Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

The amount of hyaluronic acid and airway remodelling increase with the severity of inflammation in neutrophilic equine asthma.

BACKGROUND: Equine asthma (EA) is a chronic lower airway inflammation that leads to structural and functional changes. Hyaluronic acid (HA) has crucial functions in the extracellular matrix homeostasis and inflammatory mediator activity. HA concentration in the lungs increases in several human airway diseases. However, its associations with naturally occurring EA and airway remodelling have not been previously studied. Our aim was to investigate the association of equine neutrophilic airway inflammation (NAI) severity, airway remodelling, and HA concentration in horses with naturally occurring EA. We hypothesised that HA concentration and airway remodelling would increase with the severity of NAI. HA concentrations of bronchoalveolar lavage fluid supernatant (SUP) and plasma of 27 neutrophilic EA horses, and 28 control horses were measured. Additionally, remodelling and HA staining intensity were assessed from endobronchial biopsies from 10 moderate NAI horses, 5 severe NAI horses, and 15 control horses. RESULTS: The HA concentration in SUP was higher in EA horses compared to controls (p = 0.007). Plasma HA concentrations were not different between the groups. In the endobronchial biopsies, moderate NAI horses showed epithelial hyperplasia and inflammatory cell infiltrate, while severe NAI horses also showed fibrosis and desquamation of the epithelium. The degree of remodelling was higher in severe NAI compared to moderate NAI (p = 0.048) and controls (p = 0.016). Intense HA staining was observed in bronchial cell membranes, basement membranes, and connective tissue without significant differences between the groups. CONCLUSION: The release of HA to the airway lumen increases in naturally occurring neutrophilic EA without clear changes in its tissue distribution, and significant airway remodelling only develops in severe NAI.

Increased n-6 Polyunsaturated Fatty Acids Indicate Pro- and Anti-Inflammatory Lipid Modifications in Synovial Membranes with Rheumatoid Arthritis.

Emerging evidence suggests that fatty acids (FAs) and their lipid mediator derivatives can induce both beneficial and detrimental effects on inflammatory processes and joint degradation in osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA). The present study characterized the detailed FA signatures of synovial membranes collected during knee replacement surgery of age- and gender-matched OA and RA patients (n = 8/diagnosis). The FA composition of total lipids was determined by gas chromatography and analyzed with univariate and multivariate methods supplemented with hierarchical clustering (HC), random forest (RF)-based classification of FA signatures, and FA metabolism pathway analysis. RA synovium lipids were characterized by reduced proportions of shorter-chain saturated FAs (SFAs) and elevated percentages of longer-chain SFAs and monounsaturated FAs, alkenyl chains, and C20 n-6 polyunsaturated FAs compared to OA synovium lipids. In HC, FAs and FA-derived variables clustered into distinct groups, which preserved the discriminatory power of the individual variables in predicting the RA and OA inflammatory states. In RF classification, SFAs and 20:3n-6 were among the most important FAs distinguishing RA and OA. Pathway analysis suggested that elongation reactions of particular long-chain FAs would have increased relevance in RA. The present study was able to determine the individual FAs, FA groups, and pathways that distinguished the more inflammatory RA from OA. The findings suggest modifications of FA elongation and metabolism of 20:4n-6, glycerophospholipids, sphingolipids, and plasmalogens in the chronically inflamed RA synovium. These FA alterations could have implications in lipid mediator synthesis and potential as novel diagnostic and therapeutic tools.

Dihomo-γ-Linolenic Acid (20:3n-6)-Metabolism, Derivatives, and Potential Significance in Chronic Inflammation.

Dihomo-γ-linolenic acid (DGLA) has emerged as a significant molecule differentiating healthy and inflamed tissues. Its position at a pivotal point of metabolic pathways leading to anti-inflammatory derivatives or via arachidonic acid (ARA) to pro-inflammatory lipid mediators makes this n-6 polyunsaturated fatty acid (PUFA) an intriguing research subject. The balance of ARA to DGLA is probably a critical factor affecting inflammatory processes in the body. The aim of this narrative review was to examine the potential roles of DGLA and related n-6 PUFAs in inflammatory conditions, such as obesity-associated disorders, rheumatoid arthritis, atopic dermatitis, asthma, cancers, and diseases of the gastrointestinal tract. DGLA can be produced by cultured fungi or be obtained via endogenous conversion from γ-linolenic acid (GLA)-rich vegetable oils. Several disease states are characterized by abnormally low DGLA levels in the body, while others can feature elevated levels. A defect in the activity of ∆6-desaturase and/or ∆5-desaturase may be one factor in the initiation and progression of these conditions. The potential of GLA and DGLA administrations as curative or ameliorating therapies in inflammatory conditions and malignancies appears modest at best. Manipulations with ∆6- and ∆5-desaturase inhibitors or combinations of long-chain PUFA supplements with n-3 PUFAs could provide a way to modify the body's DGLA and ARA production and the concentrations of their pro- and anti-inflammatory mediators. However, clinical data remain scarce and further well-designed studies should be actively promoted.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Synovial Fluid Fatty Acid Profiles Are Differently Altered by Inflammatory Joint Pathologies in the Shoulder and Knee Joints.

