Nutrigenomics approach elucidates health-promoting effects of high vegetable intake in lean and obese men

We aimed to explore whether vegetable consumption according to guidelines has beneficial health effects determined with classical biomarkers and nutrigenomics technologies. Fifteen lean (age 36 ± 7 years; BMI 23.4 ± 1.7 kg m⁻²) and 17 obese (age 40 ± 6 years; BMI 30.3 ± 2.4 kg m⁻²) men consumed 50- or 200-g vegetables for 4 weeks in a randomized, crossover trial. Afterward, all subjects underwent 4 weeks of energy restriction (60 % of normal energy intake). Despite the limited weight loss of 1.7 ± 2.4 kg for the lean and 2.1 ± 1.9 kg for the obese due to energy restriction, beneficial health effects were found, including lower total cholesterol, LDL cholesterol and HbA1c concentrations. The high vegetable intake resulted in increased levels of plasma amino acid metabolites, decreased levels of 9-HODE and prostaglandin D3 and decreased levels of ASAT and ALP compared to low vegetable intake. Adipose tissue gene expression changes in response to vegetable intake were identified, and sets of selected genes were submitted to network analysis. The network of inflammation genes illustrated a central role for NFkB in (adipose tissue) modulation of inflammation by increased vegetable intake, in lean as well as obese subjects. In obese subjects, high vegetable intake also resulted in changes related to energy metabolism, adhesion and inflammation. By inclusion of sensitive omics technologies and comparing the changes induced by high vegetable intake with changes induced by energy restriction, it has been shown that part of vegetables’ health benefits are mediated by changes in energy metabolism, inflammatory processes and oxidative stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0343-9) contains supplementary material, which is available to authorized users.     

Response of Asthmatics to Isoprenaline and Salbutamol Aerosols Administered by Intermittent Positive-pressure Ventilation

The bronchodilator and cardiac effects produced by aerosols of 0·5% isoprenaline and of 0·25, 0·5, and 1% salbutamol administered in 40% oxygen by intermittent positive-pressure ventilation were compared in 24 asthmatic patients. Isoprenaline and salbutamol in concentrations of 0·5% were equipotent in peak bronchodilator effect; salbutamol was superior in total bronchodilator effect and duration of average effect, but the peak bronchodilator effect occurred earlier after isoprenaline. Significantly greater tachycardia was produced by 0·5% isoprenaline than by the same concentration of salbutamol. The 0·25, 0·5, and 1% concentrations of salbutamol had about the same peak bronchodilator effect, but there was a stepwise increase in total effect and duration of average effect in relation to the concentration used. A similar stepwise increase in heart rate was also noted, but with all concentrations this was significantly less than with 0·5% isoprenaline. It was concluded that a 0·5% solution of salbutamol, which provided maximal bronchodilatation without important tachycardia, was therapeutically superior to the other three treatments.     

VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.     

Female Mice are Protected against High-Fat Diet Induced Metabolic Syndrome and Increase the Regulatory T Cell Population in Adipose Tissue

Sex differences in obesity-induced complications such as type 2 diabetes have been reported. The aim of the study was to pinpoint the mechanisms resulting in different outcome of female and male mice on a high-fat diet (HFD). Mice fed control or HFD were monitored for weight, blood glucose, and insulin for 14 weeks. Circulating chemokines, islet endocrine function and blood flow, as well as adipose tissue populations of macrophages and regulatory T-lymphocytes (T_reg) were thereafter assessed. Despite similar weight (43.8±1.0 and 40.2±1.5 g, respectively), male but not female mice developed hyperinsulinemia on HFD as previously described (2.5±0.7 and 0.5±0.1 pmol/l, respectively) consistent with glucose intolerance. Male mice also exhibited hypertrophic islets with intact function in terms of insulin release and blood perfusion. Low-grade, systemic inflammation was absent in obese female but present in obese male mice (IL-6 and mKC, males: 77.4±17 and 1795±563; females: 14.6±4.9 and 240±22 pg/ml), and the population of inflammatory macrophages was increased in intra-abdominal adipose tissues of high-fat-fed male but not female mice. In contrast, the anti-inflammatory T_reg cell population increased in the adipose tissue of female mice in response to weight gain, while the number decreased in high-fat-fed male mice. In conclusion, female mice are protected against HFD-induced metabolic changes while maintaining an anti-inflammatory environment in the intra-abdominal adipose tissue with expanded T_reg cell population, whereas HFD-fed male mice develop adipose tissue inflammation, glucose intolerance, hyperinsulinemia, and islet hypertrophy.     

