Hormonal signaling and transcriptional control of adipocyte differentiation.

Recent advances regarding the biology of adipose tissue have identified the adipocyte as an important mediator in many physiologic and pathologic processes regarding energy metabolism. Consideration for a central role of adipose tissue in the development of obesity, cardiovascular disease and noninsulin-dependent diabetes mellitus has resulted in new incentives toward understanding the complexities of adipocyte differentiation. Current knowledge of this process includes a cascade of transcriptional events that culminate in the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha). These prominent adipogenic transcription factors have been shown to regulate, directly or indirectly, the gene expression necessary for the development of the mature adipocyte. Hormonal and nutritional signaling that impinges on these trans-acting factors provides a molecular link between lipids and lipid-related compounds and the gene expression important for glucose and lipid homeostasis. Knowledge concerning the transcriptional events mediating adipocyte differentiation provides a basis for understanding the physiologic processes associated with adipose tissue as well as for the development of therapeutic interventions in obesity and its related disorders.

Regulation of signal transducers and activators of transcription (STATs) by effectors of adipogenesis: coordinate regulation of STATs 1, 5A, and 5B with peroxisome proliferator-activated receptor-gamma and C/AAAT enhancer binding protein-alpha.

We have recently demonstrated that three signal transducers and activators of transcription (STAT) family members are induced during adipocyte differentiation (Stephens et al., J. Biol. Chem. 271 (1996) 10441-10444). Since STATs 1, 5A, and 5B are induced during adipocyte differentiation, we have examined the ability of these proteins to be regulated by components of the differentiation cocktail. In addition, we have examined the effects of potent effectors of differentiation on STAT protein expression during adipogenesis. A negative effector, tumor necrosis factor-alpha (TNFalpha), and a positive effector, a thiazolidinedione, were used in these experiments. Our results demonstrate that the expression of STATs 1, 5A, and 5B is not dramatically influenced by individual components of the differentiation cocktail. However, the expression of these three STAT family members tightly correlates with lipid accumulation. Moreover, the expression of STATs 1, 5A, and 5B, but not STATs 3 and 6, are regulated in an identical fashion to both C/AAAT enhancer binding proteins alpha and peroxisome proliferator-activated receptor-gamma by TNFalpha and a thiazolidinedione. Furthermore, the expression of adipocyte-expressed JAK kinases are unaffected by effectors of differentiation. These findings suggest that three STAT family members may play a role in the regulation of adipocyte gene expression.

PPARgamma ligand-dependent induction of STAT1, STAT5A, and STAT5B during adipogenesis.

We have recently demonstrated that STAT1, STAT5A, and STAT5B are induced during adipogenesis of cultured preadipocytes in a differentiation-dependent manner. Members of the C/EBP and PPAR families of transcription factors have also been shown to be induced during adipocyte differentiation and to play a significant role in the regulation of fat-specific genes. In this investigation, we have examined the ability of C/EBPs and PPARs to contribute to STAT protein expression during conversion of non-precursor fibroblasts to functionally mature adipocytes. For this study, NIH-3T3 fibroblasts engineered to ectopically co-express C/EBPbeta and C/EBPdelta under the control of a tetracycline-responsive, inducible expression system were utilized to assess STAT expression during controlled adipogenesis. Data presented here demonstrate that STAT1, STAT5A, and STAT5B, but not STAT3 and STAT6, were induced in a tetracycline-responsive manner during the differentiation of these engineered fibroblasts. The STAT protein accumulation resulting from C/EBP expression was tightly coupled to the morphological conversion of fibroblasts to adipocytes and represents an expression profile identical to that reported for mature adipocytes in vivo. Data are also presented demonstrating that STAT protein accumulation and adipocyte conversion occurred only during controlled conditions leading to the expression of PPARgamma and that the expression of these three STATs was tightly regulated in a PPARgamma ligand dose-response fashion. These data illustrate that the cascade of transcriptional events leading to adipogenesis regulate the STAT family of transcription factors and that the differentiation-dependent upregulation of STAT protein expression is regulated downstream of PPARgamma in a ligand-dependent manner.

Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21(Waf1/Cip1), during adipogenesis.

Molecular mechanisms coupling growth arrest and cell differentiation were examined during adipogenesis. Data are presented that document a cascade expression of members of two independent families of cyclin-dependent kinase inhibitors that define distinct states of growth arrest during 3T3-L1 preadipocyte differentiation. Exit from the cell cycle into a pre-differentiation state of post-mitotic growth arrest was characterized by significant increases in p21 and p27. During onset of irreversible growth arrest associated with terminal differentiation, the level of p21 declined with a concomitant, dramatic increase in p18 and a sustained level of p27. The expression of p18 and p21, regulated at the level of protein and mRNA accumulation, was directly coupled to differentiation. Stable cell lines were engineered to express adipogenic transcription factors to examine the active role of trans-acting elements in regulating these cell cycle inhibitors. Ectopic expression of peroxisome proliferator-activated receptor (PPAR) gamma in non-precursor fibroblastic cell lines resulted in conversion to adipocytes and a coordinated increase in p18 and p21 mRNA and protein expression in a PPARgamma ligand-associated manner. These data demonstrate a role for PPARgamma in mediating the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors, thereby providing a molecular mechanism coupling growth arrest and adipocyte differentiation.

