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1.
J Endocrinol ; 191(1): 129-36, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17065396

ABSTRACT

Orexin-A and orexin-B, via their receptors orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R) have been shown to play a role in the regulation of feeding, body weight, and energy expenditure. Adipose tissue also contributes significantly to the maintenance of body weight by interacting with a complex array of bioactive peptides; however, there are no data as yet on the expression of orexin components in adipose tissue. We, therefore, analyzed the expression of OX1R and OX2R in human adipose tissue and determined functional responses to orexin-A and orexin-B. OX1R and OX2R mRNA expression was detected in subcutaneous (s.c.) and omental adipose tissue and in isolated adipocytes. Protein for OX1R and OX2R was also detected in whole adipose tissue sections and lysates. Treatment with orexin-A, and orexin-B (100 nM, 24 h) resulted in a significant increase in peroxisome proliferator-activated receptors gamma-2 mRNA expression in s.c. adipose tissue (P < 0.05). Hormone sensitive lipase mRNA was significantly reduced in omental adipose tissue with orexin-A and orexin-B treatment (P < 0.05). Glycerol release from omental adipose tissue was also significantly reduced with orexin-A treatment (P < 0.05). These findings demonstrate for the first time the presence of functional orexin receptors in human adipose tissue and suggest a role for orexins in adipose tissue metabolism and adipogenesis.


Subject(s)
Adipocytes/chemistry , Adipose Tissue/chemistry , Intracellular Signaling Peptides and Proteins/pharmacology , Neuropeptides/pharmacology , Receptors, G-Protein-Coupled/analysis , Receptors, Neuropeptide/analysis , Adult , Cell Separation , Cells, Cultured , Female , Gene Expression/drug effects , Humans , Immunohistochemistry/methods , Orexin Receptors , Orexins , RNA, Messenger/analysis , Receptors, G-Protein-Coupled/genetics , Receptors, Neuropeptide/genetics , Reverse Transcriptase Polymerase Chain Reaction
2.
Diabetologia ; 49(11): 2723-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17001470

ABSTRACT

AIMS/HYPOTHESIS: Polycystic ovary syndrome (PCOS) is a multifaceted metabolic disease linked with insulin resistance (IR) and obesity. Adiponectin, which is lower in IR states, exerts its glucose-lowering and anti-inflammatory effects by activating two receptors, ADIPOR1 and ADIPOR2. There are no data on the relative expression of these receptors in adipose tissue of PCOS women. METHODS: We investigated the expression of adiponectin receptors from corresponding s.c. and omental (o.m.) adipose tissue in women with PCOS compared with matched non-PCOS women. As there is a disturbance in the steroid milieu in PCOS women, we also assessed the effects of testosterone and oestradiol on adiponectin receptors using adipocytes and adipocyte explants. Real-time RT-PCR and western blotting were used to assess the relative adiponectin receptor mRNA expression and protein production, respectively. Biochemical measurements were performed in our hospital's laboratory. RESULTS: We are the first to describe adiponectin receptor expression and production, in corresponding s.c. and o.m. human adipose tissues at the mRNA and protein level. We demonstrate the upregulation of mRNA expression and protein production of adiponectin receptors in women with PCOS, in s.c. and o.m. adipose tissue. Treatment of adipose tissue explants and adipocytes with testosterone and oestradiol induced the expression of adiponectin receptor mRNA and protein. There was a significant positive association between ADIPOR1/R2 expression and homeostasis model assessment, testosterone, oestradiol and triglycerides and a negative relationship with sex hormone-binding globulin. CONCLUSIONS/INTERPRETATION: The precise reason for the upregulation of adiponectin receptors seen in PCOS women, a pro-diabetic state, is unknown, but it appears that sex steroids may play a role in their regulation in adipose tissue.