Deletion of Hypoxia-Inducible Factor-1α in Adipocytes Enhances Glucagon-Like Peptide-1 Secretion and Reduces Adipose Tissue Inflammation

It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.     

Erythropoietin Over-Expression Protects against Diet-Induced Obesity in Mice through Increased Fat Oxidation in Muscles

Erythropoietin can be over-expressed in skeletal muscles by gene electrotransfer, resulting in 100-fold increase in serum EPO and significant increases in haemoglobin levels. Earlier studies have suggested that EPO improves several metabolic parameters when administered to chronically ill kidney patients. Thus we applied the EPO over-expression model to investigate the metabolic effect of EPO in vivo.At 12 weeks, EPO expression resulted in a 23% weight reduction (P<0.01) in EPO transfected obese mice; thus the mice weighed 21.9±0.8 g (control, normal diet,) 21.9±1.4 g (EPO, normal diet), 35.3±3.3 g (control, high-fat diet) and 28.8±2.6 g (EPO, high-fat diet). Correspondingly, DXA scanning revealed that this was due to a 28% reduction in adipose tissue mass.The decrease in adipose tissue mass was accompanied by a complete normalisation of fasting insulin levels and glucose tolerance in the high-fat fed mice. EPO expression also induced a 14% increase in muscle volume and a 25% increase in vascularisation of the EPO transfected muscle. Muscle force and stamina were not affected by EPO expression. PCR array analysis revealed that genes involved in lipid metabolism, thermogenesis and inflammation were increased in muscles in response to EPO expression, while genes involved in glucose metabolism were down-regulated. In addition, muscular fat oxidation was increased 1.8-fold in both the EPO transfected and contralateral muscles.In conclusion, we have shown that EPO when expressed in supra-physiological levels has substantial metabolic effects including protection against diet-induced obesity and normalisation of glucose sensitivity associated with a shift to increased fat metabolism in the muscles.     

Relationship between the tissue level of cyclic AMP and the fat cell size of human adipose tissue.

The relationship between mean fat cell size, maximal tissue cyclic AMP concentration, and glycerol release was investigated in human subcutaneous adipose tissue incubated in vitro with or without isoprenaline or noradrenaline added at maximal effective concentrations. Basal and stimulated glycerol release and cyclic AMP concentration were each related to the fat cell size. Whether or not the phosphodiesterase inhibitor theophylline was present in the incubation system, basal and noradrenaline-induced cyclic AMP levels were significantly correlated with the fat cell size. The noradrenaline-induced cyclic AMP levels resulted in twice as rapid glycerol release as could be expected from the basal ratio between glycerol release and cyclic AMP. Furthermore, both basal and noradrenaline-induced glycerol release in relation to the cyclic AMP levels were more rapid in enlarge fat cells. It is concluded that basal and catecholamine-induced production of cyclic AMP is related to the fat cell size and that a quantitative relationship exists between rate of lipolysis and maximal tissue levels of cyclic AMP in human adipose tissue. Basal and noradrenaline-induced lipolysis are probably regulated by different mechanisms and the lipolytic sensitivity to cyclic AMP seems increased in large fat cells.     

Effects of Proportions of Dietary Macronutrients on Glucocorticoid Metabolism in Diet-Induced Obesity in Rats

Tissue glucocorticoid levels in the liver and adipose tissue are regulated by regeneration of inactive glucocorticoid by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and inactivation by 5α- and 5β-reductases. A low carbohydrate diet increases hepatic 11β-HSD1 and reduces glucocorticoid metabolism during weight loss in obese humans. We hypothesized that similar variations in macronutrient proportions regulate glucocorticoid metabolism in obese rats. Male Lister Hooded rats were fed an obesity-inducing ad libitum ‘Western’ diet (37% fat, n = 36) for 22 weeks, then randomised to continue this diet (n = 12) or to switch to either a low carbohydrate (n = 12) or a moderate carbohydrate (n = 12) diet for the final 8 weeks. A parallel lean control group were fed an ad libitum control diet (10% fat, n = 12) throughout. The low and moderate carbohydrate diets decreased hepatic 11β-HSD1 mRNA compared with the Western diet (both 0.7±0.0 vs 0.9±0.1 AU; p<0.01), but did not alter 11β-HSD1 in adipose tissue. 5α-Reductase mRNA was increased on the low carbohydrate compared with the moderate carbohydrate diet. Compared with lean controls, the Western diet decreased 11β-HSD1 activity (1.6±0.1 vs 2.8±0.1 nmol/mcg protein/hr; p<0.001) and increased 5α-reductase and 5β-reductase mRNAs (1.9±0.3 vs 1.0±0.2 and 1.6±0.1 vs 1.0±0.1 AU respectively; p<0.01) in the liver, and reduced 11β-HSD1 mRNA and activity (both p<0.01) in adipose tissue. Although an obesity-inducing high fat diet in rats recapitulates the abnormal glucocorticoid metabolism associated with human obesity in liver (but not in adipose tissue), a low carbohydrate diet does not increase hepatic 11β-HSD1 in obese rats as occurs in humans.     

