A novel kinase regulates dietary restriction-mediated longevity in Caenorhabditis elegans.

Although dietary restriction (DR) is known to extend lifespan across species, from yeast to mammals, the signalling events downstream of food/nutrient perception are not well understood. In Caenorhabditis elegans, DR is typically attained either by using the eat-2 mutants that have reduced pharyngeal pumping leading to lower food intake or by feeding diluted bacterial food to the worms. In this study, we show that knocking down a mammalian MEKK3-like kinase gene, mekk-3 in C. elegans, initiates a process similar to DR without compromising food intake. This DR-like state results in upregulation of beta-oxidation genes through the nuclear hormone receptor NHR-49, a HNF-4 homolog, resulting in depletion of stored fat. This metabolic shift leads to low levels of reactive oxygen species (ROS), potent oxidizing agents that damage macromolecules. Increased beta-oxidation, in turn, induces the phase I and II xenobiotic detoxification genes, through PHA-4/FOXA, NHR-8 and aryl hydrocarbon receptor AHR-1, possibly to purge lipophilic endotoxins generated during fatty acid catabolism. The coupling of a metabolic shift with endotoxin detoxification results in extreme longevity following mekk-3 knock-down. Thus, MEKK-3 may function as an important nutrient sensor and signalling component within the organism that controls metabolism. Knocking down mekk-3 may signal an imminent nutrient crisis that results in initiation of a DR-like state, even when food is plentiful.

Augmentation of postswelling surgical sealant potential of adhesive hydrogels.

Two-component hydrogels formed with star polyethylene glycol amine and linear dextran aldehyde polymers (PEG:dextran) show promise as tissue-specific surgical sealants. However, there is a significant loss of adhesion strength to soft tissues following PEG:dextran swelling, which may limit material ability to appose disjoined tissues and prevent leakage from surgical sites. We covalently incorporated the modified amino acid L-3,4-dihydroxyphenylalanine (L-DOPA) into PEG:dextran to enhance postswelling sealant performance. L-DOPA is an essential component of marine animal adhesive plaques and has been used to confer wet adhesion in synthetic materials. As both PEG:dextran cohesion and adhesion are mediated by aldehyde-amine interactions, L-DOPA side-groups make it a potent network modulator with potential to affect multiple material properties. Following 1-h submersion in aqueous media, PEG:dextran doped with 3 mM L-DOPA/M aldehyde on average swelled 50.3% less, had 287.4% greater stiffness, and had 53.6% greater functional adhesion strength compared to the neat hydrogel. Increased concentrations of L-DOPA up to 11 mM L-DOPA/M aldehyde similarly curtailed swelling and mitigated property loss with hydration, but sacrificed initial functional adhesion strength, material modulus, and biocompatibility. Taken together, these data support tailored L-DOPA conjugation as a promising approach to enhance the clinical performance of PEG:dextran sealants.

A murine experimental anthracycline extravasation model: pathology and study of the involvement of topoisomerase II alpha and iron in the mechanism of tissue damage.

The bisdioxopiperazine topoisomerase II catalytic inhibitor dexrazoxane has successfully been introduced into the clinic as an antidote to accidental anthracycline extravasation based on our preclinical mouse studies. The histology of this mouse extravasation model was investigated and found to be similar to findings in humans: massive necrosis in the subcutis, dermis and epidermis followed by sequestration and healing with granulation tissue, and a graft-versus-host-like reaction with hyperkeratotic and acanthotic keratinocytes, occasional apoptoses, epidermal invasion by lymphocytes and healing with dense dermal connective tissue. The extension of this fibrosis was quantified, and dexrazoxane intervention resulted in a statistically significant decrease in fibrosis extension, as also observed in the clinic. Several mechanisms have been proposed in anthracycline extravasation cytotoxicity, and we tested two major hypotheses: (1) interaction with topoisomerase II alpha and (2) the formation of tissue damaging reactive oxygen species following redox cycling of an anthracycline Fe(2+) complex. Dexrazoxane could minimise skin damage via both mechanisms, as it stops the catalytic activity of topoisomerase II alpha and thereby prevents access of anthracycline to the enzyme and thus cytotoxicity, and also acts as a strong iron chelator following opening of its two bisdioxopiperazine rings. Using the model of extravasation in a dexrazoxane-resistant transgenic mouse with a heterozygous mutation in the topoisomerase II alpha gene (Top2a(Y165S/+)), we found that dexrazoxane provided a protection against anthracycline-induced skin wounds that was indistinguishable from that found in wildtype mice. Thus, interaction with topoisomerase II alpha is not central in the pathogenesis of anthracycline-induced skin damage. In contrast to dexrazoxane, the iron-chelating bisdioxopiperazine ICRF-161 do not inhibit the catalytic cycle of topoisomerase II alpha. This compound was used to isolate and test the importance of iron in the wound pathogenesis. ICRF-161 was found ineffective in the treatment of anthracycline-induced skin damage, suggesting that iron does not play a dominant role in the genesis of wounds.

