Islet vascularization is regulated by primary endothelial cilia via VEGF-A-dependent signaling.

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to ß-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.

Translational assessment of a genetic engineering methodology to improve islet function for transplantation.

BACKGROUND: The functional quality of insulin-secreting islet beta cells is a major factor determining the outcome of clinical transplantations for diabetes. It is therefore of importance to develop methodological strategies aiming at optimizing islet cell function prior to transplantation. In this study we propose a synthetic biology approach to genetically engineer cellular signalling pathways in islet cells. METHODS: We established a novel procedure to modify islet beta cell function by combining adenovirus-mediated transduction with reaggregation of islet cells into pseudoislets. As a proof-of-concept for the genetic engineering of islets prior to transplantation, this methodology was applied to increase the expression of the V1b receptor specifically in insulin-secreting beta cells. The functional outcomes were assessed in vitro and in vivo following transplantation into the anterior chamber of the eye. FINDINGS: Pseudoislets produced from mouse dissociated islet cells displayed basic functions similar to intact native islets in terms of glucose induced intracellular signalling and insulin release, and after transplantation were properly vascularized and contributed to blood glucose homeostasis. The synthetic amplification of the V1b receptor signalling in beta cells successfully modulated pseudoislet function in vitro. Finally, in vivo responses of these pseudoislet grafts to vasopressin allowed evaluation of the potential benefits of this approach in regenerative medicine. INTERPRETATION: These results are promising first steps towards the generation of high-quality islets and suggest synthetic biology as an important tool in future clinical islet transplantations. Moreover, the presented methodology might serve as a useful research strategy to dissect cellular signalling mechanisms of relevance for optimal islet function.

Integrative microendoscopic system combined with conventional microscope for live animal tissue imaging.

Intravital optical imaging technology is essential for minimally invasive optical diagnosis and treatment in small animal disease models. High-resolution imaging requires high-resolution optical probes, and high-resolution optical imaging systems based on highly precise and advanced technologies and therefore, associated with high-system costs. Besides, in order to acquire small animal live images, special types of animal imaging setups are indispensable. In this paper, a microendoscopic system is designed as an add-on to existing conventional imaging microscopes, reducing the price of complete confocal endomicroscopic systems. The proposed attachable system can be configured for confocal microscopes from common manufacturers and this enables users to acquire live animal cellular images from a conventional system. It features a 4f optical plane relay system, a rotary stage for side-view endoscopic probes, and an endoscopic probe mount which swings between the horizontal and the vertical. The system could be widely useful for biological studies of animal physiology and disease models.

Kinetics of functional beta cell mass decay in a diphtheria toxin receptor mouse model of diabetes.

Functional beta cell mass is an essential biomarker for the diagnosis and staging of diabetes. It has however proven technically challenging to study this parameter during diabetes progression. Here we have detailed the kinetics of the rapid decline in functional beta cell mass in the RIP-DTR mouse, a model of hyperglycemia resulting from diphtheria toxin induced beta cell ablation. A novel combination of imaging modalities was employed to study the pattern of beta cell destruction. Optical projection tomography of the pancreas and longitudinal in vivo confocal microscopy of islets transplanted into the anterior chamber of the eye allowed to investigate kinetics and tomographic location of beta cell mass decay in individual islets as well as at the entire islet population level. The correlation between beta cell mass and function was determined by complementary in vivo and ex vivo characterizations, demonstrating that beta cell function and glucose tolerance were impaired within the first two days following treatment when more than 50% of beta cell mass was remaining. Our results illustrate the importance of acquiring quantitative functional and morphological parameters to assess the functional status of the endocrine pancreas.

Biochemical profiling of diabetes disease progression by multivariate vibrational microspectroscopy of the pancreas.

