The effect of vitamin MK-7 on bone mineral density and microarchitecture in postmenopausal women with osteopenia, a 3-year randomized, placebo-controlled clinical trial.

We conducted a randomized placebo-controlled double-blinded clinical trial of MK-7 or placebo daily for 3 years in postmenopausal women with osteopenia. BMD decreased at all sites without differences between the MK-7 and placebo-treated women. Changes in bone turnover markers and microstructure were similar between the two groups. INTRODUCTION: Vitamin K is a cofactor in the carboxylation of osteocalcin (OC) and carboxylated OC promotes mineralization of bone. Clinical studies suggest that vitamin K2 prevents bone loss. The aim of the study was to investigate the effect of vitamin K2 as an add-on to calcium and vitamin D supplementation on osteocalcin, bone mass, and microarchitecture in postmenopausal women. METHODS: We conducted a randomized placebo-controlled double-blinded clinical trial, including 142 postmenopausal women with osteopenia who received vitamin K2 (375 µg MK-7) or placebo daily for 3 years. Both groups received vitamin D3 (38 µg/day) and calcium (800 mg/day). We measured bone turnover markers in serum and bone mineral density and microarchitecture by DXA and HRpQCT. RESULTS: Undercarboxylated osteocalcin decreased in the MK-7-group (- 65.2 ± 23.5%) (mean ± SD) compared with the placebo group (- 0.03 ± 38.5%), p < 0.01 after 1 year. After 3 years, aBMD decreased at all sites without differences between the MK-7 and placebo-treated women (p > 0.09). aBMD decreased at the total hip by 1.5 ± 2.5% and 2.4 ± 2.7% in the MK-7 and the placebo groups, respectively, at the femoral neck by 1.5 ± 3.5% and 1.0 ± 5.0% in the MK-7 and the placebo groups, respectively, and at the lumbar spine by 1.8 ± 3.9% and 1.1 ± 3.1% in the MK-7 and the placebo groups, respectively. Changes in bone turnover markers were also similar between the two groups.We have previously reported improved microarchitecture with MK-7 after 1 year. However, changes in microstructure over 3 years were similar between the two groups, as assessed by both HRpQCT and DXA trabecular bone score. CONCLUSION: Treatment with MK-7 375 µg daily as an add-on to calcium and vitamin D increased carboxylation of osteocalcin. However, treatment of postmenopausal women with osteopenia for 3 years did not affect biochemical markers of bone turnover, bone mineral density, or bone microarchitecture. TRIAL REGISTRATION: The study was registered at Clinicaltrial.gov : NCT01922804 .

Oral 3-hydroxybutyrate ingestion decreases endogenous glucose production, lipolysis, and hormone-sensitive lipase phosphorylation in adipose tissue in men: a human randomized, controlled, crossover trial.

AIMS: To test whether oral administration of D/L-3-hydroxybutyrate as a sodium salt inhibits lipolysis and intracellular lipid signalling, in particular, hormone-sensitive lipase, and whether D/L-3-hydroxybutyrate alters endogenous glucose production. METHODS: We studied six young men in a randomized, controlled, crossover study after ingestion of Na-D/L-3-hydroxybutyrate (hyperketotic condition) or saline (placebo control). We quantified lipolysis and endogenous glucose production using [9,10-3 H]-palmitate and [3-3H]glucose tracers, and adipose tissue biopsies were collected to investigate key lipolytic enzymes. RESULTS: After ingestion, D/L-3-hydroxybutyrate increased by more than 2.5 mmol/l, free fatty acid concentrations decreased by >70%, and palmitate rate of appearance was halved. Protein kinase A phosphorylation of perilipin was reduced and hormone-sensitive lipase 660 phosphorylation in adipose tissue biopsies was 70-80% decreased in the hyperketotic condition and unchanged in the control. Compared to the control, endogenous glucose production was reduced by close to 20% (P<0.05) after 3-hydroxybutyrate ingestion. CONCLUSION: We conclude that oral D/L-Na-3-hydroxybutyrate increases D/L-3-hydroxybutyrate concentrations within half an hour, decreases free fatty acid concentrations, lowers lipolysis and endogenous glucose production, and dephosphorylates hormone-sensitive lipase. Collectively these phenomena may be viewed as an orchestrated feedback loop, controlling endogenous glucose production, lipolysis and ketogenesis. Such effects would be beneficial in insulin-resistant states. (www.clinicaltrials.gov ID number: NCT02917252).

