Up-regulated autophagy: as a protective factor in adipose tissue of WOKW rats with metabolic syndrome.

BACKGROUND: Wistar Ottawa Karlsburg W (RT1u) rats (WOKW) are a model of the metabolic syndrome (MetS). Adipose tissue (AT) and peripheral nerves of WOKW rats exhibit up-regulated autophagy and inflammation corresponding with decreased apoptosis rate. The aim of this study was to characterize AT in WOKW rats in relation to autophagic activity. METHODS: mRNA and protein expression of adiponectin, pro-inflammatory and pro-apoptotic markers including MCP1, TNFα, cleaved caspase-3 and RNF157, a new candidate gene regulated through autophagy, were analyzed in adipocytes isolated from visceral and subcutaneous AT of 5-month old WOKW rats with MetS and LEW.1W controls in response to pharmacological inhibition of autophagy. Immunohistochemistry was performed to detect adiponectin and RNF157 protein in cultured adipocytes. RESULTS: Inhibition of autophagy by LY294002 was associated with a fourfold up-regulation of adiponectin expression and a decrease of RNF157 protein and pro-inflammatory markers-MCP-1 and TNFα predominantly in visceral adipocytes of obese WOKW rats compared to LEW.1W rats. Moreover, inhibition of autophagic activity correlates with an activation of cleaved caspase-3 apoptotic signaling pathway. CONCLUSIONS: Up-regulated autophagy in obese WOKW rats contributes to the regulation of visceral AT function and involves an altered balance between pro-inflammatory and protective adipokine expression. Our data suggest that activation of AT autophagy protects against adipocyte apoptosis at least under conditions of obesity related MetS in WOKW rats.

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.

Increased autophagy in peripheral nerves may protect Wistar Ottawa Karlsburg W rats against neuropathy.

OBJECTIVE: Wistar Ottawa Karlsburg W (RT1(u)) rats (WOKW) develop obesity, dyslipidemia, moderate hypertension, hyperinsulinemia and impaired glucose tolerance prone to induce peripheral neuropathy (PN). Autophagy has been shown to prevent neurodegeneration in the central and peripheral nervous system. We analyzed the potential protective role of autophagy in an established rat model in preventing PN. METHODS: We examined electrophysiology (motor-and sensory/mixed afferent conduction velocities and the minimal F-wave latency) and morphology, including ultrathin sections, myelin sheath thickness (g-ratio) and immunohistochemical markers of autophagy and inflammation in the sciatic nerve of five-month-old, male WOKW as compared to Wistar derived, congenic LEW.1W control rats, characterized by the same major histocompatibility complex as WOKW rats (RT1(u)). Moreover, the expression of axonal and synaptic proteins (NF68, GAP43, MP0), autophagy- (Atg5, Atg7, LC3), and apoptosis (cleaved caspase-3)-related markers was measured using Western blot. RESULTS: No abnormalities in nerve electrophysiology and morphology were found in WOKW compared to LEW.1W rats. However, autophagosomes were more frequently apparent in sciatic nerves of WOKW rats. In Western blot analyses no significant differences in expression of neuronal structural proteins were found, but autophagy markers were up-regulated in WOKW compared to LEW.1W sciatic nerves. Immunostaining revealed a greater infiltration of Iba1/ED-1-positive macrophages, CD-3-positive T-cells and LC3-expression in sciatic nerves of WOKW rats. CONCLUSIONS: Our results indicate that WOKW rats show an up-regulated autophagy and a mild inflammatory response but do not develop overt neuropathy. We suggest that autophagy and inflammatory cells may exert a protective role in preventing neuropathy in this rat model of the metabolic syndrome but the mechanism of action is still unclear.

Leptin-deficient (ob/ob) mouse ovaries show fatty degeneration, enhanced apoptosis and decreased expression of steroidogenic acute regulatory enzyme.