Despite the dramatic increase in the prevalence of diabetes, techniques for in situ studies of the underlying pancreatic biochemistry are lacking. Such methods would facilitate obtaining mechanistic understanding of diabetes pathophysiology and aid in prognostic and/or diagnostic assessments. In this report we demonstrate how a multivariate imaging approach (orthogonal projections to latent structures - discriminant analysis) can be applied to generate full vibrational microspectroscopic profiles of pancreatic tissues. These profiles enable extraction of known and previously unrecorded biochemical alterations in models of diabetes, and allow for classification of the investigated tissue with regards to tissue type, strain and stage of disease progression. Most significantly, the approach provided evidence for dramatic alterations of the pancreatic biochemistry at the initial onset of immune-infiltration in the Non Obese Diabetic model for type 1 diabetes. Further, it enabled detection of a previously undocumented accumulation of collagen fibrils in the leptin deficient ob/ob mouse islets. By generating high quality spectral profiles through the tissue capsule of hydrated human pancreata and by in vivo Raman imaging of pancreatic islets transplanted to the anterior chamber of the eye, we provide critical feasibility studies for the translation of this technique to diagnostic assessments of pancreatic biochemistry in vivo.

Apolipoprotein CIII links islet insulin resistance to ß-cell failure in diabetes.

Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

Reporter islets in the eye reveal the plasticity of the endocrine pancreas.

The islets of Langerhans constitute the endocrine part of the pancreas and are responsible for maintenance of blood glucose homeostasis. They are deeply embedded in the exocrine pancreas, limiting their accessibility for functional studies. Understanding regulation of function and survival and assessing the clinical outcomes of individual treatment strategies for diabetes requires a monitoring system that continuously reports on the endocrine pancreas. We describe the application of a natural body window that successfully reports on the properties of in situ pancreatic islets. As proof of principle, we transplanted "reporter islets" into the anterior chamber of the eye of leptin-deficient mice. These islets displayed obesity-induced growth and vascularization patterns that were reversed by leptin treatment. Hence, reporter islets serve as optically accessible indicators of islet function in the pancreas, and also reflect the efficacy of specific treatment regimens aimed at regulating islet plasticity in vivo.

Donor islet endothelial cells in pancreatic islet revascularization.

OBJECTIVE: Freshly isolated pancreatic islets contain, in contrast to cultured islets, intraislet endothelial cells (ECs), which can contribute to the formation of functional blood vessels after transplantation. We have characterized how donor islet endothelial cells (DIECs) may contribute to the revascularization rate, vascular density, and endocrine graft function after transplantation of freshly isolated and cultured islets. RESEARCH DESIGN AND METHODS: Freshly isolated and cultured islets were transplanted under the kidney capsule and into the anterior chamber of the eye. Intravital laser scanning microscopy was used to monitor the revascularization process and DIECs in intact grafts. The grafts' metabolic function was examined by reversal of diabetes, and the ultrastructural morphology by transmission electron microscopy. RESULTS: DIECs significantly contributed to the vasculature of fresh islet grafts, assessed up to 5 months after transplantation, but were hardly detected in cultured islet grafts. Early participation of DIECs in the revascularization process correlated with a higher revascularization rate of freshly isolated islets compared with cultured islets. However, after complete revascularization, the vascular density was similar in the two groups, and host ECs gained morphological features resembling the endogenous islet vasculature. Surprisingly, grafts originating from cultured islets reversed diabetes more rapidly than those originating from fresh islets. CONCLUSIONS: In summary, DIECs contributed to the revascularization of fresh, but not cultured, islets by participating in early processes of vessel formation and persisting in the vasculature over long periods of time. However, the DIECs did not increase the vascular density or improve the endocrine function of the grafts.

ApoB100-LDL acts as a metabolic signal from liver to peripheral fat causing inhibition of lipolysis in adipocytes.

BACKGROUND: Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown. METHODS AND FINDINGS: We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr(-/-)Apob(100/100)). CONCLUSIONS: Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome.

Mechanism of increased lipolysis in cancer cachexia.