Subject(s)
Adipocytes/physiology , Adipose Tissue/physiopathology , Insulin Resistance , Polycystic Ovary Syndrome/genetics , RNA, Messenger/genetics , Receptors, Cell Surface/genetics , Adult , Female , Gene Expression Regulation , Humans , Polycystic Ovary Syndrome/physiopathology , Polycystic Ovary Syndrome/surgery , Protein Biosynthesis , Receptors, Adiponectin , Transcription, Genetic
3.
Biochem Biophys Res Commun ; 348(3): 832-8, 2006 Sep 29.
Article in English | MEDLINE | ID: mdl-16899222

ABSTRACT

Obesity is a risk factor for prostate cancer, and plasma levels of the adipokine, adiponectin, are low in the former but high in the latter. Adiponectin has been shown to modulate cell proliferation and apoptosis, suggesting that adiponectin and its receptors (Adipo-R1, Adipo-R2) may provide a molecular association between obesity and prostate carcinogenesis. We show for the first time, the protein distribution of Adipo-R1 and Adipo-R2 in LNCaP and PC3 cells, and in human prostate tissue. Using real-time RT-PCR we provide novel data demonstrating the differential regulation of Adipo-R1 and Adipo-R2 mRNA expression by testosterone, 5-alpha dihydrotestosterone, beta-estradiol, tumour necrosis factor-alpha, leptin, and adiponectin in LNCaP and PC3 cells. Our findings suggest that adiponectin and its receptors may contribute to the molecular association between obesity and prostate cancer through a complex interaction with other hormones and cytokines that also play important roles in the pathophysiology of obesity and prostate cancer.


Subject(s)
Prostatic Neoplasms/metabolism , Receptors, Cell Surface/biosynthesis , Adiponectin/metabolism , Aged , Aged, 80 and over , Cell Line, Tumor , Cytokines/metabolism , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Obesity/genetics , Obesity/metabolism , Prostate/metabolism , RNA, Messenger/metabolism , Receptors, Adiponectin , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiology
4.
Int J Obes Relat Metab Disord ; 24(5): 585-92, 2000 May.
Article in English | MEDLINE | ID: mdl-10849580

ABSTRACT

OBJECTIVES: Uncoupling protein 2 (UCP2) is a recently described homologue of the uncoupling protein of brown adipocytes (UCP1), which is expressed at high levels in human white adipose tissue. Studies were undertaken (1) to establish whether the expression of UCP2 mRNA varies in a depot-related manner in isolated human adipocytes, (2) to determine whether thiazolidinedione exposure influences the expression of UCP2 mRNA in cultured human pre-adipocytes, and (3) to determine whether human UCP2 is targeted to mitochondria when transfected into mammalian cells. SUBJECTS: Abdominal subcutaneous and omental adipose tissue biopsies were obtained from adult patients undergoing elective intra-abdominal surgical procedures. MEASUREMENTS: A competitive reverse transcriptase-polymerase chain reaction (RT-PCR) was used to quantify UCP2 mRNA expression in human omental and subcutaneous adipocytes, and in cultured human preadipocytes differentiated in vitro using the thiazolidinedione, BRL49653. Chinese hamster ovary cells were transfected with a vector expressing human UCP2, and its cellular localization was determined by confocal immunofluorescence microscopy. RESULTS: Adipocytes isolated from human omentum consistently expressed more UCP2 mRNA than did subcutaneous adipocytes from the same subjects (mean fold difference 2.92+/-0.44 P<0.001, n=11) with no effect of gender or body mass index being seen. BRL49653 treatment of subcutaneously, but not omentally, derived preadipocytes stimulated expression of UCP2 mRNA (5.1+/-1.1 fold). Transfected human UCP2 was detected exclusively in mitochondria of CHO cells. CONCLUSIONS: Increased expression of UCP2 in human omental adipose tissue relative to subcutaneous adipose tissue is related to the expression levels in adipocytes per se, a finding which may relate to the particular functional attributes of this sub-population of adipocytes. Furthermore, BRL 49653 has site-specific effects of on the expression of UCP2 in human preadipocytes, a finding which may be relevant to the therapeutic effects of such compounds. Finally we present evidence for the mitochondrial localisation of human UCP2.