Anomalies of fatty acid (FA) metabolism characterize osteoarthritis (OA) and rheumatoid arthritis (RA) in the knee joint. No previous study has investigated the synovial fluid (SF) FA manifestations in these aging-related inflammatory diseases in the shoulder. The present experiment compared the FA alterations between the shoulder and knee joints in patients with end-stage OA or end-stage RA. SF samples were collected during glenohumeral or knee joint surgery from trauma controls and from OA and RA patients (n = 42). The FA composition of SF total lipids was analyzed by gas chromatography with flame ionization and mass spectrometric detection and compared across cohorts. The FA signatures of trauma controls were mostly uniform in both anatomical locations. RA shoulders were characterized by elevated percentages of 20:4n-6 and 22:6n-3 and with reduced proportions of 18:1n-9. The FA profiles of OA and RA knees were relatively uniform and displayed lower proportions of 18:2n-6, 22:6n-3 and total n-6 polyunsaturated FAs (PUFAs). The results indicate location- and disease-dependent differences in the SF FA composition. These alterations in FA profiles and their potential implications for the production of PUFA-derived lipid mediators may affect joint lubrication, synovial inflammation and pannus formation as well as cartilage and bone degradation and contribute to the pathogeneses of inflammatory joint diseases.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism.

BACKGROUND: Orotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena. METHODS: In our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography-mass spectrometry. RESULTS: Our data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles. CONCLUSIONS: The present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

Distinct fatty acid signatures in infrapatellar fat pad and synovial fluid of patients with osteoarthritis versus rheumatoid arthritis.

BACKGROUND: Infrapatellar fat pad (IFP) has recently emerged as a potential source of inflammation in knee arthropathies. It has been proposed to be one source of adipocytokines, fatty acids (FA), and FA-derived lipid mediators that could contribute to the pathophysiological processes in the knee joint. Alterations in synovial fluid (SF) lipid composition have been linked to both osteoarthritis (OA) and rheumatoid arthritis (RA). The aim of the present study was to compare the FA signatures in the IFP and SF of RA and OA patients. METHODS: Pairs of IFP and SF samples were collected from the same knees of RA (n = 10) and OA patients (n = 10) undergoing total joint replacement surgery. Control SF samples (n = 6) were harvested during diagnostic or therapeutic arthroscopic knee surgery unrelated to RA or OA. The FA composition in the total lipids of IFP and SF was determined by gas chromatography with flame ionization and mass spectrometric detection. RESULTS: Arthropathies resulted in a significant reduction in the SF proportions of n-6 polyunsaturated FA (PUFA), more pronouncedly in OA than in RA. OA was also characterized with reduced percentages of 22:6n-3 and lower product/precursor ratios of n-3 PUFA. The proportions of total monounsaturated FA increased in both RA and OA SF. Regarding IFP, RA patients had lower proportions of 20:4n-6, total n-6 PUFA, and 22:6n-3, as well as lower product/precursor ratios of n-3 PUFA compared to OA patients. The average chain length of SF FA decreased in both diagnoses and the double bond index in OA. CONCLUSIONS: The observed complex alterations in the FA signatures could have both contributed to but also limited the inflammatory processes and cartilage destruction in the RA and OA knees.

Hyaluronan histochemistry-a potential new tool to assess the progress of liver disease from simple steatosis to hepatocellular carcinoma.