Association between Serum Irisin Levels and Non-Alcoholic Fatty Liver Disease in Health Screen Examinees

Irisin is a recently found myokine that aids obesity control and improves glucose homeostasis by acting on white adipose tissue cells and increases total energy consumption. The aim of this study was to evaluate serum irisin levels in patients with non-alcoholic fatty liver disease (NAFLD) and to compare these levels with those of normal controls. Among 595 health screen examinees who had visited our institute between January 2013 to March 2013, 355 patients (84 NAFLD patients and 271 normal controls) were enrolled depending on whether they gave written informed consents and their history of alcohol intake, blood tests, and abdominal ultrasonographic findings. Age; sex; laboratory test parameters; homeostasis model assessment-insulin resistance; and levels of leptin, adiponectin, and irisin were assessed. Serum irisin levels (ng/ml) were significantly higher in the NAFLD group than in normal controls (63.4±32.6 vs. 43.0±29.7, p<0.001) and higher in the mild fatty liver group than in the moderate-to-severe fatty liver group (68.3±38.2 vs. 56.6±21.2, p<0.001). Additionally, serum irisin levels were not different between the non-obese and obese groups (48.4±34.2 vs. 45.8±22.9, p = 0.492); however, the levels were significantly lowest in normal controls and highest in the mild fatty liver group in the non-obese (44.9±31.7 vs. 73.1±48.5 vs 59.7±18.0, p<0.001) and obese groups (35.0±17.0 vs. 62.9±21.2 vs. 54.6±23.3, p<0.001). Serum irisin levels were significantly higher in NAFLD patients, which is not consistent with the results of previously published studies. Therefore, more studies are needed to confirm the role of irisin in NAFLD.     

Free fatty acid release from human adipose tissue in relation to obesity -- effect of isoprenaline.

The capacity to release non-esterified acids and glycerol from the abdominal subcutaneous adipose tissue into a medium containing different concentrations of isoprenaline was studied in 21 adults with different body weight (14 obese, 7 controls). The obese had a significantly lower maximum adipokinetic capacity (expressed as pD2) than controls. The significant decrease in pD2 in the obese was found to depend on the stabilization of the weight while the dynamic pD2 stage values of the obese did not differ from those of controls. The hypothesis is expressed that lower pD2 values may be related to the smaller decrease of body fat during weight reduction.     

Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.     

Plasma TSH and Serum T-4 Levels in Long-term Follow-up of Patients Treated with 131I for Thyrotoxicosis

In February 1972 58% of patients euthyroid after iodine-131 therapy given for thyrotoxicosis between 1954 and 1966 had a high plasma TSH (>7·4 μU/ml) and 42% a normal plasma TSH level. A group of 69 of the euthyroid patients with high plasma TSH levels (25·0±2·0 μU/ml) in 1972 were re-examined 15 and 24 months later. The mean plasma TSH in the 66 patients remaining euthyroid at 15 months was 22·6±1·8 μU/ml, while three patients had become hypothyroid. At 24 months 64 of the patients were still available for study, of whom 61 remained euthyroid with a mean plasma TSH of 21·6±2·0 μU/ml, and a further three had become hypothyroid.All of a group of 61 of the euthyroid patients with normal plasma TSH levels (4·0±0·2 μU/ml) in 1972 remained euthyroid at 24 months with a mean plasma TSH of 4·1±0·3 μU/ml, though the plasma TSH level had become slightly raised in three.The mean serum T-4 level in the euthyroid patients with a high plasma TSH was significantly lower, though still in the normal range, than that in the euthyroid patients with a normal plasma TSH both in 1972 and in 1974.Since no patient with a normal plasma TSH level after iodine-131 treatment six to 18 years earlier for thyrotoxicosis developed hypothyroidism over a two-year period, the follow-up of such patients need not be so rigorous as that of similarly treated euthyroid patients with raised plasma TSH levels in whom hypothyroidism developed at the rate of 5% per year.     

