Joanna Kosacka

Adipocyte-derived angiopoietin-1 supports neurite outgrowth and synaptogenesis of sensory neurons

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin‐1 (Ang‐1). We further considered initial hints that Ang‐1‐induced neuritogenesis involves transactivation of the high‐affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3‐L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang‐1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang‐1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high‐affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang‐1 and NGF in the control of neuritogenesis. DRG‐adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD‐95 correlated with Ang‐1 levels in culture. It is concluded that adipocyte‐secreted Ang‐1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang‐1 appears to play a physiological role in WAT plasticity.

Autophagy in adipose tissue of patients with obesity and type 2 diabetes.

BACKGROUND Pathophysiology of obesity is closely associated with enhanced autophagy in adipose tissue (AT). Autophagic process can promote survival or activate cell death. Therefore, we examine the occurrence of autophagy in AT of type 2 diabetes (T2D) patients in comparison to obese and lean individuals without diabetes. METHODOLOGY/PRINCIPAL FINDINGS Numerous autophagosomes accumulated within adipocytes were visualized by electron transmission microscopy and by immunofluorescence staining for autophagy marker LC3 in obese and T2D patients. Increased autophagy was demonstrated by higher LC3-II/LC3-I ratio, up-regulated expression of LC3 and Atg5 mRNA, along with decreased p62 and mTOR protein levels. Increased autophagy occurred together with AT inflammation. CONCLUSIONS Our data suggest fat depot-related differences in autophagy regulation. In subcutaneous AT, increased autophagy is accompanied by increased markers of apoptosis in patients with obesity independently of T2D. In contrast, in visceral AT only in T2D patients increased autophagy was related to higher markers of apoptosis.

Oxidized low-density lipoprotein (oxLDL)-induced cell death in dorsal root gangion cell cultures depends not on the lectin-like oxLDL receptor-1 but on the toll-like receptor-4

DRG cells have been found to undergo apoptosis and necrosis after oxidized low‐density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL‐induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) and toll‐like receptor‐4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL‐treated DRG cell cultures, the increase of cleaved caspase‐3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL‐treated DRG cell cultures expressed LOX‐1 and TLR4 at similar levels. The LOX‐1 expression remained unchanged after receptor blockade. However, the inhibition of LOX‐1 caused a significant increase of cleaved caspase‐3 and a decrease of TLR4 levels. The TLR4‐inhibited DRG cell cultures lacked changes in LOX‐1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N‐terminal kinase (JNK) and a significant decrease of cleaved caspase‐3 but did not change the TLR4 level. We conclude that LOX‐1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL‐induced cell death in DRG cell cultures does not depend on the LOX‐1 but on the TLR4.

Altered sciatic nerve fiber morphology and endoneural microvessels in mouse models relevant for obesity, peripheral diabetic polyneuropathy, and the metabolic syndrome

The morphology of sciatic nerves from leptin‐deficient ob/ob mice and leptin receptor‐deficient db/db mice, both models for obesity, peripheral diabetic neuropathy, and the metabolic syndrome, has yet to be examined for changes in nerve fibers and in endoneural microvessels. Sciatic nerves from three groups of 4‐month‐old mice (WT C57BL6, ob/ob, and db/db) were investigated. In ultrathin sections, the thickness of myelin sheaths was significantly reduced in small, medium‐sized, and large axons of db/db mice compared with WT mice. In ob/ob mice, only large fibers showed a decrease in myelin sheath thickness. The number of nonmyelinated nerve fibers was lower in ob/ob mice than in the db/db group. A thickened basal lamina of Schwann cells occurred in the ob/ob group only. In contrast, the basement membrane of endoneural microvessels was thickened in both obese groups. For this reason, laminin expression in Western blot analysis was lower in the db/db group than in the ob/ob one. Endoneural microvessels, which had been injected with fluorescein isothiocyanate, depicted signs of vasodilatation in the ob/ob and vasoconstriction in db/db mice. Endoneural vessels displayed two receptors of oxLDL. LOX‐1 was strongly expressed in db/db mice, whereas TLR4 was at its maximum in the ob/ob group. We conclude that changes in nerve fibers and in endoneural microvessels are present in sciatic nerve of both mouse models of type 2 diabetes. Upregulation of oxLDL‐dependent receptors in endoneural microvessels might be connected to different degrees of oxidative stress in severe diabetic db/db mice and in the mild diabetic ob/ob group.