Nonalcoholic fatty liver disease is among the most common liver diseases worldwide and one cause of cirrhosis that can result in the development of hepatocellular carcinoma (HCC). Hyaluronan (HA) is a high-molecular-mass glycosaminoglycan with diverse functions in tissue injury and repair, for instance, in inflammation and fibrogenesis. The aim of the present study was to investigate the relationships between the HA synthesizing and degrading enzymes in a spectrum of liver pathologies. This was realized by histological staining of liver sections from controls and patients with simple steatosis, steatohepatitis, cirrhosis and HCC (n = 90). HA-positive staining intensified in connective tissue in all liver pathologies, and staining of CD44, the major HA receptor, similarly increased in steatohepatitis and cirrhosis. HA synthase 1 (HAS1)-positive staining was reduced in steatosis, steatohepatitis and HCC. Staining of HAS3, which produces HA of a lower molecular mass, promotes inflammation and is pathogenic in animal models, increased in all diagnoses. The responses in staining intensity of HAS2 and hyaluronidases 1-2 were specific for different cell types. These findings suggest that HAS1-2 are responsible for HA synthesis in healthy livers, while HAS3 increases in importance in liver diseases. It is noteworthy that the pathological changes in HA metabolism are already visible in simple steatosis and, thus, precede the histological changes of inflammation and fibrosis. It could be possible to intervene in disease progression at an early stage by influencing HA metabolism. The results could have potential clinical applications with HAS3 immunostaining supplementing the existing HCC diagnostics.

Extracellular vesicles are integral and functional components of the extracellular matrix.

Extracellular vesicles (EV) are small plasma membrane-derived particles released into the extracellular space by virtually all cell types. Recently, EV have received increased interest because of their capability to carry nucleic acids, proteins, lipids and signaling molecules and to transfer their cargo into the target cells. Less attention has been paid to their role in modifying the composition of the extracellular matrix (ECM), either directly or indirectly via regulating the ability of target cells to synthesize or degrade matrix molecules. Based on recent results, EV can be considered one of the structural and functional components of the ECM that participate in matrix organization, regulation of cells within it, and in determining the physical properties of soft connective tissues, bone, cartilage and dentin. This review addresses the relevance of EV as specific modulators of the ECM, such as during the assembly and disassembly of the molecular network, signaling through the ECM and formation of niches suitable for tissue regeneration, inflammation and tumor progression. Finally, we assess the potential of these aspects of EV biology to translational medicine.

First in vivo detection and characterization of hyaluronan-coated extracellular vesicles in human synovial fluid.

Extracellular vesicles (EVs) function in intercellular signaling by transporting different membrane and cytosolic molecules, including hyaluronan (HA) and its synthesis machinery. As both EVs and HA are abundant in synovial fluid, we hypothesized that HA synthesized in synovial membrane would be carried on the surface of EVs. Synovial fluid (n = 15) and membrane samples (n = 5) were obtained from knee surgery patients. HA concentrations were analyzed in synovial fluid and HA and its synthesis machinery were examined with histochemical stainings in synovial membrane. To assess the size distribution of EVs in synovial fluid and to visualize HA on EVs, nanoparticle tracking analysis (NTA), confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were utilized. The average HA concentration in synovial fluid was 2.0 ± 0.21 mg/ml without significant differences between the patients with trauma/diagnostic arthroscopy and primary or post-traumatic osteoarthritis. Positive stainings of HA synthases (HAS1-3), HA and its receptor CD44 in synovial cells indicated active HA secretion in synovial membrane. According to NTA, EVs were abundant in synovial fluid and their main populations were ≤300 nm in diameter after differential centrifugation. There were no significant differences in the EV counts between the patients with primary or post-traumatic osteoarthritis. TEM verified that HA-positive particles detected by CLSM were lipid membrane vesicles surrounded by a HA coat. Our results provide the first in vivo evidence that human synovial fluid contains HA-positive EVs, one source of which presumably is the long HAS-positive protrusions of synovial fibroblasts. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1960-1968, 2016.

Evidence of endoplasmic reticulum stress and liver inflammation in the American mink Neovison vison with benign hepatic steatosis.

We investigated the presence of inflammatory signs in the progression of fatty liver disease induced by fasting. Sixty standard black American mink (Neovison vison) were fasted for 0, 1, 3, 5, or 7 days and one group for 7 days followed by re-feeding for 28 days. Liver sections were evaluated histologically and liver mRNA levels indicating endoplasmic reticulum (ER) stress, adipogenic transformation, and inflammation were assessed by quantitative real-time PCR. After 3 days of fasting, the mink had developed moderate liver steatosis. Increased hyaluronan reactivity in lymphocytic foci but no Mallory-Denk bodies were seen in livers of the mink fasted for 5-7 days. Up-regulation of glucose-regulated protein, 78 kDa was observed on day 7 indicating ER stress, especially in the females. Liver lipoprotein lipase and monocyte chemoattractant protein 1 mRNA levels increased in response to 5-7 days of food deprivation, while tumor necrosis factor α (TNF-α) was the highest in the mink fasted for 5 days. The expression of the genes of interest, except for TNF-α, correlated with each other and with the liver fat content. The mRNA levels were found to change more rapidly below n-3/n-6 polyunsaturated fatty acid ratio threshold of 0.15. Following re-feeding, hepatocyte morphology and mRNA abundance returned to pre-fasting levels. Within the studied timeframe, evidence for ER stress, adipogenic transformation, and liver inflammation suggested incipient transition from steatosis to steatohepatitis with potential for development of more severe liver disease. This may present a possibility to influence disease progression before histologically observable steatohepatitis.