Fracture-induced changes in biomarkers CTX, PINP, OC, and BAP-a systematic review.

To assess the time from fracture until bone turnover markers (BTM), which are biochemical markers reflecting in vivo bone formation and resorptive activity, have returned to a stable level since BTM have been shown to be at least as good as bone mineral density in monitoring the effect of anti-resorptive treatment in osteoporosis. This study searched for articles in PUBMED, CINAHL, Medline, EM-BASE, and Cochrane, and identified 3486 unique articles. These articles were screened based on predefined inclusion and exclusion criteria. Seven articles addressing time to normalization of either CTX, PINP, osteocalcin, or bone-specific alkaline phosphatase after a recent fracture were identified and these were analyzed qualitatively. CTX appeared to return to baseline within 6 months. PINP appeared to return to baseline within 6 months and interestingly dip below baseline after a year. Osteocalcin was elevated throughout the first year after a fracture, with most changes in the first 6 months. Bone-specific alkaline phosphatase (BAP) was increased for up to a year, however with a discrepancy between used assays. Seven studies were identified, showing CTX and PINP to return to baseline within 6 months. OC was elevated for 12 months. BAP was increased for up to a year. However, none of these studies had fasting patients and a long follow-up period with regular measurements. The studies could indicate that the BTM CTX and PINP have returned to baseline within 6 months; however, further studies are needed assessing pre-analytical factors while having a long follow-up. Bone turnover markers appear as good as or better than bone mineral density in monitoring the effect of anti-resorptive medication in osteoporosis. This study tries to identify the time from fracture until BTM are back at baseline. Most studies did not however take pre-analytical variation into consideration. Further research is therefore needed.

Chylomicronemia risk factors ranked by importance for the individual and community in 108 711 women and men.

BACKGROUND: Hypertriglyceridemia prevalence is increasing as more individuals become obese, and chylomicronemia risk factors for the individual and community have not been described previously. OBJECTIVE: To describe chylomicronemia risk factors in the general population for individuals and community. METHODS: A total of 108 711 individuals from the Copenhagen General Population Study were grouped as unlikely chylomicronemia (nonfasting triglycerides <2 mmol L-1 (177 mg dL-1 )), possible chylomicronemia (2-4.99 mmol L-1 (177-442 mg dL-1 )), probable chylomicronemia (5-9.99 mmol L-1 (443-885 mg dL-1 )) and definite chylomicronemia (≥10 mmol L-1 (≥ 886 mg dL-1 )). Relative risk (RR) from Poisson regression ranked dichotomized chylomicronemia risk factors for individuals, and population attributable fractions (PAF) for the community: type 2 diabetes, alcohol intake, obesity, fat intake, hypothyroidism, kidney function, education, sedentary lifestyle, menopause and hormone replacement (women). RESULTS: For women and men, chylomicronemia was unlikely in 81% and 64%, possible in 18% and 33%, probable in 1% and 3% and definite in 0.03% and 0.14%, respectively. For the individual, the three top-ranked risk factors for probable/definite versus unlikely chylomicronemia in women were type 2 diabetes (RR: 4.21; 95% confidence interval: 3.30-5.36), menopause (RR: 3.74; 2.62-5.36) and obesity (RR: 3.44; 2.81-4.21). Corresponding top-ranked risk factors in men were obesity (RR: 3.86; 3.46-4.30), type 2 diabetes (RR: 1.88; 1.61-2.19) and reduced kidney function (RR: 1.86; 1.48-2.34). For the community, top-ranked risk factors in women were menopause (PAF: 63%), obesity (PAF: 29%) and type 2 diabetes (PAF: 15%). Corresponding top-ranked risk factors in men were obesity (PAF: 29%), type 2 diabetes (PAF: 6.4%) and sedentary lifestyle (PAF: 6.0%). CONCLUSIONS: Obesity and type 2 diabetes were the most important modifiable chylomicronemia risk factors in women and men, both for the individual and community. This could influence chylomicronemia prevention and help design randomized trials aimed at reducing triglycerides.

