Studies on the compartmentation of lipid in adipose cells. II. Cholesterol accumulation and distribution in adipose tissue components

Adipose tissue was shown to contain 0.6-1.6 mg of cholesterol per gram wet weight. When expressed per unit of protein or organ mass, fat tissue contains more cholesterol than most other organs or membranes. The cholesterol content of fat tissue increased with the age and weight of the rat. Over 95% of adipose tissue sterols was cholesterol, and most of it was free. In young (150-165 g) rats two-thirds of fat tissue cholesterol was in collagenase-derived adipocytes while in older rats (450-480 g) 90% of fat tissue cholesterol was in adipocytes and the remainder was in stromal-vascular elements. Age-related differences in subcellular cholesterol distribution were also observed. The cholesterol/phospholipid molar ratios of purified plasma membrane fractions from small and large fat cells were identical (0.22-0.25), thus resembling muscle and liver membranes. 7.5 days after intravenous administration of [4-(14)C]cholesterol the specific activity of adipose cholesterol exceeded that of plasma cholesterol. At 28 days the specific activity of adipose and muscle cholesterol exceeded that of plasma three- to fivefold. The t((1/2)) disappearance of adipose tissue cholesterol was approximately 27 days, which is consistent with its function as a slowly turning over storage pool. Thus, fat tissue is a major cholesterol storage organ. This may well account for the marked expansion of the slowly exchangeable cholesterol pool (pool B) observed in obesity.     

Changes in N-acetylaspartate and myo-inositol detected in the cerebral cortex of hamsters with Creutzfeldt-Jakob disease

Thiazolidinediones are potent antidiabetic compounds, which act by enhancing peripheral insulin sensitivity. They are also activators of the peroxisome proliferator activated receptor gamma in adipose tissue. Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat and hence the capacity of adipose tissue to utilize glucose. Nevertheless, muscles are the major site for insulin-mediated glucose disposal. The increase of muscle glucose utilization under thiazolidinedione treatment could be secondary to local adipose tissue differentiation. This possibility is supported by the fact that a thiazolidinedione-induced myoblast conversion into adipocytes has been described in vitro. To address this problem, we have studied the in vivo effect of a pioglitazone treatment on insulin-induced glucose utilization and the expression of genes exclusively expressed in mature adipocytes in three muscles differing by their fibre composition in Zucker (fa/fa) rats. Whereas pioglitazone treatment increased insulin-stimulated glucose utilization to the same extent in all muscle types, an adipocyte differentiation was only present in the oxidative muscle, the soleus. Soleus muscle was also the only one in which the presence of genes specific for adipose tissue could be detected before the pioglitazone treatment. There was no detectable expression of adipocyte specific genes in the extensor digitorum longus or in the epitrochlearis muscles before or after the drug treatment. We conclude that pioglitazone effects on muscle glucose metabolism cannot be due to a local adipocyte differentiation, and that the conversion of myoblasts into adipocytes under thiazolidinedione stimulation observed in vitro is, if it exists, a marginal phenomenon in vivo.     

Uterine bizarre lipoleiomyoma

A case of uterine bizarre lipoleiomyoma occurring in a 58-year-old woman is described. A subserosal 8.5 cm mass in the posterior wall of the body had a lipoma-like appearance with strands of fibrous tissue. Histologically, the tumor predominantly consisted of adipocytes which varied in size and shape. Some cells had the appearance of signet ring cells, some had hyperchromatic nuclei, and multivacuolated lipoblasts with hyperchromatic nuclei were observed throughout the lesion. The fibrous areas showed a proliferation of smooth muscle cells with and without atypical nuclei. There was no mitotic figure. Flow cytometric analysis indicated aneuploidy. The patient was well without disease 10 months after hysterectomy. Patients with this type of tumor need a close and long-term follow-up because of the paucity of clinical information.     

