Identification of omentin mRNA in human epicardial adipose tissue: comparison to omentin in subcutaneous, internal mammary artery periadventitial and visceral abdominal depots.

OBJECTIVE: The purpose of this study was to determine the relative distribution of omentin and visfatin mRNA in human epicardial, peri-internal mammary, upper thoracic, upper abdominal and leg vein subcutaneous adipose tissue as well as the distribution of omentin in the nonfat cells and adipocytes of human omental adipose tissue. BACKGROUND: Omentin is found in human omentum but not subcutaneous fat. Omentin and visfatin are considered markers of visceral abdominal fat. RESEARCH DESIGN AND METHODS: The mRNA content of omentin and visfatin was measured by qRT-PCR analysis of fat samples removed from humans undergoing cardiac or bariatric surgery. RESULTS: Omentin mRNA in internal mammary fat was 3.5%, that in the upper thoracic subcutaneous fat was 4.7% while that in the other subcutaneous fat depots was less than 1% of omentin in epicardial fat. The distribution of visfatin mRNA did not vary between the five depots. Omentin mRNA was preferentially expressed in the nonfat cells of omental adipose tissue since the omentin mRNA content of isolated adipocytes was 9% of that in nonfat cells, and similar results were seen for visfatin. The amount of omentin mRNA in differentiated adipocytes was 0.3% and that of visfatin 4% of that in nonfat cells. The amount of omentin mRNA in preadipocytes was virtually undetectable while that of visfatin was 3% of that in freshly isolated nonfat cells from omental adipose tissue. CONCLUSION: Omentin mRNA is predominantly found in epicardial and omental human fat whereas visfatin mRNA is found to the same extent in epicardial, subcutaneous and omental fat.

Regulation of adiponectin release and demonstration of adiponectin mRNA as well as release by the non-fat cells of human omental adipose tissue.

OBJECTIVE: Adiponectin is an adipokine produced by adipose tissue. The present studies examined the in vitro release of adiponectin by human omental adipose tissue explants as well as the mRNA content of freshly isolated non-fat cells and adipocytes plus cultured preadipocytes and adipocytes derived from omental fat. RESULTS: The release of adiponectin was reduced while that of interleukin-8 (IL-8) was enhanced in tissue explants from morbidly obese women. The release of adiponectin was also reduced by one-third in explants from morbidly obese diabetic women while that of IL-8 was unaffected by diabetes. The release of adiponectin was enhanced by insulin and by inhibition of endogenous tumor necrosis factor (TNFalpha) using etancercept. Adiponectin was released in appreciable amounts by the undigested matrix obtained by collagenase digestion of adipose tissue. The release of adiponectin by non-fat cells (matrix+SV cells) was comparable to that by the adipocytes derived from the same amount of tissue while the adiponectin mRNA content of the pooled matrix and SV cell fractions was 40% of that in intact tissue. The adiponectin mRNA content was 48-fold greater in isolated adipocytes than in non-fat cells derived from adipose tissue. In contrast, the amount of adiponectin mRNA in vitro differentiated omental adipocytes was 1 x l0(6)-fold greater than that in cultured preadipocytes while that of leptin was 3 x 10(4)-fold greater. CONCLUSION: Adiponectin mRNA is present in the non-fat cells of freshly isolated adipose tissue and release by the non-fat cells derived from a gram of adipose tissue is comparable to that by adipocytes isolated from the same amount of tissue. While leptin is only released by mature adipocytes, adiponectin is released by both the non-fat cells and the fat cells derived from human omental adipose tissue.

Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.

Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.

Coordination of the mating and cell integrity mitogen-activated protein kinase pathways in Saccharomyces cerevisiae.

Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner. The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1. In this report, we show that Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis. Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade. Additionally, Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation. Both proteins also affect the timing and extent of Mpk1 activation. This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation. Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2. Surprisingly, Mpk1 activation by pheromone was only partially impaired in cells lacking the MEK kinase Bck1. This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions.

Protein kinase C-dependent regulation of human erythroleukemia (HEL) cell sphingosine kinase activity.