Loss of fat mass is a key feature of cancer cachexia and has been attributed to increased adipocyte lipolysis. The mechanism behind this alteration is unknown and was presently investigated. We studied mature s.c. fat cells and differentiated preadipocytes from 26 cancer patients with and without cachexia. Hormone-induced lipolysis and expression of lipolysis-regulating genes were determined together with body composition and in vivo lipolytic activity (fasting plasma glycerol or fatty acids related to body fat). Body fat was reduced by 40% and in vivo lipolytic activity was 2-fold increased in cachexia (P = 0.001). In mature adipocytes, the lipolytic effects of catecholamines and natriuretic peptide were 2- to 3-fold increased in cachexia (P < 0.001). This was completely counteracted by inhibiting the rate-limiting lipolysis enzyme hormone-sensitive lipase (HSL). In cachexia, the expression levels of HSL mRNA and protein were increased by 50% and 100%, respectively (P = 0.005-0.03), which strongly correlated with in vitro lipolytic stimulation (r = 0.7-0.9). The antilipolytic effect of insulin in mature fat cells and the stimulated lipolytic effect in differentiated preadipocytes were unaltered in cachexia. Patients who lost weight due to other factors than cancer cachexia had no change in adipocyte lipolysis. In conclusion, adipocyte lipolysis is increased in cancer cachexia not due to nonepigenic factors or to weight loss per se, but most probably because of enhanced expression and function of adipocyte HSL. The selective inhibition of this enzyme may prevent fat loss in cancer patients.

A common haplotype in the G-protein-coupled receptor gene GPR74 is associated with leanness and increased lipolysis.

The G-protein-coupled receptor GPR74 is a novel candidate gene for body weight regulation. In humans, it is predominantly expressed in brain, heart, and adipose tissue. We report a haplotype in the GPR74 gene, ATAG, with allele frequency ~4% in Scandinavian cohorts, which was associated with protection against obesity in two samples selected for obese and lean phenotypes (odds ratio for obesity 0.48 and 0.62; nominal P=.0014 and .014; n=1,013 and 1,423, respectively). In a population-based sample, it was associated with lower waist (P=.02) among 3,937 men and with obesity protection (odds ratio 0.36; P=.036) among those selected for obese or lean phenotypes. The ATAG haplotype was associated with increased adipocyte lipid mobilization (lipolysis) in vivo and in vitro. In human fat cells, GPR74 receptor stimulation and inhibition caused a significant and marked decrease and increase, respectively, of lipolysis, which could be linked to catecholamine stimulation of adipocytes through beta -adrenergic receptors. These findings suggest that a common haplotype in the GPR74 gene protects against obesity, which, at least in part, is caused by a relief of inhibition of lipid mobilization from adipose tissue. The latter involves a cross-talk between GPR74 and beta -adrenoceptor signaling to lipolysis in fat cells.

Differential lipolytic regulation in human embryonic stem cell-derived adipocytes.

OBJECTIVE: Human embryonic stem cells (hESCs) have raised great hopes for future clinical applications. Several groups have succeeded in differentiating hESCs into adipocytes, as determined by morphology, mRNA expression, and protein secretion. However, determination of lipolytic response, the most important characteristic of adipocytes, has not been performed. This work was intended to study adipogenic conversion of hESCs by functional assessment of differentiation. RESEARCH METHODS AND PROCEDURES: Single undifferentiated colonies were allowed to transform into embryonic bodies. mRNA expression for a set of adipocyte-specific genes and leptin/adiponectin secretion and lipolysis were assessed at different time-points after differentiation. RESULTS: In contrast to primary human adipocytes, hESC-derived adipocytes showed a very small response to classical beta-adrenergic agonists, although they expressed the major genes in the lipolytic cascade. In contrast, there was a significant lipolytic response to atrial natriuretic peptide. DISCUSSION: Although hESC-derived adipocytes seem to be morphologically and expressionally similar to mature adipocytes, there are important functional differences that could depend on their early developmental origin. We conclude that, in contrast to mature adipocytes, hESC-derived adipocytes display a differential response to atrial natriuretic peptide and catecholamines.

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis.

