of adipocytokines, especially focusing on adiponectin is discussed with respect to cardiovascular diseases.     

JUP/plakoglobin is regulated by salt-inducible kinase 2, and is required for insulin-induced signalling and glucose uptake in adipocytes

BackgroundSalt-inducible kinase 2 (SIK2) is abundant in adipocytes, but downregulated in adipose tissue from individuals with obesity and insulin resistance. Moreover, SIK isoforms are required for normal insulin signalling and glucose uptake in adipocytes, but the underlying molecular mechanisms are currently not known. The adherens junction protein JUP, also termed plakoglobin or γ-catenin, has recently been reported to promote insulin signalling in muscle cells.ObjectiveThe objective of this study was to analyse if JUP is required for insulin signalling in adipocytes and the underlying molecular mechanisms of this regulation.MethodsCo-expression of SIK2 and JUP mRNA levels in adipose tissue from a human cohort was analysed. siRNA silencing and/or pharmacological inhibition of SIK2, JUP, class IIa HDACs and CRTC2 was employed in 3T3-L1- and primary rat adipocytes. JUP protein expression was analysed by western blot and mRNA levels by qPCR. Insulin signalling was evaluated by western blot as levels of phosphorylated PKB/Akt and AS160, and by monitoring the uptake of ³H-2-deoxyglucose.ResultsmRNA expression of SIK2 correlated with that of JUP in human adipose tissue. SIK2 inhibition or silencing resulted in downregulation of JUP mRNA and protein expression in 3T3-L1- and in primary rat adipocytes. Moreover, JUP silencing reduced the expression of PKB and the downstream substrate AS160, and consequently attenuated activity in the insulin signalling pathway, including insulin-induced glucose uptake. The known SIK2 substrates CRTC2 and class IIa HDACs were found to play a role in the SIK-mediated regulation of JUP expression.ConclusionsThese findings identify JUP as a novel player in the regulation of insulin sensitivity in adipocytes, and suggest that changes in JUP expression could contribute to the effect of SIK2 on insulin signalling in these cells.     

A highly sensitive enzyme-linked immunosorbent assay for quantification of adipocytokines secreted by mouse adipocytes

The mouse preadipocyte 3T3-L1 line is the most useful cell line for the study of adipocytes. Adipocytes secrete adipocytokines, and abnormal adipocytokine production can cause the metabolic syndrome. Although it is important to understand the characteristics of mouse adipocytokine secretion, it is difficult to quantify these products because they are produced in low concentrations. Here, we developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for detecting the concentrations of mouse adipocytokines, such as TNFα and leptin. In this method, the antigen was sandwiched by using goat- and rabbit-derived polyclonal antibodies, and the fluorescence intensity produced in the reaction with 4-methylumbelliferyl-β-galacto-sidase pyranosidase (MUG) was measured. TNFα and leptin could be measured at concentrations as low as approximately 1 pg/ml. By using our ELISA method, we also measured the concentrations of TNFα and leptin in mouse 3T3-L1 preadipocytes and adipocytes. The differentiation of preadipocytes into adipocytes enhanced TNFα production and secretion and reduced the leptin production. The amount of TNFα produced in the adipocytes was 3.0 ng/mg-protein; this amount was considerably higher than that of leptin.