Insights into the transcriptional control of adipocyte differentiation.

The adipocyte is now known to play an active role in many physiological and pathological processes regarding energy metabolism. Consideration of adipose tissue as an endocrine organ that secretes a variety of unrelated bioactive molecules has broadened our appreciation of adipocyte function to exceed the once considered passive role in lipid metabolism. Growing interest in this tissue has lead to significant advances regarding the molecular basis for adipocyte differentiation. Several diverse families of transcription factors are currently under active investigation for their roles in mediating this complex process. Knowledge concerning the sequence of transcriptional events during adipogenesis and the interplay among adipogenic transcription factors provides a basis for understanding the physiological processes associated with adipose tissue as well as for the development of therapeutic intervention of adipocyte related diseases. J. Cell. Biochem. Suppls. 32/33:59-67, 1999.

PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes.

To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression.

The expression and regulation of STATs during 3T3-L1 adipocyte differentiation.

STATs (Signal Transducers and Activators of Transcription) comprise a family of transcription factors that reside in the cytoplasm of resting cells. In response to a variety of stimuli, STATs become tyrosine-phosphorylated and translocate to the nucleus where they mediate transcriptional regulation. We have used the 3T3-L1 murine cell line to examine the expression of STAT proteins as a function of their differentiation into adipocytes. The expression of STATs 1, 3, and 5, but not of STAT 6, is markedly elevated in adipocytes as compared with their fibroblast precursors. Exposure of 3T3-L1 preadipocytes to tumor necrosis factor alpha (TNF alpha) blocks their differentiation into adipocytes. Therefore, we examined STAT expression as a function of differentiation in the presence of this cytokine. The expression of STATs 1 and 5 is markedly attenuated in the presence of TNF alpha, whereas STAT 3 expression is unaffected by this treatment. Only STAT 1 is down-regulated by TNF alpha in fully differentiated cells. Thus, although the expression of STATs 1, 3, and 5 is markedly enhanced upon differentiation, only STAT 5 expression is tightly correlated with the adipocyte phenotype. These data suggest that STAT 5, and possibly STAT 1, could be potential inducers of tissue-specific genes, which contribute to the development and maintenance of the adipocyte phenotype.

Cell spreading and the regulation of ornithine decarboxylase.

The aim of this study was to investigate the effect of cell spreading on the induction of ornithine decarboxylase and the rate of putrescine uptake in anchorage-dependent and anchorage-independent cells. Plating non-transformed IEC-6 epithelial cells at high versus low cell density restricted cell spreading from 900 microns 2 to approximately 140 microns 2, blunted the transient induction of ornithine decarboxylase activity from 202 to 32 pmol 14CO2/mg protein per hour and reduced the rate of [14C] putrescine uptake from 46 to 23 pmol/10(5) cells per hour. The mean spreading area of the cell population was controlled by coating tissue culture dishes with the nonadhesive polymer, polyHEMA. Ornithine decarboxylase activity and putrescine uptake correlated with cell spreading with minimal spreading (263 microns 2) corresponding to an 83% decrease in ornithine decarboxylase activity and 51% decrease in the rate of putrescine uptake. Adding the RGD peptide, Gly-Arg-Gly-Glu-Ser-Pro to the medium of sparsely plated cells resulted in rapid reductions in cell spreading concomitant with dose-dependent decreases in ornithine decarboxylase activity and putrescine uptake. Finally, minimizing cell spreading by depriving cells of substratum contact completely abolished serum-induced increases in ornithine decarboxylase and reduced the rate of putrescine uptake by 47%. In contrast to IEC-6 cells, ornithine decarboxylase of neoplastic HTC-116 cells was constitutively expressed with basal and stimulated activity (193 and 982 pmol 14CO2/mg protein per hour, respectively) completely independent of cell adhesion. Putrescine uptake, however, was abolished in the absence of cell adhesion. These data suggest that the induction of ornithine decarboxylase activity and the rate of putrescine uptake correlate with spreading of anchorage-dependent IEC-6 cells and that ornithine decarboxylase activity but not putrescine uptake, appears to be independent of spreading of neoplastic HTC-116 cells.

Allopurinol improves myocardial reperfusion injury in a xanthine oxidase-free model.