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.

NF-kappaB is important for TNF-alpha-induced lipolysis in human adipocytes.

Tumor necrosis factor-alpha (TNF-alpha) promotes lipolysis in mammal adipocytes via the mitogen-activated protein kinase (MAPK) family, resulting in reduced expression/function of perilipin (PLIN). The role of another pivotal intracellular messenger activated by TNF-alpha, nuclear factor-kappaB (NF-kappaB), has not been recognized. We explored the role of NF-kappaB in TNF-alpha-induced lipolysis of human fat cells. Primary cultures of human adipocytes were incubated in the presence of a cell-permeable peptide that inhibits NF-kappaB signaling (WP). Incubation with WP, but not with a biologically inactive peptide (MP), abolished the nuclear translocation of NF-kappaB and effectively abrogated TNF-alpha-induced lipolysis in a concentration-dependent manner. Western blot analysis demonstrated that although TNF-alpha per se reduced mainly PLIN protein expression, TNF-alpha in the presence of WP resulted in a pronounced combined reduction of both hormone-sensitive lipase (HSL) and PLIN protein. The expression of a set of other lipolytic or adipocyte-specific proteins was not affected. The regulation was presumably at the transcriptional level, because mRNA expression for HSL and PLIN was markedly reduced with TNF-alpha in the presence of NF-kappaB inhibition. This was confirmed in gene reporter assays using human PLIN and HSL promoter constructs. We conclude that in the presence of NF-kappaB inhibition, TNF-alpha-mediated lipolysis is reduced, which suggests that NF-kappaB is essential for retained human fat cell lipolysis.

Adipocyte lipases and defect of lipolysis in human obesity.

The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine- and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine- and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity.

Functional studies of mesenchymal stem cells derived from adult human adipose tissue.

Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal-vascular portion of subcutaneous adipose tissue from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation with catecholamines, including a pronounced antilipolytic effect mediated through alpha2A-adrenoceptors, a typical trait in human but not rodent fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable cellular source for studies of adipocyte biology.

Targets for TNF-alpha-induced lipolysis in human adipocytes.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha)-induced lipolysis may be important for insulin resistance in both obesity and cachexia. In rodent cells TNF-alpha enhances lipolysis through down-regulation of the expression of the membrane proteins Galpha(i) and the lipid droplet-associated protein perilipin (PLIN). In human (but not murine) adipocytes TNF-alpha stimulates lipolysis through the mitogen activated protein kinases (MAPKs) p44/42 and JNK although it is unclear whether this is mediated via PLIN and/or Galpha(i). METHODS: Galpha(i) and PLIN as down-stream effectors of MAPKs were assessed in human adipocytes stimulated with TNF-alpha in the absence or presence of specific MAPK inhibitors. RESULTS: A 48-h incubation with TNF-alpha resulted in a pronounced increase in lipolysis, which was paralleled by a decrease in the mRNA and protein expression of PLIN. Both these effects were inhibited in a concentration-dependent manner in the presence of MAPK inhibitors specific for p44/42 (PD98059) and JNK (SP600125). However, TNF-alpha did not affect Galpha(i) mRNA or protein expression. Furthermore, experiments with pertussis toxin demonstrated that inhibition of Galpha(i) signaling did not affect TNF-alpha-mediated lipolysis. CONCLUSIONS: Our results suggest that TNF-alpha-mediated lipolysis is dependent on down-regulation of PLIN expression via p44/42 and JNK. This could be an important mechanism for the development of insulin resistance in both obesity and cachexia. However, in contrast to findings in rodent cells, Galpha(i) does not appear to be essential for TNF-alpha-induced lipolysis in human adipocytes.

Functional characterization of human mesenchymal stem cell-derived adipocytes.