Subject(s)
Adipocytes/metabolism , Membrane Transport Proteins , Mitochondrial Proteins , Proteins/genetics , Thiazolidinediones , Adipocytes/drug effects , Adult , Animals , CHO Cells , Cells, Cultured , Cricetinae , Female , Humans , Hypoglycemic Agents/pharmacology , Ion Channels , Male , Microscopy, Fluorescence , Middle Aged , Omentum , Polymerase Chain Reaction , Protein Biosynthesis , RNA, Messenger/biosynthesis , Rosiglitazone , Thiazoles/pharmacology , Transfection , Uncoupling Protein 2
5.
J Endocrinol ; 163(1): 33-8, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10495404

ABSTRACT

Tumour necrosis factor-alpha (TNF-alpha), secreted by cells of the macrophage-monocyte lineage, has a well established role in inflammation and host-defence. The more recent discovery that adipocytes also secrete TNF-alpha has led to a substantial body of research implicating this molecule in the insulin resistance of obesity. However, little is known about the normal regulation of TNF-alpha release from human adipose tissue. In particular, it is not known whether adipocyte production of TNF-alpha is responsive to similar or different molecular regulators than those relevant to macrophages. TNF-alpha release from cultured human adipose tissue and isolated adipocytes was examined using an ELISA. Insulin, cortisol or the thiazolidinedione, BRL 49653, did not have a significant effect on TNF-alpha release from adipose tissue or isolated adipocytes. In contrast, lipopolysaccharide (LPS), a major stimulus of TNF-alpha protein production in monocytes and macrophages, resulted in a fivefold stimulation of TNF-alpha release from human adipose tissue. Significant stimulation of TNF-alpha release was also seen from isolated adipocytes, indicating that the increase in TNF-alpha release from adipose tissue in the presence of LPS is unlikely to be entirely attributable to contaminating monocytes or macrophages. Consistent with this observation was the finding that mRNA for CD14, a known cellular receptor for LPS, is expressed in human adipocytes. The increase in TNF-alpha protein release in response to LPS was blocked by an inhibitor of the matrix metalloproteinase responsible for the cleavage of the membrane-bound proform of TNF-alpha, indicating that this release represented regulated secretion and was not due to cell lysis. In conclusion, the regulation of TNF-alpha protein release from human adipose tissue and isolated adipocytes appears to be similar to its regulation in cell types more traditionally implicated in host defence. The production by the adipocyte of a range of molecules involved in host defence-TNF-alpha, factors D, B and C3, interleukin-6, and macrophage colony-stimulating factor--suggest that this cell type may make a significant contribution to innate immunity.


Subject(s)
Adipose Tissue/metabolism , Immunity/physiology , Tumor Necrosis Factor-alpha/metabolism , Adipocytes/drug effects , Adipocytes/immunology , Adipocytes/metabolism , Adipose Tissue/drug effects , Adipose Tissue/immunology , Adult , Aged , Cells, Cultured , Culture Techniques , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lipopolysaccharide Receptors/genetics , Lipopolysaccharides/pharmacology , Male , Middle Aged , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric , Stimulation, Chemical
6.
Mol Pathol ; 52(1): 1-10, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10439832

ABSTRACT

Since its initial description over twenty years ago the PCR has become one of the most valuable and flexible tools available to biomedical research. Subsequently, refinements and modifications to the basic approach, many of which have been described in this review, have enabled the application of the PCR to many areas of diagnostic medicine and have ensured its rapid acceptance as a routine test in many pathology disciplines. The growing importance of molecular approaches to the diagnosis of disease, particularly in histopathology, will continue to secure an ever expanding role for the PCR in diagnostic pathology.