Tissue-specific increases in 11beta-hydroxysteroid dehydrogenase type 1 in normal weight postmenopausal women.

With age and menopause there is a shift in adipose distribution from gluteo-femoral to abdominal depots in women. Associated with this redistribution of fat are increased risks of type 2 diabetes and cardiovascular disease. Glucocorticoids influence body composition, and 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) which converts inert cortisone to active cortisol is a putative key mediator of metabolic complications in obesity. Increased 11betaHSD1 in adipose tissue may contribute to postmenopausal central obesity. We hypothesized that tissue-specific 11betaHSD1 gene expression and activity are up-regulated in the older, postmenopausal women compared to young, premenopausal women. Twenty-three pre- and 23 postmenopausal, healthy, normal weight women were recruited. The participants underwent a urine collection, a subcutaneous adipose tissue biopsy and the hepatic 11betaHSD1 activity was estimated by the serum cortisol response after an oral dose of cortisone. Urinary (5alpha-tetrahydrocortisol+5beta-tetrahydrocortisol)/tetrahydrocortisone ratios were higher in postmenopausal women versus premenopausal women in luteal phase (P<0.05), indicating an increased whole-body 11betaHSD1 activity. Postmenopausal women had higher 11betaHSD1 gene expression in subcutaneous fat (P<0.05). Hepatic first pass conversion of oral cortisone to cortisol was also increased in postmenopausal women versus premenopausal women in follicular phase of the menstrual cycle (P<0.01, at 30 min post cortisone ingestion), suggesting higher hepatic 11betaHSD1 activity. In conclusion, our results indicate that postmenopausal normal weight women have increased 11betaHSD1 activity in adipose tissue and liver. This may contribute to metabolic dysfunctions with menopause and ageing in women.

Expression and activity of 11beta-hydroxysteroid dehydrogenase type 1 enzyme in subcutaneous and visceral adipose tissue of prepubertal children.

BACKGROUND: Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Ligand availability to the glucocorticoid receptor is controlled by isoforms of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) which converts endogenous cortisone to active cortisol. AIM: To evaluate the expression and activity of 11beta-HSD1 in subcutaneous adipose tissue (SC) and visceral adipose tissue (VAT) in prepubertal children with normal weight. METHODS: Fourteen patients (11 female/3 male) with a mean age of 6.9+/-0.9 years and a body mass index (BMI) of 17.4+/-0.61 underwent elective open abdominal surgery. RESULTS: Expression of 11beta-HSD1 mRNA in SC and VAT was similar (0.8+/-0.15 vs. 0.61+/-0.12 AU). The activity of this enzyme in SC was significantly lower compared to VAT (1.42+/-0.39 vs. 2.79+/-0.61 ng cortisol/g tissue/24 h, p<0.05). In addition, we observed a significant direct correlation with the expression of 11beta-HSD1 in VAT adipose tissue with the patient's BMI (r=0.825, p=0.002). CONCLUSIONS: This correlation together with the increased activity of this enzyme in visceral adipose tissue might contribute to decreased hepatic insulin sensitivity due to increased portal cortisol when BMI increases. These observations appear to be particularly important in children born with low birth weight who develop rapid early weight gain.

Localization of stilbene synthase in Vitis vinifera L. during berry development.

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.

Evolutionarily conserved WNK and Ste20 kinases are essential for acute volume recovery and survival after hypertonic shrinkage in Caenorhabditis elegans.