Manifestations of fasting-induced fatty liver and rapid recovery from steatosis in voles fed lard or flaxseed oil lipids.

Long-chain n-3 polyunsaturated fatty acids (PUFA) can have beneficial effects against fat deposition, cardiovascular diseases, and liver steatosis. We investigated how diets based on lard (predominantly saturated and monounsaturated fatty acids) or flaxseed oil (rich in 18:3n-3) affect liver fat-% and fatty acid profiles of tundra voles (Microtus oeconomus). We also studied potential participation of hyaluronan (HA) in the pathology of fatty liver and whether the development and recovery of fasting-induced steatosis are influenced by n-3 PUFA. The dietary fatty acid composition was manifested in the liver fatty acid signatures. Fasting for 18 h induced macrovesicular steatosis and the liver fat-% increased to 22% independent of the preceding diet. Fasting-induced steatosis did not involve inflammation or connective tissue activation indicated by the absence of both leukocyte accumulation and increased HA. Food deprivation modified the liver fatty acid signatures to resemble more closely the diets. Fasting reduced the proportions of long-chain n-3 PUFA in both dietary regimes and n-3/n-6 PUFA ratios in the lard-fed voles. Decreases in long-chain n-3 PUFA may promote lipid accumulation by modulating the expression of lipid-metabolizing genes. Dietary 18:3n-3 did not prevent the development or attenuate the manifestation of steatosis in the fasted voles or promote the recovery.

Lessons from a non-domestic canid: joint disease in captive raccoon dogs (Nyctereutes procyonoides).

The purpose of this study was to describe pathological changes of the shoulder, elbow, hip and stifle joints of 16 museum skeletons of the raccoon dog (Nyctereutes procyonoides). The subjects had been held in long-term captivity and were probably used for fur farming or research, thus allowing sufficient longevity for joint disease to become recognisable. The prevalence of disorders that include osteochondrosis, osteoarthritis and changes compatible with hip dysplasia, was surprisingly high. Other changes that reflect near-normal or mild pathological conditions, including prominent articular margins and mild bony periarticular rim, were also prevalent. Our data form a basis for comparing joint pathology of captive raccoon dogs with other mammals and also suggest that contributing roles of captivity and genetic predisposition should be explored further in non-domestic canids.

Freezing tolerance and low molecular weight cryoprotectants in an invasive parasitic fly, the deer ked (Lipoptena cervi).

Insect cold hardiness is often mediated by low molecular weight cryoprotectants, such as sugars, polyols, and amino acids (AA). While many free-living northern insects must cope with extended periods of freezing ambient temperatures (Ta), the ectoparasitic deer ked Lipoptena cervi imago can encounter subfreezing Ta only during a short autumnal period between hatching and host location. Subsequently, it benefits from the body temperature of the cervid host for survival in winter. This study investigated the cold tolerance of the species by determining its lower lethal temperature (100% mortality, LLT100) during faster and slower cold acclimation, by determining the supercooling point (SCP) and by measuring the concentrations of potential low molecular weight cryoprotectants. The LLT100 of the deer ked was approximately -16 ° C, which would enable it to survive freezing nighttime Ta not only in its current area of distribution but also further north. The SCP was -7.8 ° C, clearly higher than the LLT100 , indicating that the deer ked displays freezing tolerance. The concentrations of free AA, especially nonessential AA, were higher in the cold-acclimated deer keds similar to several other insects. The concentrations of proline increased together with γ-aminobutyrate, arginine, asparagine, cystine, glutamate, glutamine, hydroxylysine, sarcosine, serine, and taurine. AA could be hypothesized to act as cryoprotectants by, e.g., protecting enzymes and lipid membranes from damage caused by cold.

Adaptations to fasting in a terrestrial mustelid, the sable (Martes zibellina).