Subject(s)
Polymerase Chain Reaction/methods , DNA/isolation & purification , DNA Mutational Analysis/methods , Humans , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods
7.
Diabetes ; 47(1): 138-41, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9421389

ABSTRACT

Thiazolidinediones (TZDs) are a novel class of insulin-sensitizing agents used in the treatment of NIDDM and are potent agonists for the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma). The thiazolidinedione BRL 49653 has been shown to promote the differentiation of the HIB-1B brown preadipocyte cell line and to increase rat interscapular brown adipose tissue (BAT) mass. Given the importance of brown fat in the control of energy metabolism in rodents, this may represent an important therapeutic effect of this class of compound. To date, however, no studies examining the effects of TZDs on human brown fat have been reported. In the present study, we have measured uncoupling protein 1 (UCP-1) mRNA, a specific marker for BAT, in isolated adipocytes and subcultured preadipocytes prepared from different adult human adipose tissue depots. Consistent with previous studies of adult human whole adipose tissue, UCP-1 mRNA was detectable in isolated human adipocytes prepared from all depots studied with a rank order of perirenal, omental, and subcutaneous. BRL 49653 treatment of subcultured human pre-adipocytes prepared from all depots resulted in increased levels of UCP-1 mRNA, compared with those of the vehicle-treated cells. When exposed to BRL 49653 for 5 days, preadipocytes from the human perirenal depot accumulated lipid, and a proportion of cells showed clear mitochondrial staining for UCP-1 protein by confocal microscopy. Thus, cells of the brown fat lineage were detectable in all human adipose depots studied, and cultured human pre-adipocytes, particularly from the perirenal depot, showed a marked increase in UCP-1 expression in response to thiazolidinediones. Given the role of brown adipocytes in the enhancement of energy expenditure, promotion of brown fat adipogenesis by thiazolidinediones could contribute to the beneficial effects of these drugs on insulin resistance in humans.


Subject(s)
Adipocytes/metabolism , Carrier Proteins/biosynthesis , Hypoglycemic Agents/pharmacology , Membrane Proteins/biosynthesis , Stem Cells/metabolism , Thiazoles/pharmacology , Thiazolidinediones , Adipocytes/cytology , Adipocytes/drug effects , Base Sequence , Carrier Proteins/analysis , Carrier Proteins/genetics , Cells, Cultured , DNA Primers/analysis , DNA Primers/chemistry , DNA Primers/genetics , Female , Gene Expression Regulation , Humans , Ion Channels , Male , Membrane Proteins/analysis , Membrane Proteins/genetics , Mitochondrial Proteins , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/chemistry , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear/agonists , Rosiglitazone , Stem Cells/cytology , Stem Cells/drug effects , Transcription Factors/agonists , Uncoupling Protein 1
8.
Nature ; 387(6636): 903-8, 1997 Jun 26.
Article in English | MEDLINE | ID: mdl-9202122

ABSTRACT

The extreme obesity of the obese (ob/ob) mouse is attributable to mutations in the gene encoding leptin, an adipocyte-specific secreted protein which has profound effects on appetite and energy expenditure. We know of no equivalent evidence regarding leptin's role in the control of fat mass in humans. We have examined two severely obese children who are members of the same highly consanguineous pedigree. Their serum leptin levels were very low despite their markedly elevated fat mass and, in both, a homozygous frame-shift mutation involving the deletion of a single guanine nucleotide in codon 133 of the gene for leptin was found. The severe obesity found in these congenitally leptin-deficient subjects provides the first genetic evidence that leptin is an important regulator of energy balance in humans.