Regional differences in triacylglycerol synthesis in adipose tissue and in cultured preadipocytes

The initial suspicion that obesity increases coronary risk has been much sharpened with the demonstration that risk is more tightly linked to abdominal than to peripheral obesity, and tighter yet again when the mass of omental adipose tissue is taken into account. These data suggest that important metabolic differences might exist between adipocytes from different regions, and indeed, it has long been appreciated that triacylglycerol hydrolysis can be stimulated to a greater extent in omental than in subcutaneous adipocytes. The present study focuses on triacylglycerol synthesis in human subcutaneous and omental adipocytes, a process which, by contrast, has received relatively little attention. Experiments were done on adipose tissue removed at laparotomy and on cultured preadipocytes. With the former, triacylglycerol synthesis was measured in the presence and absence of oleate added to the medium using radiolabeled glucose and oleate as tracers. The results demonstrate that under all conditions examined triacylglycerol synthesis in subcutaneous adipose tissue exceeded that in deep omental adipose tissue. To study the cells in more detail, preadipocytes were cultured and triacylglycerol synthesis was examined again under basal conditions and with stimulation with insulin and acylation stimulating protein (ASP). Under basal conditions, particularly when oleate was added to the medium, clear differences were present such that triacylglycerol synthesis was substantially greater in subcutaneous preadipocytes than in omentally derived preadipocytes. These differences were more pronounced when the cells were stimulated with either insulin or acylation stimulating protein. Overall, triacylglycerol synthetic capacity in subcutaneous tissue exceeded that in omental tissue. As a consequence, omental tissue as compared to subcutaneous adipose tissue would have a limited capacity to prevent fatty acids from reaching the liver and stimulating hepatic lipoprotein synthesis.     

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.     

Uterine angiomyolipoma associated with pregnancy

Uterine angiomyolipomas are rare lesions composed of mature adipose tissue, smooth muscle, fibrous connective tissue, and blood vessels in varying proportions. We reported the first case of angiomyolipoma associated with a normal pregnancy. Initially, the tumor developed intramurally and could have been confused with a partial molar gestation. After delivery, tumor development was extensive and subserosal, making differential diagnosis from a sarcoma difficult. The question of histological diagnosis, as well as that of immunocytochemical analysis which seems to be helpful in such cases, is discussed here.     

Tumor necrosis factor-alpha induces apoptosis of human adipose cells

Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.     

Enterotoxigenicity as an attribute of virulence in Yersinia enterocolitica

As previously reported, we have discovered that a novel compound, NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl)carbamoyl] benzylphosphonate) has a powerful lipoprotein lipase (LPL) stimulating activity. Oral administration of NO-1886 increased LPL activity in postheparin plasma of experimental animals, resulting in the reduction of plasma triglyceride with concomitant elevation of high density lipoprotein cholesterol. However, the mechanism of NO-1886 on LPL activity is not clearly understood. To address this problem, we examined the effect of NO-1886 on LPL activity in primary rat cell culture isolated from adipose and skeletal muscle tissue. NO-1886 increased total LPL activity 18% and 23% in adipocytes at a dose of 3 and 10 micrograms/ml, respectively, and 43% at a dose of 10 micrograms/ml in skeletal muscle cells. These results indicate that NO-1886 may act directly on LPL-producing cells such as adipose and skeletal muscle.     

Evaluation of alternative methods to prepare porcine adipocytes for measurement with an electronic particle number and size determination apparatus