Members of the germinal center kinase (GCK)-VI subfamily of Ste20 kinases regulate a Caenorhabditis elegans ClC anion channel and vertebrate SLC12 cation-Cl(-) cotransporters. With no lysine (K) (WNK) protein kinases interact with and activate the mammalian GCK-VI kinases proline-alanine-rich Ste20-related kinase (PASK) and oxidative stress-responsive 1 (OSR1). We demonstrate here for the first time that GCK-VI kinases play an essential role in whole animal osmoregulation. RNA interference (RNAi) knockdown of the single C. elegans GCK-VI kinase, GCK-3, dramatically inhibits systemic volume recovery and survival after hypertonic shrinkage. Tissue-specific RNAi suggests that GCK-3 functions primarily in the hypodermis and intestine to mediate volume recovery. The single C. elegans WNK kinase, WNK-1, binds to GCK-3, and wnk-1 knockdown gives rise to a phenotype qualitatively similar to that of gck-3(RNAi) worms. Knockdown of the two kinases together has no additive effect, suggesting that WNK-1 and GCK-3 function in a common pathway. We postulate that WNK-1 functions upstream of GCK-3 in a manner similar to that postulated for its mammalian homologs. Phylogenetic analysis of kinase functional domains suggests that the interaction between GCK-VI and WNK kinases first occurred in an early metazoan and therefore likely coincided with the need of multicellular animals to tightly regulate transepithelial transport processes that mediate systemic osmotic homeostasis.

Induction of type 3 deiodinase activity in inflammatory cells of mice with chronic local inflammation.

During illness, changes in thyroid hormone metabolism occur, so-called nonthyroidal illness (NTI). NTI has been characterized by a fall of serum T(3) due to decreased extrathyroidal conversion of T(4) into T(3) by liver type 1 deiodinase (D1), without an increase in serum TSH. Type 3 deiodinase (D3) was thought not to play an important role during NTI, but recently it has been shown that D3 activity is up-regulated in liver and skeletal muscle of critically ill patients related to hypoxia. We studied D3 gene expression and activity in liver and muscle/subcutis of mice during illness, which was induced by two different stimuli: bacterial endotoxin (lipopolysaccharide) administration, resulting in an acute systemic response, and a turpentine injection in each hindlimb, resulting in a local sc abscess. Lipopolysaccharide induced a rapid decrease in liver D1 and D3 activity but not skeletal muscle of hindlimb. In contrast, local inflammation induced by turpentine did not decrease liver D1 and D3 activity but increased markedly D3 activity in the muscle/subcutis sample containing the abscess, associated with strongly increased IL-1beta and IL-6 mRNA expression. Inflammatory cells, surrounding the abscess showed D3 and T(3)-transporter monocarboxylate transporter-8 immunoreactivity, whereas muscle cells did not show any immunoreactivity. In conclusion, local inflammation strongly induces D3 activity in inflammatory cells, especially in invading polymorphonuclear granulocytes, suggesting enhanced local degradation of T(3).

C. elegans serine-threonine kinase KIN-29 modulates TGFbeta signaling and regulates body size formation.

BACKGROUND: In C. elegans there are two well-defined TGFbeta-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFbeta signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. RESULTS: kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase) family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. CONCLUSION: kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFbeta outputs, such as tail morphogenesis.

Ovarian hormone status and abdominal visceral adipose tissue metabolism.

We examined abdominal sc and visceral adipose tissue metabolism in a sample of 19 regularly cycling premenopausal women (age 46.3 +/- 3.7 yr) and 10 women with natural menopause or pharmacological ovarian suppression (age 51.1 +/- 9.2 yr). Subcutaneous and visceral (omental, epiploic) adipose tissue biopsies were obtained during abdominal hysterectomies. Body composition and adipose tissue distribution were measured before the surgery by dual x-ray absorptiometry and computed tomography, respectively. Ovarian hormone-deficient women tended to be older (P = 0.08) and were characterized by increased visceral adipose tissue area (P < 0.05). Subcutaneous adipocyte size, lipoprotein lipase (LPL) activity, and basal lipolysis were not significantly different between groups. On the other hand, omental fat cell size was significantly higher in ovarian hormone-deficient women, compared with premenopausal women (P < 0.05). The omental/sc LPL activity ratio and omental adipocyte basal lipolysis were also significantly higher in ovarian hormone-deficient women (P < 0.05 for both comparisons). Significant positive correlations were found between visceral adipose tissue area and omental LPL activity (r = 0.54, P < 0.003), omental adipocyte basal lipolysis (r = 0.66, P < 0.0001), and omental fat cell size (r = 0.81, P < 0.0001). In multivariate analyses, ovarian status was no longer a significant predictor of adipose cell metabolism variables after visceral adipose tissue area was entered into the model, with the exception of the omental/sc LPL activity ratio, which remained independently associated with ovarian status. In conclusion, although the size of the visceral adipose tissue compartment was an important determinant of adipocyte metabolism in this depot, the increased omental/sc LPL activity ratio in ovarian hormone-deficient women supports the notion of a predominant visceral fat storage in these women.