The function of adipocytes derived from human mesenchymal stem cells (hMSC) was investigated for the first time in hMSC from fetal liver (FL) and adult bone marrow (BM) and compared with preadipocytes from human subcutaneous adipose tissue differentiated according to adipocyte-specific protocols. FL- and BM-derived adipocytes displayed both morphological and functional characteristics of mature adipocytes including specific intracellular signaling pathways for tumor necrosis factor-alpha, catecholamine-regulated lipolysis as well as secretion of adiponectin and leptin. Similar to differentiated preadipocytes, hMSC adipocytes displayed lipolytic effects mediated by beta-adrenoceptors and antilipolytic effects mediated by the alpha 2A-adrenoceptor (alpha 2A-AR) and expressed proteins with a pivotal role in human lipolysis, including beta 2-AR, alpha 2A-AR, and hormone-sensitive lipase. We conclude that hMSC-derived adipocytes are morphologically and functionally similar to preadipocytes and display an intact lipolytic signaling pathway and endocrine function. These systems could be of great value in adipocyte research as a renewable source of adipocytes.

Increased lipolysis and decreased leptin production by human omental as compared with subcutaneous preadipocytes.

Site differences in adipose tissue function may have implications for insulin-resistant conditions. In mature adipose tissue, subcutaneous adipocytes have higher leptin secretion, similar tumor necrosis factor (TNF)-alpha secretion, and lower catecholamine-stimulated lipolysis as compared with omental adipocytes. In this study, lipolysis and leptin and TNF-alpha secretion were compared between human omental and subcutaneous preadipocytes. After 16 days of incubation in a minimal differentiation medium, leptin mRNA and secretion were found to be two to eight times higher in subcutaneous than omental preadipocytes (P < 0.05). On the other hand, norepinephrine-induced lipolysis was about two times higher in the omental than in the subcutaneous preadipocytes, whereas basal lipolysis did not differ between the two regions. TNF-alpha secretion was marginally but significantly higher in the omental than in the subcutaneous preadipocytes. Preadipocyte differentiation was equal in both regions and was augmented to the same extent by different thiazolidinediones (rosiglitazone, pioglitazone, or troglitazone) in the two depots. In the presence of rosiglitazone, leptin secretion remained about three times higher and norepinephrine-induced lipolysis about six times lower in subcutaneous as compared with omental preadipocytes (P < 0.05), whereas TNF-alpha secretion and basal lipolysis were similar in preadipocytes from the two regions. These findings remained unaltered even if rosiglitazone was removed from the medium. However, leptin mRNA showed no regional differences in rosiglitazone-treated cells. Thus, regional differences in adipocyte leptin secretion as well as in norepinephrine-induced lipolysis are marked and present during different stages of preadipocyte differentiation and seem to be determined by intrinsic (i.e., primary) factors.

Mapping of early signaling events in tumor necrosis factor-alpha -mediated lipolysis in human fat cells.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine with a proposed role in obesity-related insulin resistance. This could be mediated by increased lipolysis in adipose tissue resulting in elevated free fatty acid levels. The early intracellular signals entailed in TNF-alpha-mediated lipolysis are unknown but may involve members of the mitogen-activated protein kinase (MAPK) family. We investigated the possible contribution of MAPK in TNF-alpha-induced lipolysis in human preadipocytes. TNF-alpha activated the three mammalian MAPK, p44/42, JNK, and p38, in a distinct time- and concentration-dependent manner. TNF-alpha also induced a concentration-dependent stimulation of lipolysis with a more than 3-fold increase at the maximal dose. Lipolysis was completely inhibited by blockers specific for p44/42 (PD98059) and JNK (dimetylaminopurine) but was not affected by the p38 blocker SB203580. Use of receptor-specific TNF-alpha mutants showed that activation of MAPK is entirely mediated by the TNFR1 receptor. The results in human preadipocytes differed from those obtained in murine 3T3-L1 adipocytes in which all three MAPK were constitutively active. Thus, studies of intracellular signaling pathways obtained in different cellular contexts should be interpreted with caution. In conclusion, although TNF-alpha activates all three known MAPK in human preadipocytes, only p44/42 and JNK appear to be involved in the regulation of lipolysis.