Experimental investigations with mammalian adipose tissue require a determination of adipocyte number as a basis for expression of metabolic and growth data. Determination of cell size is also important in adipose tissue because the fivefold or greater variation in adipocyte diameter in most growing and adult mammals precludes simple determination of cell number to interpret the biological observations. There are two approaches to determine adipocyte size and number: microscopic methods and electronic particle counter methods. Microscopic methods use embedded sections, frozen sections, or isolated cells, whereas electronic particle number and size instrumental methods use adipocytes released from fixed tissue fragments or adipocytes fixed after isolation. The advantage of the electronic approach is that it evaluates thousands of particles, although the standard fixative, osmium, is quite toxic. Consequently, we evaluated a number of alternative fixation methods to prepare isolated porcine adipocytes for number and size determination by electronic instrumentation. Fixation in 3, 4, or 5% glutaraldehyde or in 4% formaldehyde were not acceptable procedures for porcine adipocytes. The 4% glutaraldehyde fixation procedure was acceptable for isolated rat adipocytes (Stewart and Kaplan, 1993); porcine adipocytes seem to be much more susceptible to breakage using these procedures than rat adipocytes. We also added urea or Triton X-100 to glutaraldehyde- and osmium-fixed cells to decrease clumping and adhesion of individual cells; none of these additions was beneficial. Ability to store samples would improve the logistics for these time-consuming analyses. Samples of osmium-fixed adipocytes were stored in osmium, in .9% NaCl (saline) after removal of osmium, in 8 M urea after osmium removal with saline, or in .01% Triton X-100 after osmium removal with saline. Storage in urea or Triton was inappropriate because of irreversible clumping of individual cells. Storage in osmium was acceptable for at least 30 d. and storage in saline was marginally acceptable. The variability of the size determination process for osmium-fixed adipocytes was evaluated.     

Effects of honey consumption on enamel microhardness in normal versus xerostomic patients

BACKGROUND: This report describes the pathology of a myolipoma which occurred in the eyelid. Myolipoma is a benign hamartomatous tumour in which smooth muscle cells are interspersed with adipocytes. PATIENT DETAILS: An irregular yellowish tumour (30 x 25 mm) with illdefined borders had been present for 50 years in the medial part of the left lower eyelid of a 67-year-old woman. The tumour was excised and studied by conventional histology, immunohistochemistry and transmission electron microscopy. RESULTS: The tumour was formed by bundles of spindle-shaped cells with cigarshaped nuclei intermingled with multiloculated clear cells containing small eccentric nuclei. By immunohistochemistry, positive staining of the spindle cells was restricted to smooth muscle actin and desmin; the clear cells were non-reactive with the immunohistochemical panel, but fat was identified within the cytoplasm. The ultrastructural features of the spindle cells were those of a leiomyoma, while the clear cells were classified as adipocytes. CONCLUSION: This tumour was considered to originate from the media of blood vessels within the tumour.     

Pivotal role of leptin in insulin effects

The OB protein, also known as leptin, is secreted by adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks regulating ingestive behavior and energy balance. The two forms of leptin receptors (long and short forms) have been identified in various peripheral tissues, a fact that makes them possible target sites for a direct action of leptin. It has been shown that the OB protein interferes with insulin secretion from pancreatic islets, reduces insulin-stimulated glucose transport in adipocytes, and increases glucose transport, glycogen synthesis and fatty acid oxidation in skeletal muscle. Under normoglycemic and normoinsulinemic conditions, leptin seems to shift the flux of metabolites from adipose tissue to skeletal muscle. This may function as a peripheral mechanism that helps control body weight and prevents obesity. Data that substantiate this hypothesis are presented in this review.     

Desensitization of the beta-adrenergic response in human brown adipocytes

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.     

Involvement of human interleukin 6 in experimental cachexia induced by a human uterine cervical carcinoma xenograft

A human tumor xenograft model for cancer cachexia was established by growing a uterine cervical carcinoma, Yumoto, in nude mice. The tumor transplanted into the mice induced severe body weight loss (30% of body weight) when the tumor weight was only 1 g. In addition, other indicators for cachexia, such as adipose tissue and muscle wasting and hypoglycemia, were also observed in the tumor-bearing mice, suggesting that this is a proper model for experimental cachexia induced by a human tumor. We then examined the association of this model with various cytokines, such as tumor necrosis factor alpha, interleukin (IL)-1alpha, IL-1beta, IFN-gamma, IL-6, and leukemia inhibitory factor, and identified human IL-6, which was produced by the tumor cells, as a mediator of cachexia. A neutralizing antibody against hIL-6 administered to the mice after the development of cachexia symptoms significantly improved body weight loss, adipose tissue wasting, hypoglycemia, acute phase reaction, and leukocytosis, although it did not suppress the tumor growth. These results demonstrate that the hIL-6 produced by the tumor cells is an essential mediator of the cachexia induction in this model.     