The aim of this study was to investigate whether the actively wintering Palearctic sable Martes zibellina has evolved physiological adaptations to tolerate nutritional scarcity. Sixteen farm-bred male sables were divided into a fed control group and an experimental group fasted for 4 days. The rate of weight loss in the sable was similar to other medium-sized mustelids. Fasting led to hypoglycaemia and to a decreased lymphocyte percentage. The sable derived metabolic energy from both subcutaneous and intraabdominal white adipose tissues and the relative decrease in fat mass was the largest for the retroperitoneal and subcutaneous depots. Metabolic energy derived partly from body proteins indicated by the increased plasma levels of urea, uric acid and total essential amino acids. Triacylglycerols accumulated in the livers of the fasted sables and the increased plasma aminotransferase activities suggested hepatic dysfunction. The decreased plasma insulin concentrations and the elevated cortisol levels probably contributed to stimulated lipolysis and protein catabolism. Moreover, fasting increased the plasma ghrelin concentrations of the sables and down-regulated the thyroid activity.

Endocrine and metabolic alterations in the mink (Mustela vison) due to chronic phytoestrogen exposure.

Phytoestrogens are natural components of plant-based food items with beneficial health effects. The aim of the present study was to investigate the chronic effects of dietary phytoestrogens, genistein (8 mg kg(-1) day(-1)) and beta-sitosterol (50 mg kg(-1) day(-1)), on the weight regulation of the mink (Mustela vison). The parental generation was exposed from August 2002 to May-June 2003 to either beta-sitosterol or genistein, while the kits were exposed through gestation and lactation. Food consumption and body masses were monitored monthly. Plasma lipid, glucose, total protein and hormone (ghrelin, leptin, triiodothyronine and thyroxine) concentrations were measured from the parents in August 2002, January 2003 and at the end of the experiment in May-June 2003 when the kits were 21 days of age. Relative food intake was higher in the beta-sitosterol-exposed minks than in the control or genistein minks in September 2002. Plasma leptin and total protein concentrations were lower in the beta-sitosterol kits compared to the control kits. Furthermore, plasma ghrelin levels and liver phosphorylase activities of the mink kits were higher due to genistein exposure. In mink kits, exposure to both phytoestrogens reduced the plasma thyroxine concentrations. The kidney glycogen concentrations and the muscle phosphorylase activities of phytoestrogen-treated adult minks were elevated. The results of this study suggest that minks are sensitive to perinatal phytoestrogen exposure.

Adaptations of the raccoon dog (Nyctereutes procyonoides) to wintering--effects of restricted feeding or periodic fasting on lipids, sex steroids and reproduction.

The raccoon dog (Nyctereutes procyonoides) is an omnivorous canid with autumnal hyperphagia and fattening followed by mid-winter passivity and fasting in boreal latitudes with seasonal snow cover. The effects of two different feeding levels (400 or 200 kcal/animal/d) or fasting (5-week fasting+1-week feeding+3-week fasting) on plasma lipids, sex steroids and reproductive success of farm-bred raccoon dogs (n=60 females and 24 males) were studied in winter. The body masses, body mass indices (BMIs) and levels of plasma triacylglycerols (TG), total cholesterol and low- and high-density lipoprotein cholesterol did not differ between the fed and the restrictively fed animals. During fasting, the plasma TG concentrations increased and the BMIs decreased, indicating the release of fatty acids from adipose tissue. After the fasting periods, the levels of plasma cholesterol and high-density lipoprotein cholesterol increased, whereas the TG levels decreased indicating the rebuilding of energy reserves. The fact that the different wintertime feeding regimes had no impact on the plasma glucose, total protein, cortisol, estradiol, progesterone or testosterone levels, or on the reproductive success, indicates versatile adaptive capacity in the species.

Phytoestrogens alter the reproductive organ development in the mink (Mustela vison).

The aim of the present study was to examine the reproductive effects of two perorally applied phytoestrogens, genistein (8 mg/kg/day) and beta-sitosterol (50 mg/kg/day), on the mink (Mustela vison) at human dietary exposure levels. Parental generations were exposed over 9 months to these phytoestrogens and their offspring were exposed via gestation and lactation. Parents and their offspring were sampled 21 days after the birth of the kits. Sex hormone levels, sperm quality, organ weights, and development of the kits were examined. The exposed females were heavier than the control females at the 1st postnatal day (PND). The control kits were heavier than the exposed kits from the 1st to the 21st PND. Phytoestrogens did not affect the organ weights of the adult minks, but the relative testicular weight of the exposed kits was higher than in the control kits. The relative prostate weight was higher and the relative uterine weight lower in the beta-sitosterol-exposed kits than in the control kits. Moreover, the plasma dihydrotestosterone levels were lower in the genistein-exposed male kits compared to the control male kits. This study could not explain the mechanisms behind these alterations. The results indicate that perinatal phytoestrogen exposures cause alterations in the weight of the reproductive organs of the mink kits.