Enhanced glycerol 3-phosphate dehydrogenase activity in adipose tissue of obese humans

The primary purpose of this investigation was to determine whether adipose tissue glycerol 3-phosphate dehydrogenase activity is associated with human obesity. The data presented in this paper indicate that the glycerol 3-phosphate dehydrogenase activity in adipose tissue from morbidly obese subjects is approximately 2-fold higher than from lean individuals. Moreover, positive correlation between adipose tissue glycerol 3-phosphate dehydrogenase activity and body mass index (BMI) (r = 0.5; p < 0.01) was found. In contrast, the adipose tissue fatty acid synthase (FAS) and ATP-citrate lyase (ACL) activities in morbidly obese patients are significantly lower than in lean subjects. Furthermore, negative correlation between adipose tissue FAS activity and BMI (r = –0.3; p < 0.05) as well as between ACL activity and BMI (r = –0.3; p < 0.05) was found.These data indicate that elevated glycerol 3-phosphate dehydrogenase might contribute to the increase of triacylglycerol (TAG) synthesis in obese subjects, however, fatty acids necessary for glycerol 3-phosphate esterification must be derived (because of lower FAS and ACL activities) mainly from TAG in circulating lipoproteins formed in liver (VLDL), and/or from the intake with food (chylomicrons).The conclusion is, that the enhanced activity of glycerol 3-phosphate dehydrogenase, and hence the generation of more glycerol 3-phosphate in adipose tissue offers a novel explanation for increased TAG production in adipose tissue of obese subjects.     

Effects of Salbutamol and Isoprenaline Phenylephrine in Reversible Airways Obstruction

Ventolin (salbutamol) and Medihaler-Duo (isoprenaline/phenylephrine combination) standard pressurized inhalers were used to administer doses of two or six “puffs” to 16 patients with known reversible airways obstruction. The doses were administered in random order over two days. Both the Ventolin and Medihaler-Duo inhalers substantially increased FEV₁, but in the doses used salbutamol was more effective than isoprenaline/phenylephrine (P < 0·01). There was no significant difference between two and six puffs of salbutamol, though there seemed to be an advantage of six puffs of isoprenaline/phenylephrine over two puffs (P < 0·05). Adrenaline (1/1,000) 0·5 ml and atropine 0·6 mg produced similar increases in FEV₁ to those produced by salbutamol.The Pao₂ fell more than 5 mm Hg in three patients after salbutamol and in three after isoprenaline/phenylephrine. There was no significant fall in mean Pao₂ in any of the treatment groups. It is concluded that the Ventolin inhalant, administered in the conventional dose of two puffs, is as effective a bronchodilator as subcutaneous adrenaline and atropine, is more effective than the Medihaler-Duo, and is without detectable side effects.     

Blood Angiotensin II Levels of Normal and Hypertensive Subjects

A specific radioimmunoassay for angiotensin II has shown that its normal concentration in arterial blood is 2·4±1·2 (S.D.) mμg./l00 ml.; the venous level is consistently below this value, being usually 50–75% of it. Definite rises in blood angiotensin II levels were found in some patients with hypertension, both essential and secondary to renal disease. Extremely low levels were observed in two anephric women, and in one patient with Conn''s syndrome. This radioimmunoassay offers a valuable alternative to renin bioassay in evaluation of the role of the renal pressor system in clinical disorders associated with hypertension and aldosteronism.     

Coupling of lipolysis and de novo lipogenesis in brown,beige, and white adipose tissues during chronic β3-adrenergic receptor activation

Chronic activation of β3-adrenergic receptors (β3-ARs) expands the catabolic activity of both brown and white adipose tissue by engaging uncoupling protein 1 (UCP1)-dependent and UCP1-independent processes. The present work examined de novo lipogenesis (DNL) and TG/glycerol dynamics in classic brown, subcutaneous “beige,” and classic white adipose tissues during sustained β3-AR activation by CL 316,243 (CL) and also addressed the contribution of TG hydrolysis to these dynamics. CL treatment for 7 days dramatically increased DNL and TG turnover similarly in all adipose depots, despite great differences in UCP1 abundance. Increased lipid turnover was accompanied by the simultaneous upregulation of genes involved in FAS, glycerol metabolism, and FA oxidation. Inducible, adipocyte-specific deletion of adipose TG lipase (ATGL), the rate-limiting enzyme for lipolysis, demonstrates that TG hydrolysis is required for CL-induced increases in DNL, TG turnover, and mitochondrial electron transport in all depots. Interestingly, the effect of ATGL deletion on induction of specific genes involved in FA oxidation and synthesis varied among fat depots. Overall, these studies indicate that FAS and FA oxidation are tightly coupled in adipose tissues during chronic adrenergic activation, and this effect critically depends on the activity of adipocyte ATGL.     