Circulating sCD36 levels in patients with non-alcoholic fatty liver disease and controls.

BACKGROUND AND OBJECTIVE: CD36 is implicated in fatty-acid uptake in multiple tissues, including hepatocytes and adipocytes. Circulating CD36 (sCD36) is increased in non-alcoholic fatty liver disease (NAFLD). We explored this association further by investigating correlations between sCD36 levels, intrahepatic lipid content and markers of obesity in NAFLD patients and controls. METHODS: In total, 111 NAFLD patients and 33 normal/overweight controls were included. Intrahepatic lipid content was measured by magnetic resonance spectroscopy; and subgroups of participants had a dual-energy X-ray absorptiometry (n=99), magnetic resonance imaging (n=94, subcutaneous and visceral adipose tissue) and liver biopsy (n=28 NAFLD patients) performed. Plasma sCD36 was assessed by enzyme-linked immunosorbent assay. RESULTS: NAFLD patients had elevated sCD36 levels compared with controls (0.68 (0.12-2.27) versus 0.43 (0.10-1.18), P<0.01). sCD36 correlated with intrahepatic lipid (rs=0.30), alanine transaminase (ALT) (r=0.31), homeostasis model assessment index-insulin resistance (r=0.24), high-density lipoprotein (r=-0.32) and triglyceride (r=0.44, all P<0.01). Intrahepatic lipid and plasma triglyceride were independent predictors of sCD36 levels in a multiple regression analysis. Further, sCD36 and body mass index were weakly correlated (r=0.17, P=0.04); yet, we found no correlations between sCD36 and other measures of fat distribution except an inverse relation to visceral adipose tissue (rs=-0.21, P<0.05). We observed a trend for correlation between sCD36 and hepatic CD36 mRNA expression (r=0.37, P=0.07). CONCLUSIONS: sCD36 levels increased with the level of intrahepatic lipid, insulin resistance and dyslipidemia. The weak association with markers of obesity and the association with hepatic CD36 mRNA expression suggest that excess sCD36 in NAFLD patients is derived from the hepatocytes, which may support that CD36 is involved in NAFLD development. An unhealthy and unbalanced CD36 expression in adipose and hepatic tissue may shift the fatty-acid load to the liver.

Chronic maternal inflammation or high-fat-feeding programs offspring obesity in a sex-dependent manner.

BACKGROUND/OBJECTIVES: The current world-wide obesity epidemic partially results from a vicious circle whereby maternal obesity during pregnancy predisposes the offspring for accelerated weight gain and development of metabolic syndrome. Here we investigate whether low-grade inflammation, characteristic of the obese state, provides a causal role for this disastrous fetal programming in mice. METHODS: We exposed pregnant and lactating C57BL/6JBom female mice to either high-fat diet (HFD), or continuous infusion of lipopolysaccharide (LPS), a potent trigger of innate immunity, and studied offspring phenotypes. RESULTS: Both maternal LPS or HFD treatments rendered the offspring hyperphagic and inept of coping with a HFD challenge during adulthood, increasing their adiposity and weight gain. The metabolic effects were more pronounced in female offspring, while exposed male offspring mounted a larger inflammatory response to HFD at adulthood. CONCLUSIONS: This supports our hypothesis and highlights the programming potential of inflammation in obese pregnancies.

Chronic adrenergic stimulation induces brown adipose tissue differentiation in visceral adipose tissue.

BACKGROUND: Recruitment of brown adipose tissue is a promising strategy to treat obesity and Type 2 diabetes, but the physiological effects of a large amount of metabolically active brown adipose tissue in humans are unknown. CASE REPORT: In the present paper, we report a case of massive brown adipose tissue infiltration of the visceral adipose tissue depot in a person with Type 2 diabetes with a catecholamine-secreting paraganglioma. The patient was evaluated with [18F]-fludeoxyglucose positron emission tomography/computed tomography on three occasions: pre-therapy, during α-blockade and postoperatively. During surgery, biopsies of visceral and subcutaneous adipose tissue were obtained and evaluated for brown adipose tissue. At diagnosis, brown adipose tissue glucose uptake, assessed by [18F]-fludeoxyglucose-positron emission tomography, was massively increased. [18F]-fludeoxyglucose uptake was confined to known locations for brown adipose tissue, with additional uptake in the visceral adipose tissue. As a result of increased thermogenesis, resting energy expenditure was doubled. After surgical removal of the tumour, antidiabetic medicine was no longer needed, despite an 8.2-kg weight gain. CONCLUSION: These results show that human visceral adipose tissue holds an unprecedented potential for brown adipogenic differentiation; however, a detrimental effect on glucose metabolism persisted despite massive brown adipose tissue activity, with a doubling of resting energy expenditure.