The ability of allopurinol to protect against reperfusion injury in the heart has usually been attributed to its xanthine oxidase (XO)-inhibiting properties. Human myocardium however, has exhibited low levels of XO activity. To investigate the effects of allopurinol in an XO-free model and determine whether pretreatment is necessary, 12 domestic pigs (15 kg to 20 kg) underwent occlusion of the left circumflex for 8 minutes followed by reperfusion for 4 hours. One group received allopurinol infusion (5 mg/kg IV) at occlusion over 45 minutes and a control group (n = 6) received a saline infusion (same volume). Left ventricular and aortic pressure, electrocardiograms, and regional wall motion (sonomicrometry) were monitored throughout the process. Regional blood flow (microspheres) were obtained before, during, and 5, 10, and 30 minutes after ischemia. Occlusion decreased transmural flow at the midpapillary level by 75% (0.28 versus 1.10 mL/minute/g). The allopurinol-treated group exhibited a mild, generalized hyperemia at 5 minutes (ischemic zone: 1.44 versus 1.10 mL/min/g, which returned to control levels at 10 and 30 minutes. In contrast, the control group was associated with only 80% restoration of resting blood flow at 5 minutes (0.84 versus 1.10 mL/min/g), which stabilized at 63% of control levels at 10 and 30 minutes. When evaluated for the propensity of arrhythmias using an arbitrary arrhythmia score, the allopurinol group demonstrated no myocardial ectopy when compared with the focal ectopy routinely encountered in the control group at all time intervals.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular endothelial cell proliferation: regulation of cellular polyamines.

OBJECTIVE: The aims were to investigate the requirement for and regulation of cellular polyamines during vascular endothelial cell proliferation. METHODS: Proliferation of cultured bovine pulmonary artery endothelial cells was determined after cellular polyamine depletion. In addition, polyamine synthesis and uptake mechanisms were examined in the presence and absence of trophic stimuli and density dependent inhibition of cell proliferation. RESULTS: Serum stimulated, subconfluent cells ceased cell division following the inhibition of ornithine decarboxylase (ODC), a rate limiting enzyme for polyamine biosynthesis. The addition of 10 microM putrescine, the product of the enzyme reaction catalysed by ODC, completely reversed the inhibition of cell growth. Serum deprivation reduced cytosolic ODC activity to near non-detectable levels. Readdition of 10% fetal bovine serum (FBS) resulted in transient increases in ODC activity which preceded DNA synthesis and mitosis. Basic fibroblast growth factor also stimulated ODC activity in a dose dependent manner with levels approximating the maximum obtainable with FBS. In contrast, platelet derived growth factor and epidermal growth factor did not stimulate ODC activity. Finally, mitogenic stimuli did not induce ODC activity in density arrested cells. The uptake of radiolabelled putrescine from the cell medium was time dependent and saturable. Kinetic studies from dividing cells revealed a single transport component for putrescine uptake [maximum rate of uptake (Vmax) = 11.2(SEM 2.0) pmol.10(5) cells-1.h-1; Michaelis constant (Km) = 1.1(0.3) microM]. Putrescine uptake by density arrested cells was characterised by a 57% decrease in Vmax with no change in Km. Readdition of FBS to serum deprived subconfluent cells, in the presence of ODC inhibitors, resulted in a rapid increase in the rate of putrescine uptake with Vmax increasing by 533% over FBS alone by 48 h. DISCUSSION: These data suggest that polyamines are essential for endothelial cell proliferation and that synthesis and uptake mechanisms are regulated according to cell growth.

Improved protection of chronically inflow-limited myocardium with retrograde coronary sinus cardioplegia.

The effects of retrograde coronary sinus cardioplegia (CSC) and conventional aortic route cardioplegia (ARC) on reperfusion blood flow patterns were compared in 18 mongrel dogs having ameroid-induced coronary collaterals. Animals were placed on cardiopulmonary bypass and cooled systemically. The aorta was clamped, and the hearts were arrested with a bolus infusion of hypothermic (4 degrees C) hyperkalemic cardioplegic solution delivered either into the aortic root or through a balloon catheter placed in the coronary sinus. After 45 minutes of ischemic arrest, hearts were reperfused, and blood flow patterns were measured at 1, 5, 10, and 60 minutes. In the normal region, a significant hyperemia occurred with both CSC and ARC. However, the peak reactive transmural flow was 120% more with ARC than with CSC (4.55 +/- 0.45 vs. 2.12 +/- 0.19 ml/min/g, respectively; p less than 0.05) and remained elevated after 60 minutes of reperfusion, when CSC flows had returned to control levels. In the collateral-dependent region, a significant hyperemia was observed with ARC that persisted throughout reperfusion. However, with CSC, no significant changes in blood flow were detected at any time during reperfusion. Thus, the decreased hyperemic response after arrest suggests a reduced energetic debt with CSC compared with ARC and may indicate superior myocardial protection with CSC. This is particularly evident in chronically inflow-restricted myocardial regions.

Study of periodontal disease and coenzyme Q.

A 25-year old Caucasian with uniquely severe and chronic periodontal disease, requiring extraction of all teeth, was treated with hexahydrocoenzyme Q4 which functions like naturally occurring coenzyme Q10 in gingival tissue. During the fourth and fifth examinations after the seventh and eighth weeks of treatment, three dentists separately and independently scored clinical improvements of five symptoms by significances of essentially P less than 0.01 to P less than 0.001. The initial benefit of the treatment was observed at the second examination after three weeks of treatment. The administration of coenzyme Q could be an important therapeutic adjunct to periodontal therapy for certain patients.