Cytochemical and immunohistochemical evidence of direct origin of white adipocytes from mesenchymal cells.

AIM: By using histochemical, enzymohistochemical and immunohistochemical methods the present study aims to find out specific morphological markers in the precursors of the white subcutaneous adipocytes of human and rat embryos, which could be used for identification and help in clarification of the origin of that cell type. MATERIAL AND METHODS: On cryostat sections of subcutaneous tissue from the thigh region of human embryos and hind limb of rat embryos histological hematoxylineosin staining, Sudan III-hematoxylin staining and enzyme-histochemical reactions for lipoprotein lipase are performed. On paraffin section of the same material immunohistochemical reaction for leptin is performed. RESULTS: The analysis reveals that the earliest preadipocytes possess specific histochemical, enzymohistochemical and immunohistochemical features, which characterize exclusively these cells but not the fibroblasts and endothelial cells scattered around in the subcutaneous tissue. These features include lipid drops, positive enzymohistochemical reaction for lipoprotein lipase and immunohistochemical expression for leptin. CONCLUSION: The results could be used as evidence of direct origin of the white adipocytes from the embryonal mesenchyme through their own progenitor cells rather than through fibroblasts or endothelial cells.

Differences in delta9 desaturase activity between Jersey- and Limousin-sired cattle.

An experiment examined delta9 desaturase activity and FA composition in subcutaneous adipose tissue in two differing breeds of cattle. Jersey-sired cattle had significantly higher rates of desaturase activity than Limousin-sired cattle (1.55 vs. 0.75 nmol/mg protein/min). This difference was also demonstrated by a lower concentration of individual (e.g., 18:0) and total saturated FA (38.3 vs. 45.1 wt%), and a higher concentration of individual (e.g., 16:1) and total monounsaturated FA (58.2 vs. 52.7 wt%) in the Jersey animals. Other indices of desaturation calculated from the FA composition showed this same difference. The slip point of adipose tissue of Jersey cattle (36.8 degrees C) was significantly lower than that of Limousin cattle (39.2 degrees C), but Jersey adipose tissue had a greater content of beta-carotene. The positive relationship between adipose tissue beta-carotene and desaturation opposes the negative relationship between dietary beta-carotene and desaturation determined elsewhere. These results, however, lead to the hypothesis that some cattle have a reduced capacity to metabolize beta-carotene to various forms of vitamin A, a compound that can reduce delta9 desaturase enzyme activity. In addition, the higher level of intramuscular fat in Jersey cattle (6.97 vs. 3.82%) is possibly related to a lack of inhibition of the adipocyte differentiation genes by vitamin A.

Subcutaneous adipose 11 beta-hydroxysteroid dehydrogenase type 1 activity and messenger ribonucleic acid levels are associated with adiposity and insulinemia in Pima Indians and Caucasians.

Metabolic effects of cortisol may be critically modulated by glucocorticoid metabolism in tissues. Specifically, active cortisol is regenerated from inactive cortisone by the enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11-HSD1) in adipose and liver. We examined activity and mRNA levels of 11-HSD1 and tissue cortisol and cortisone levels in sc adipose tissue biopsies from 12 Caucasian (7 males and 5 females) and 19 Pima Indian (10 males and 9 females) nondiabetic subjects aged 28 +/- 7.6 yr (mean +/- SD; range, 18-45). Adipose 11-HSD1 activity and mRNA levels were highly correlated (r = 0.51, P = 0.003). Adipose 11-HSD1 activity was positively related to measures of total (body mass index, percentage body fat) and central (waist circumference) adiposity (P < 0.05 for all) and fasting glucose (r = 0.43, P = 0.02), insulin (r = 0.60, P = 0.0005), and insulin resistance by the homeostasis model (r = 0.70, P < 0.0001) but did not differ between sexes or ethnic groups. Intra-adipose cortisol was positively associated with fasting insulin (r = 0.37, P = 0.04) but was not significantly correlated with 11-HSD1 mRNA or activity or with other metabolic variables. In this cross-sectional study, higher adipose 11-HSD1 activity is associated with features of the metabolic syndrome. Our data support the hypothesis that increased regeneration of cortisol in adipose tissue influences metabolic sequelae of human obesity.