OBJECTIVE: Leptin-deficient (ob/ob) mice are obese and infertile. Dysfunctions of the ovaries are preferentially related to leptin-deficiency. DESIGN: Morphological and molecular biological obesity-dependent changes in ob/ob ovaries. SUBJECTS: Ovaries were obtained from three-month-old mice either homozygote (ob/ob) and heterozygote (ob/+) or wild-type (C57BL6, WT) for the investigation by light and electron microscopy, as well as for western blot analysis of lectin-like oxidised low density lipoprotein receptor (LOX-1), Toll-like receptor 4 (TLR4), CD36, cleaved caspase-3, microtubule-associated protein light chain 3 (LC3), and the steroidogenic acute regulatory protein (StAR). RESULTS: Compared with control ovaries with corpora lutea, ob/ob ovaries lacked corpora lutea, follicular atresia was at a higher rate; lipid droplets accumulated in follicle cells and in the oocyte with damaged mitochondria; the basement membrane of follicles was thickened. LOX-1 and CD36 expressions were comparable for all three groups. Ob/ob ovaries showed significantly higher levels of TLR4 and cleaved caspase-3 than the ones from the control groups. The high LC3-II/I ratio in the WT and ob/+ ovaries was related to the presence of corpora lutea. The StAR protein was lower in the ob/ob ovaries signifying reduced steroidogenesis. CONCLUSIONS: Excessive lipid storage causes disorders of ovarian function in ob/ob mice. The local lipid overload leads to advanced follicular atresia with apoptosis and defect steroidogenesis. We suggest that the changes in lipid metabolism lead to increased oxidative stress and thereby, they are an important reason of anovulation and infertility.

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.

Peptidergic and nitrergic innervation of the pineal gland in the domestic pig: an immunohistochemical study.

The presence and co-localization of vasoactive intestinal polypeptide (VIP), peptide N-terminal histidine C-terminal isoleucine (PHI), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM), calcitonin gene-related peptide (CGRP), substance P (SP) and the neuronal isoform of nitric oxide synthase (NOS) were studied in neuronal structures of the pig pineal gland. Paraformaldehyde-fixed pineals of 3-month-old gilts were sliced into serial cryostat sections, which were subjected to a set of double immunofluorescence stainings. Based on the co-existence patterns of neuropeptides, five populations of nerve fibres supplying the pig pineal were distinguished: (1) PHI-positive, (2) PACAP-positive, (3) SOM-positive, (4) SP/CGRP-positive and (5) SP-positive/CGRP-negative. Only a subpopulation of PHI-positive fibres contained VIP at the level detectable by immunofluorescence. NOS was found in some intrapineal PHI- and VIP-positive fibres. PHI-, VIP- and NOS-positive nerve fibres were more numerous in the peripheral than in the central part of the pineal. PACAP-positive fibres were equally distributed within the gland. The density of SOM-positive fibres was higher in the ventro-proximal than in the dorso-distal part of the pineal. SOM was also detected in some neuronal-like cells or specialized pinealocytes situated in the central region of the gland. Two populations of fibres containing SP were found: CGRP-positive, present in the distal and central parts of the pineal as well as CGRP-negative, localized in the proximal compartment of the gland.

Pituitary adenylate cyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers in the pig pineal gland.

The present study demonstrates the occurrence of PACAP-immunoreactive (PACAP-IR) nerve fibers in different compartments of the pig pineal gland, including glandular capsule (where they form a very dense network) and subependymal tissue close to the pineal recess (moderate to dense meshwork of varicose fibers). Furthermore, several varicose fibers penetrate from the capsule into the connective tissue septa and then into the parenchyma, where they form unequally distributed, fine network and, in some cases, basket-like structures around pinealocytes. Some of the PACAP-IR nerve fibers, observed both in the habenular and posterior epithalamic areas, extend to the pineal gland. PACAP-IR cells could be demonstrated neither in the pineal gland, nor in epithalamic areas.