Sphingosine kinase functions in both the catabolism of sphingosine and in signal transduction pathways utilizing sphingosine-1-phosphate. The regulation of sphingosine kinase activity in human erythroleukemia (HEL) cells was investigated by treatment with several bioactive agents. Treatment of HEL cells with phorbol 12-myristate 13-acetate (PMA) caused a time- and concentration-dependent increase in sphingosine kinase activity measured in vitro. Sphingosine kinase activity increased in a phorbol ester- and diacylglycerol-specific manner. Staurosporine and calphostin C, protein kinase C (PKC) inhibitors, blocked the increased in sphingosine kinase activity, suggesting a PKC-dependent regulation. The effects of PMA on sphingosine kinase were dependent on transcription and translation. Purified PKC had no direct effect on sphingosine kinase activity. However, these studies led to the observation that HEL cell sphingosine kinase activity is stimulated in vitro by phosphatidylserine. Interestingly, other inducers of HEL cell differentiation, dimethylsulfoxide and retinoic acid, did not affect sphingosine kinase activity. These results indicate a separate and distinct pathway of PKC-dependent sphingosine kinase activation, and suggest a role for sphingosine kinase in regulation of cell function.

A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway.

Role of ceramide in mitogenesis induced by exogenous sphingoid bases.

Dihydrosphingosine, an intermediate in the de novo synthesis of ceramide, induced proliferation of Swiss 3T3 cells. The proliferative effects of this lipid were much more potent than those of sphingosine, a break-down product of ceramide. The maximal proliferative response to dihydrosphingosine occurred at relatively low concentrations (1 microM), while sphingosine produced its maximal effect at much higher concentrations (15 microM). The cell-permeable ceramide, N-hexanoylsphingosine (C6-ceramide), which was also a mitogen in these cells (at 1 microM), caused a striking morphological change when added to the cells at concentrations of 5-10 microM. This shape change was reversible with the removal of ceramide. Exogenous dihydrosphingosines and sphingosines have at least two metabolic fates in Swiss 3T3 cells, conversion to ceramide or to sphingosine 1-phosphate. Surprisingly, both the synthetic threo- isomer and the naturally occurring erythro- isomer of dihydrosphingosine and sphingosine (D-erythro-sphingosine, L-threo-sphingosine, DL-threo-dihydrosphingosine, and DL-erythro-dihydrosphingosine) were readily phosphorylated in intact Swiss 3T3 cells. This substrate specificity may be an indication of a sphingosine kinase activity which is distinct from that of platelets or rat brain. Although sphingosine 1-phosphate and ceramide were both produced upon the addition of sphingosine and dihydrosphingosine, no sphingosine 1-phosphate was produced when Swiss 3T3 cells were treated with mitogenic concentrations of C6-ceramide. These data are consistent with the formation of ceramide and not sphingosine 1-phosphate being required for the mitogenesis produced by exogenous sphingoid bases.

Inhibition of sphingosine kinase in vitro and in platelets. Implications for signal transduction pathways.

Sphingosine kinase was partially purified and characterized from rat brain microsomes. A new assay, utilizing octyl-beta-D-glucopyranoside and sphingosine mixed micelles, was developed to quantitate formation of the sphingosine-1-phosphate product. The assay was proportional with respect to time and protein, displayed Michaelis-Menten kinetics, and was subject to surface dilution in regard to the sphingosine substrate. Investigations into substrate specificity showed that the enzyme is specific for the erythro-enantiomers of sphingosine and dihydrosphingosine. Neither of the threo-enantiomers were phosphorylated in this system, but both were found to be potent competitive inhibitors of sphingosine kinase activity. Human platelet sphingosine kinase activity displayed substrate and inhibitor specificities similar to the rat brain enzyme. A mixture of DL-threo-dihydrosphingosine competitively inhibited sphingosine kinase activity in a dose dependent manner in isolated platelets. DL-Threo-dihydrosphingosine caused a prolongation of the inhibition of thrombin-induced protein kinase C-dependent 40 (47)-kDa protein phosphorylation in platelets. D-, L-, or DL-Threo-dihydrosphingosine may be useful as a tool to investigate D-Erythrosphingosine metabolism and the function of sphingosine-1-phosphate in signal transduction processes.