Modulation of 11 beta-hydroxysteroid dehydrogenase type 1 in mature human subcutaneous adipocytes by hypothalamic messengers.

Glucocorticoids are regulated at the prereceptor level by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which interconverts inactive cortisone and active cortisol. In a previous study, we noted that patients with hypothalamic obesity had an increased ratio of cortisol/cortisone metabolites, suggesting enhanced 11 beta-HSD-1 activity. In this in vitro study, we tested the hypothesis that adipose 11 beta-HSD-1 is regulated by the hypothalamus via circulating hormones, sympathetic nervous system innervation, and/or cytokines. Preadipocytes were retrieved from sc fat from healthy nonobese individuals and differentiated in vitro to mature adipocytes. Cells were incubated with several potential effectors, and the activity of 11 beta-HSD-1 was assayed by measuring conversion of added 500 nM cortisone to cortisol. Expression of 11 beta-HSD-1 mRNA was determined by real-time PCR, whereas lipolytic effects were determined by measuring glycerol concentration in the culture medium. CRH down-regulated 11 beta-HSD-1 activity with maximal effect at 10(-9)M (65 +/- 10% of control; P < 0.001) and caused a reduction in lipolysis. Likewise, ACTH down-regulated 11 beta-HSD-1 activity with maximal effect at 10(-9) M (65 +/- 20%; P < 0.05) and reduced medium glycerol. Neither CRH nor ACTH affected 11 beta-HSD-1 mRNA expression. TNF alpha up-regulated 11 beta-HSD-1 activity maximally at 0.6 x 10(-9) M (140 +/- 20%; P < 0.001); the same cytokine increased 11 beta-HSD-1 mRNA levels to 3-fold of control (P < 0.05) and increased medium glycerol levels to 165 +/- 14% of control (P < 0.01). IL-1 beta also up-regulated 11 beta-HSD-1 activity maximally at 0.6 x 10(-9) M (160 +/- 33%; P < 0.001) and caused an increase in glycerol levels (159 +/- 11% of control; P < 0.001). Of the adrenergic agonists, salbutamol up-regulated 11 beta-HSD-1 activity maximally at 10(-7) M (162 +/- 46%; P < 0.02), and clonidine down-regulated it at 10(-7) M (82 +/- 15%; P < 0.005). We conclude that possible distinct hypothalamic mediators regulating adipose tissue 11 beta-HSD-1 might include down-regulation of 11 beta-HSD-1 activity by CRH, ACTH, and alpha 2 sympathetic stimulation, and up-regulation of the enzyme by beta 2 sympathetic stimulation and by the cytokines TNFalpha and IL-1 beta.

Cytochemical study of in situ differentiation of white adipose cells in rat.

AIM: The aim of the present study was to follow up the histochemical and enzyme-histochemical characteristics of differentiating white adipose cells of rat in situ. METHOD: Nine rat fetuses aged 15-21 day of gestation were used. Fragments from the subcutaneous tissue of lower limb were frozen at -18 degrees C. On consecutive cryostat sections Sudan III-hematoxylin staining and enzyme-histochemical reactions for NADH2-cytochrome C-reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and lipoprotein lipase were performed. RESULTS: The onset of the rat adipocyte differentiation was detected at days 15-16 of gestation. It was seen as appearance and accumulation of lipid droplets, rounding of the cells and displacement of the nuclei to the cell periphery. Gradually, with the advance of gestational age the number of differentiating adipose cells increased and lipid packages, composed of mature-like unilocular adipocytes and multilocular adipocytes that had not yet completed their differentiation, were formed. The differentiating embryonal adipose cells expressed positive enzyme-histochemical reactions for NADH2-cytochrome C-reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and lipoprotein lipase. CONCLUSION: The described histochemical and enzyme-histochemical characteristics could be used as markers for distinguishing the earliest embryonal adipose cells in rat, which is impossible with the classic histological techniques. At the same time they might be accepted as the primary morphological criteria of adipocyte differentiation in situ.