Apolipoproteins D and E3 exert neurotrophic and synaptogenic effects in dorsal root ganglion cell cultures.

Co-cultures of 3T3-L1 adipocytes with neurons from the rat dorsal root ganglia (DRG) showed enhanced neuritogenesis and synaptogenesis. Microarray analysis for upregulated genes in adipocyte/DRG co-cultures currently points to apolipoproteins D and E (ApoD, ApoE) as influential proteins. We therefore tested adipocyte-secreted cholesterol and the carrier proteins ApoD and ApoE3. Cholesterol, ApoD, and ApoE3 each increased neurite outgrowth and upregulated the expression of presynaptic synaptophysin and synaptotagmin, as well as the postsynaptic density protein 95. The neurotrophic effects of ApoD and ApoE3 were associated with an increased expression of the low-density lipoprotein receptor and apolipoprotein E receptor 2. Simultaneous treatment with receptor-associated protein, an apolipoprotein receptor antagonist, inhibited the neurotrophic function of both apolipoproteins. The application of ApoD, ApoE3, and cholesterol to DRG cell cultures corresponded with increased expression of the chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4). Surprisingly, the inhibition of CXCR4 by the antagonistic drug AMD3100 decreased the apolipoprotein/cholesterol dependent neurotrophic effects. We thus assume that apolipoprotein-induced neuritogenesis in DRG cells interferes with CXCR4 signaling, and that adipocyte-derived apolipoproteins might be helpful in nerve repair.

COMP-Angiopoietin-1 Recovers Molecular Biomarkers of Neuropathy and Improves Vascularisation in Sciatic Nerve of ob/ob Mice

Background Leptin-deficient ob/ob mice are a model of type 2 diabetes induced peripheral neuropathy. Ob/ob mice exhibit obesity, insulin resistance, hyperglycaemia, and alterations of peripheral nerve fibres and endoneural microvessels. Here we test the hypothesis that cartilage oligomeric matrix protein (COMP)-Ang-1, a soluble and stabile form of Ang-1 which promotes angiogenesis and nerve growth, improves regeneration of nerve fibres and endoneural microvessels in ob/ob mice. Methods and Findings COMP-Ang-1 (100 ng/ml) or NaCl were intraperitoneally (i.p.) injected into male (N = 184), 3-month old, ob/ob or ob/+ mice for 7 and 21 days. We measured expression of Nf68, GAP43, Cx32, Cx26, Cx43, and TNFα in sciatic nerves using Western blot analysis. To investigate the inflammation in sciatic nerves, numbers of macrophages and T-cells were counted after immunofluorescence staining. In ultrathin section, number of myelinated/non-mylinated nerve fibers, g-ratio, the thickness of Schwann cell basal lamina and microvessel endothelium were investigated. Endoneural microvessels were reconstructed with intracardial FITC injection. Treatment with COMP-Ang-1 over 21 days significantly reduced fasting blood glucose and plasma cholesterol concentrations compared to saline treated ob/ob mice. In addition, COMP-Ang-1 treatment: 1) up-regulated expression of Nf68 and GAP43; 2) improved expression of gap junction proteins including connexin 32 and 26; 3) suppressed the expression of TNFα and Cx43 and 4) led to decreased macrophage and T-cell infiltration in sciatic nerve of ob/ob mice. The significant changes of sciatic nerve ultrastructure were not observed after 21-day long COMP-Ang-1 treatment. COMP-Ang-1 treated ob/ob mice displayed regeneration of small-diameter endoneural microvessels. Effects of COMP-Ang-1 corresponded to increased phosphorylation of Akt and p38 MAPK upon Tie-2 receptor. Conclusions COMP-Ang-1 recovers molecular biomarkers of neuropathy, promotes angiogenesis and suppresses inflammation in sciatic nerves of ob/ob mice suggesting COMP-Ang-1 as novel treatment option to improve morphologic and protein expression changes associated with diabetic neuropathy.

Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.