Comparison of adipose glycerol kinase of hyperglycemic obese mice and lean litter-mates

Summary Glycerol kinase activity found in the epididymal adipose tissue of lean litter-mates of hyperglycemic obese mice exhibits two distinct Km values. The Km(s) obtained graphically by a Hoftsee plot are 40 and 637 m. The glycerol kinase of obese mice showed 29 times more total activity per fat pad or 9 times more activity per mg of protein as compared to that of the lean controls. The increased glycerol kinase activity found in the obese mice predominantly exhibited low Km value. Increase in the activity with the high Km value was minimal. The apparent molecular weight of adipose glycerol kinase is approximately 60,000–65,000. A higher activity of glycerol kinase in the epididymal adipose tissue of the obese mice (ob/ob) has been reported by Treble and Mayer ¹. The significance of this increased enzyme in obesity² has been investigated by many investigators^3–6, using either whole epididymal fat pad or isolated fat cells as the source of glycerol kinase. However, little is known regarding the properties of this enzyme, and even the characterization of the liver glycerol kinase is not yet complete^{7, 8}.We have recently shown that adipose glycerol kinase of Sprague-Dawly rat and of Swiss mice exhibit two Km values, and the apparent molecular weight is approximately 55,000–60,000⁹. The present study was undertaken in order to establish whether the enzyme in ob/+ mice also exhibits two Km values and if so, whether the activity associated with both Km(s) is increased. We found that the specific conditions for enzyme activity determination and the basis on which the activity is expressed are quantitatively important in clarifying the difference in glycerol kinase activity of ob/ob and ob/+ mice. The increased activity in ob/ob mice is predominantly the low Km enzyme, and the apparent molecular weight is approximately 60,000–65,000.Supported in part by the Juvenile Diabetes Foundation. We thank Mr. CHARLES ROSENTHAL for technical assistance.     

Role of NKG2D in Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance

The early events that initiate inflammation in the adipose tissue during obesity are not well defined. It is unclear whether the recruitment of CD8 T cells to the adipose tissue during onset of obesity occurs through antigen-dependent or -independent processes. We have previously shown that interaction between NKG2D (natural-killer group 2, member D) and its ligand Rae-1ε is sufficient to recruit cytotoxic T lymphocytes to the pancreas and induce insulitis. Here, we tested whether NKG2D–NKG2D ligand interaction is also involved in obesity-induced adipose tissue inflammation and insulin resistance. We observed a significant induction of NKG2D ligand expression in the adipose tissue of obese mice, especially during the early stages of obesity. However, mice lacking NKG2D developed similar levels of insulin resistance and adipose tissue inflammation compared to control mice when placed on a high-fat diet. Moreover, overexpression of Rae-1ε in the adipose tissue did not increase immune cell infiltration to the adipose tissue either in the setting of a normal or high-fat diet. These results indicate that, unlike in the pancreas, NKG2D–NKG2D ligand interaction does not play a critical role in obesity-induced inflammation in the adipose tissue.     

Assessment of Upper Airways Obstruction

An indication of obstruction to the upper airways (trachea and larynx) may be obtained by calculating the ratio of the forced expired volume in one second to the peak expiratory flow rate (FEV₁/PEFR). This index was found to be usually less than 10 in normal subjects (mean 7·3), and in patients with asthma (mean 6·9), chronic bronchitis (mean 7·7), or interstitial lung disease (mean 6·3). A study of simulated upper airways obstruction showed that this index rises as the obstruction becomes more severe. All of 18 patients with proved upper airways obstruction had FEV₁/PEFR indices greater than 10 (mean 14·0). This test can be carried out with forced expiratory manoeuvres only, and it does not require the use of complicated equipment. An FEV₁/PEFR ratio greater than 10, when upper airways obstruction is suspected, indicates that significant obstruction may be present. High values suggest that the obstruction may be severe, and that further investigations are indicated.     

Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain

Introduction

Nerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.

Methods

Primary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E₂ (PGE₂), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.

Results

Unstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE₂, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.

Conclusions

These results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.