Systemic expression of a newly recognized protein, LECT2, in the human body

LECT2 (leukocyte cell-derived chemotaxin 2) is a new, recently isolated protein shown to be synthesized by hepatocytes. All hepatocytes show diffuse immunostaining for LECT2 within the cytoplasm. In the present study an attempt was made to investigate the expression of LECT2 in normal and diseased human organs and tissues, other than the liver, using indirect immunoperoxidase staining. LECT2 was found to be generally expressed in vascular, endothelial and smooth muscle cells, adipocytes, cerebral nerve cells, apical squamous epithelia, parathyroid cells, sweat and sebaceous glandular epithelia, Hassall bodies and some mononuclear cells in immunohematopoietic tissue, although some of these cells and tissues were occasionally unstained in diseased conditions. Alternatively, this protein was generally negative, although it was occasionally positively stained in osteoblasts, chondrocytes, cardiac and skeletal muscle cells, smooth muscle cells of the gastrointestinal tract, and the epithelial cells of some tissues. LECT2 seems to be related to the cell cycle or repair process following damage to a variety of cells.     

Effects of retinoic acid on lipoprotein lipase activity and mRNA level in vitro and in vivo

This study was designed to determine whether all-trans retinoic acid altered lipoprotein lipase (LPL) activity and mRNA levels in vitro and tissue LPL mRNA levels in vivo. Incubation of adipocytes or adipose tissue for up to 12 hr with 10(-6) or 10(-5) M all-trans retinoic acid did not decrease LPL activity. There was no change in LPL mRNA levels following 3 hr incubation of adipocytes with all-trans retinoic acid. Feeding all-trans retinoic acid for 4 days led to a significant decrease in adipose tissue LPL activity but no change in heart enzyme activity. Retinoic acid did not alter the increase in heart LPL activity observed with fasting. There were no changes in LPL mRNA levels in adipose tissue, heart or liver. Retinoic acid does not have an acute direct effect on adipose tissue LPL activity. The observed decrease in adipose tissue LPL activity in vivo is not due to alterations in mRNA levels and may be a secondary effect of retinoic acid.     

Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth

Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.     

Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids

The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of insulin and corticosteroids to induce PPAR gamma expression in isolated human adipocytes in culture. We conclude that: (a) human PPAR gamma mRNA expression is most abundant in adipose tissue, but lower level expression of both splice variants is seen in skeletal muscle; to an extent that is unlikely to be due to adipose contamination. (b) RNA derived from adipose tissue of obese humans has increased expression of PPAR gamma 2 mRNA, as well as an increased ratio of PPAR gamma2/gamma1 splice variants that is proportional to the BMI; (c) a low calorie diet specifically down-regulates the expression of PPAR gamma2 mRNA in adipose tissue of obese humans; (d) insulin and corticosteroids synergistically induce PPAR gamma mRNA after in vitro exposure to isolated human adipocytes; and (e) the in vivo modulation of PPAR gamma2 mRNA levels is an additional level of regulation for the control of adipocyte development and function, and could provide a molecular mechanism for alterations in adipocyte number and function in obesity.     

Myolipoma of the round ligament: report of a case with a review of the English literature

Tumours consisting of a mixture of mature adipose and smooth muscle tissues, including those designated lipoleiomyomas, fibrolipoleiomyomas and myolipomas, are exceedingly rare, but most often occur in the uterine corpus. We describe here a case of such a tumour arising in the right round ligament of a 44-year-old woman. The tumour, which measured approximately 20x15x10 cm, was well encapsulated and did not involve the intrapelvic organs. Intricate mixtures of adult adipose tissue and bland smooth muscle exhibited no cellular atypia or nuclear mitotic figures, and there was little vascular proliferation. We diagnosed the lesion as a myolipoma of soft tissue with dual differentiation, and have found only 13 cases of this tumour including our own in the English literature. The present tumour is the first reported in the round ligament. Although this tumour is rare, its recognition is important for the avoidance of erroneous diagnoses.