Identification of genetic loci associated with different responses to high-fat diet-induced obesity in C57BL/6N and C57BL/6J substrains

We have recently demonstrated that C57BL/6NTac and C57BL/6JRj substrains are significantly different in their response to high-fat diet-induced obesity (DIO). The C57BL/6JRj substrain seems to be protected from DIO and genetic differences between C57BL/6J and C57BL/6N substrains at 11 single nucleotide polymorphism (SNP) loci have been identified. To define genetic variants as well as differences in parameters of glucose homeostasis and insulin sensitivity between C57BL/6NTac and C57BL/6JRj substrains that may explain the different response to DIO, we analyzed 208 first backcross (BC1) hybrids of C57BL/6NTac and C57BL/6JRj [(C57BL/6NTac × C57BL/6JRj)F1 × C57BL/6NTac] mice. Body weight, epigonadal and subcutaneous fat mass, circulating leptin, as well as parameters of glucose metabolism were measured after 10 wk of high-fat diet (HFD). Genetic profiling of BC1 hybrids were performed using TaqMan SNP genotyping assays. Furthermore, to assess whether SNP polymorphisms could affect mRNA level, we carried out gene expression analysis in murine liver samples. Human subcutaneous adipose tissue was used to verify murine data of SNAP29. We identified four sex-specific variants that are associated with the extent of HFD-induced weight gain and fat depot mass. BC1 hybrids carrying the combination of risk or beneficial alleles exhibit the phenotypical extremes of the parental strains. Murine and human SC expression analysis revealed Snap29 as strongest candidate. Our data indicate an important role of these loci in responsiveness to HFD-induced obesity and suggest genes of the synaptic vesicle release system such as Snap29 being involved in the regulation of high-fat DIO.

Deferoxamine-induced neurite outgrowth and synapse formation in postnatal rat dorsal root ganglion (DRG) cell cultures

Deferoxamine (DFO) was granted orphan drug status for the treatment of traumatic spinal cord injury but its neuroprotective mechanism is not well understood. We therefore investigated the mode of action of DFO in serum-starved and/or iron-stressed cultures of rat dorsal root ganglion (DRG) cells. We probed for redox signaling by determining hemeoxygenase-1 activity and by measuring expression of intracellular iron metabolism-related proteins under pro-oxidative conditions. We also employed DNA microarrays to better understand the genomic response of DRG cultures to treatment with DFO thereby enabling the generation of hypotheses. Essentially, DFO treatment resulted in outgrowth of neurofilament 200-positive neurites and induction of synapse formation as determined by immunoblotting, transmission electron microscopy and immunofluorescence confocal microscopy. Furthermore, DFO treatment of DRG cell cultures activated neuroprotective and antioxidative programs such as matrix metallopeptidase 2 and apolipoprotein D to promote neurite regeneration. Indeed, DFO reduced markedly reactive oxygen species formation, increased the expression of hemeoxygenase-1 and improved iron management through regulation of transferrin receptor and ferritin. We propose DFO treatment of DRG cell cultures to completely abolish the oxidative effect of ferrous iron (Fe(2+)). Taken collectively, DFO reduced oxidative stress and induced synthesis of neuroprotective and antioxidative molecules to foster nerve repair and functional recovery. Our findings help to better understand the therapeutic benefit of DFO in the treatment of spinal cord injury.

Repin1 maybe involved in the regulation of cell size and glucose transport in adipocytes.

Replication initiator 1 (Repin1) is highly expressed in liver and adipose tissue and has been suggested as candidate gene for obesity and its related metabolic disorders in congenic and subcongenic rat strains. The cellular localization and function of Repin1 has remained elusive since its discovery in 1990. To characterize the role of Repin1 in adipocyte biology, we used siRNA knockdown technology to reduce the expression of Repin1 by electroporation of semiconfluent 3T3-L1 preadipocytes. Glucose transport, palmitate uptake as well as triglyceride content were measured. In paired samples of human visceral and subcutaneous adipose tissue, we investigated whether Repin1 mRNA expression is related to measures of fat accumulation and adipocyte size. We demonstrate that Repin1 increases during adipogenesis. RNA interference based Repin1 downregulation in mature adipocytes significantly reduces adipocyte size and causes reduced basal, but enhanced insulin stimulated glucose uptake into 3T3-L1 cells. Additionally, knockdown of Repin1 resulted in reduced palmitate uptake and significantly changed the mRNA expression of genes involved lipid droplet formation, adipogenesis, glucose and fatty acid transport. Furthermore, we found significant correlations between Repin1 mRNA expression in human paired visceral and subcutaneous adipose tissue and total body fat mass as well as adipocyte size. We have identified a potential role for Repin1 in the regulation of adipocyte size and expression of glucose transporters GLUT1 and GLUT4 in adipocytes.