Andrea Dicker

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

Tumor necrosis factor-α (TNF-α) 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-α, nuclear factor-κB (NF-κB), has not been recognized. We explored the role of NF-κB in TNF-α-induced lipolysis of human fat cells. Primary cultures of human adipocytes were incubated in the presence of a cell-permeable peptide that inhibits NF-κB signaling (WP). Incubation with WP, but not with a biologically inactive peptide (MP), abolished the nuclear translocation of NF-κB and effectively abrogated TNF-α-induced lipolysis in a concentration-dependent manner. Western blot analysis demonstrated that although TNF-α per se reduced mainly PLIN protein expression, TNF-α 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-α in the presence of NF-κB inhibition. This was confirmed in gene reporter assays using human PLIN and HSL promoter constructs. We conclude that in the presence of NF-κB inhibition, TNF-α-mediated lipolysis is reduced, which suggests that NF-κB is essential for retained human fat cell lipolysis.

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.

Primary differences in lipolysis between human omental and subcutaneous adipose tissue observed using in vitro differentiated adipocytes.

Catecholamine-induced lipolysis is elevated in omental as compared to subcutaneous adipocytes due to primary differences between the two cell types (i.e., they have different progenitor cells). Whether there is regional variation in atrial natriuretic peptide (ANP)-induced lipolysis is unknown. We studied whether beta-adrenoceptor signaling to lipolysis and ANP-induced lipolysis are involved in the primary differences in lipolysis. In vitro experiments on differentiated preadipocytes from human subcutaneous and omental adipose tissue were performed. The cells were kept in culture for a relative long duration, so any influence of local environment and circulation in the various adipose tissue depots could be excluded. Using beta1-, beta2-, and beta3-adenoceptor agonists, lipolysis was found to be significantly higher in omental as compared to subcutaneous differentiated preadipocytes. Forskolin and dibutyryl cAMP, which act at post-adrenoceptor levels, did not show any regional difference. There was no regional difference in ANP-induced lipolysis. Gene expression of beta1- and beta3-adrenoceptors was higher and beta2-adrenoceptor expression was lower in the omental cells. Omental fat cells have an increased beta-adrenoceptor-mediated lipolysis principally due to primary differences in the early event that couples beta-adrenoceptor subtypes to G-proteins. ANP-induced lipolysis is not subject to primary regional variation.

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

Significance Insulin resistance and β-cell failure are the major defects in type 2 diabetes. We now demonstrate that local insulin resistance-induced increase in apolipoprotein CIII (apoCIII) within pancreatic islets causes promotion of an intraislet inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i), and apoptosis. Decreasing apoCIII in vivo in animals with insulin resistance improves glucose tolerance, and apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of apoCIII, demonstrate a normal [Ca2+]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 β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes. Insulin resistance and β-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 β-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]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 [Ca2+]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 β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

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.

Differential function of the α2A-adrenoceptor and phosphodiesterase-3B in human adipocytes of different origin

OBJECTIVE:Human adipocytes can be obtained in vitro by differentiation of human preadipocytes or mesenchymal stem cells (hMSC). Although functionally similar to freshly isolated cells, no detailed comparison of the different cell types has been performed. The antilipolytic α2A-adrenoceptor (AR) and the cAMP-degrading enzyme Phosphodiesterase-3B (PDE3B) have been implicated in the fine-tuning of lipolysis but little is known regarding their role in human adipocytes nor whether their expression and/or function differs in fat cells from different precursors.METHODS:The effects of α2A-AR and PDE3B inhibition in mature adipocytes was determined and compared to that in differentiated preadipocytes and hMSC-derived fat cells. Gene expression was determined by real-time PCR and protein expression by Western blot.RESULTS:Noradrenaline (NA) stimulated lipolysis in preadipocytes and mature adipocytes but markedly reduced lipolysis in differentiated hMSC derived-adipocytes. This was due to a potent stimulation of α2A-AR since co-incubation with NA and the α2-AR-inhibitor yohimbine restored NA-induced lipolysis. The order of Yohimbine response was hMSC>preadipocytes>mature adipocytes. Although α2-AR mRNA expression was highest in mature adipocytes there was no difference in α2A-AR protein levels between the cell types. In contrast, Gα i2 mRNA and protein expression was significantly higher in MSC-derived adipocytes, suggesting that differences in the response to α2A-AR inhibition reside at the postreceptor level. Incubation with the cAMP-analog 8-bromo(8b) cAMP increased lipolysis in hMSC-derived fat cells while co-incubation with the PDE3-specific inhibitor OPC3911 did not alter the lipolytic effect. In contrast, OPC3911 increased 8bcAMP-induced lipolysis significantly in preadipocytes and mature adipocytes. The response to PDE3B inhibition was; mature adipocytes>preadipocytes>hMSC a finding that correlated significantly with both PDE3B mRNA expression and enzymatic activity.CONCLUSION:Although differentiated adipocytes of different origins display similar functional characteristics there are important differences in the regulation of lipolysis with a marked α2A-AR and less pronounced PDE3B effect in fat cells from MSCs.

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.

Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion.

The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status.

Effects of pain controlling neuropeptides on human fat cell lipolysis

Objective:Neuropeptides NPFF and NPSF are involved in pain control, acting through the G-protein coupled receptors (GPR)74 (high affinity for NPFF) and GPR147 (equal affinity for NPFF and NPSF). GPR74 also inhibits catecholamine-induced adipocyte lipolysis and regulates fat mass in humans. The aim of this study was to compare the effects of NPFF and NPSF on noradrenaline-induced lipolysis and to determine the expression of their receptors in human fat cells.Design:Adipose tissue was obtained during surgery. Adipocytes were prepared and kept in primary culture. Lipolysis, protein expression and gene expression were determined.Results:NPFF counteracted noradrenaline-induced lipolysis, which was more marked after 48 h than after 4 h exposure and was solely attributed to inhibition of β-adrenoceptor signalling. NPSF counteracted noradrenaline-induced lipolysis maximally after 4 h of exposure, which was attributed to a combination of inhibition of β-adrenoceptor signalling and decreased activation of the protein kinase-A hormone sensitive lipase complex by cyclic AMP. Both neuropeptides were effective in nanomolar concentrations. NPFF and NPSF had no effects on the expression of genes involved in catecholamine signal transduction. Both GPR74 and GPR147 were expressed at the protein level in fat cells from various adipose regions. GPR74 mRNA levels were higher in adipose tissue from obese as compared with non-obese subjects. High gene expression of either receptor correlated with low noradrenaline-induced lipolysis (P<0.05).Conclusions:Pain controlling neuropeptides NPFF and NPSF may be important for the regulation of lipolysis in man probably acting through GPR74 and GPR147. At low concentrations they inhibit catecholamine-induced lipolysis through rapid and long-term post-transcriptional effects at several steps in adrenoceptor signalling in fat cells.

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.