Subject(s)
Metabolism, Inborn Errors/genetics , Obesity/genetics , Proteins/metabolism , Adult , Age of Onset , Animals , Body Composition , CHO Cells , Child , Child, Preschool , Consanguinity , Cricetinae , Female , Frameshift Mutation , Homozygote , Humans , Leptin , Male , Metabolism, Inborn Errors/blood , Mice , Mice, Obese , Molecular Sequence Data , Obesity/blood , Pedigree , Polymorphism, Single-Stranded Conformational , Proteins/genetics , Sequence Analysis, DNA , Transfection
9.
Diabetes ; 46(3): 342-7, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9032087

ABSTRACT

Obese subjects with excess intra-abdominal fat deposition suffer greater adverse metabolic consequences than do similarly overweight subjects with a predominantly subcutaneous distribution of adiposity. Little is known about the factors regulating the regional distribution of body fat. Leptin is a recently characterized protein secreted by adipocytes that appears to provide a long-term hormonal feedback signal regulating fat mass. No systematic evaluation of site-related differences in human adipocyte leptin expression has been reported to date. Levels of leptin mRNA were examined by quantitative reverse transcription-polymerase chain reaction in adipocytes isolated from omental and subcutaneous adipose depots of nonobese and mildly obese individuals undergoing elective surgery. In all individuals studied (n = 24), leptin mRNA levels were higher in subcutaneous than in omental adipocytes (P < 0.0001). In contrast, there were no consistent site-specific differences in the expression of glycerol-3-phosphate dehydrogenase mRNA. The subcutaneous-to-omental ratio of leptin mRNA expression was markedly higher in women (5.5 +/- 1.1-fold) than in men (1.9 +/- 0.2-fold) (P < 0.02). A significant relationship between BMI and leptin mRNA expression was demonstrable in the subcutaneous adipocytes of women (P < 0.006). Thus, leptin mRNA appears to be expressed predominantly by subcutaneous adipocytes, particularly in women. These findings suggest a possible role for leptin in the control of adipose tissue distribution and mass.


Subject(s)
Adipocytes/metabolism , Adipose Tissue/anatomy & histology , Adipose Tissue/metabolism , Protein Biosynthesis , Sex Characteristics , Transcription, Genetic , Abdomen , Adult , Aged , Female , Humans , Leptin , Male , Middle Aged , Obesity/genetics , Omentum , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Regression Analysis , Skin
10.
J Clin Invest ; 100(12): 3149-53, 1997 Dec 15.
Article in English | MEDLINE | ID: mdl-9399962

ABSTRACT

Activation of peroxisome proliferator-activated receptor (PPAR) gamma, a nuclear receptor highly expressed in adipocytes, induces the differentiation of murine preadipocyte cell lines. Recently, thiazolidinediones (TZDs), a novel class of insulin-sensitizing compounds effective in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) have been shown to bind to PPARgamma with high affinity. We have examined the effects of these compounds on the differentiation of human preadipocytes derived from subcutaneous (SC) and omental (Om) fat. Assessed by lipid accumulation, glycerol 3-phosphate dehydrogenase activity, and mRNA levels, subcultured preadipocytes isolated from either SC or Om depots did not differentiate in defined serum-free medium. Addition of TZDs (BRL49653 or troglitazone) or 15-deoxyDelta12,14prostaglandin J2 (a natural PPARgamma ligand) enhanced markedly the differentiation of preadipocytes from SC sites, assessed by all three criteria. The rank order of potency of these agents in inducing differentiation matched their ability to activate transcription via human PPARgamma. In contrast, preadipocytes from Om sites in the same individuals were refractory to TZDs, although PPARgamma was expressed at similar levels in both depots. The mechanism of this depot-specific TZD response is unknown. However, given the association between Om adiposity and NIDDM, the site-specific responsiveness of human preadipocytes to TZDs may be involved in the beneficial effects of these compounds on in vivo insulin sensitivity.


Subject(s)
Adipocytes/cytology , Adipocytes/drug effects , Chromans/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Thiazoles/pharmacology , Thiazolidinediones , Transcription Factors/genetics , Adipocytes/metabolism , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Humans , Mice , Microbodies , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/pharmacology , Receptors, Cytoplasmic and Nuclear/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Rosiglitazone , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , Transcription Factors/biosynthesis , Transcriptional Activation/drug effects , Troglitazone
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