Expression of vitamin D-metabolizing enzymes in human adipose tissue -- the effect of obesity and diet-induced weight loss.

OBJECTIVE: Low vitamin D (VD) levels are common in obesity. We hypothesized that this may be due to metabolism of VD in adipose tissue (AT). Thus, we studied (1) whether the VD-metabolizing enzymes were expressed differently in AT of lean and obese individuals and in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and (2) whether their expression was influenced by weight loss. METHODS: Samples of SAT and VAT were analyzed for expression of the vitamin-D-25-hydroxylases CYP2R1, CYP2J2, CYP27A1 and CYP3A4, the 25-vitamin-D-1α-hydroxylase CYP27B1, the catabolic vitamin-D-24-hydroxylase CYP24A1, and the vitamin D receptor, using reverse transcriptase-PCR. Moreover, plasma 25-hydroxy-vitamin D (25OHD) level was measured and related to the expression of these enzymes. Samples of SAT and VAT from 20 lean women and 20 obese women, and samples of SAT from 17 obese subjects before and after a 10% weight loss were analyzed. RESULTS: A plasma 25OHD level <50 nmol l(-1) was highly prevalent in both lean (45%) and obese (90%) women (P<0.01). Plasma 25OHD increased by 27% after weight loss in the obese individuals (P<0.05). Expression levels of the 25-hydroxylase CYP2J2 and the 1α-hydroxylase CYP27B1 were decreased by 71% (P<0.0001) and 49% (P<0.05), respectively, in SAT of the obese. CYP24A1 did not differ between lean and obese women, but the expression was increased by 79% (P<0.05) after weight loss. CONCLUSION: Obesity is characterized by a decreased expression of the 25-hydroxylase CYP2J2 and the 1α-hydroxylase CYP27B1 in SAT, whereas the catabolic CYP24A1 does not differ between lean and obese women. However, the expression of CYP24A1 is increased after weight loss. Accordingly, AT has the capacity to metabolize VD locally, and this can be dynamically altered during obesity and weight loss.

Investigations of the anti-inflammatory effects of vitamin D in adipose tissue: results from an in vitro study and a randomized controlled trial.

Inflammation is a key feature of obesity and type 2 diabetes. The active vitamin D metabolite, 1,25-dihydroxyvitamin D [1,25(OH)2D], modulates the inflammation in vitro. We studied whether inflammation in adipose tissue (AT) cultures could be reduced by incubation with 1,25(OH)2D in vitro, or by oral treatment with vitamin D in vivo in obese subjects with low plasma levels of 25-hydroxyvitamin D. Samples of subcutaneous AT were stimulated with IL-1ß to induce inflammation. In the in vitro study, samples were concomitantly incubated with or without 1,25(OH)2D, and analyzed for mRNA and protein levels of inflammatory markers IL-6, IL-8, and MCP-1. In the in vivo study, samples of subcutaneous AT from obese subjects obtained before and after treatment with 7,000 IU of vitamin D daily or placebo in a randomized controlled trial were stimulated with IL-1ß. The samples were analyzed for AT gene expression and compared with plasma markers of inflammation. In the in vitro study, concomitant incubation with 1,25(OH)2D reduced mRNA levels of MCP-1 by 45% (p=0.01), of IL-6 by 32% (p=0.002), and of IL-8 by 34% (p=0.03), and reduced secretion of IL-8 protein by 18% (p=0.005). In vivo treatment with vitamin D did not reduce AT expression or circulating levels of MCP-1, IL-6, or IL-8. 1,25(OH)2D has significant anti-inflammatory effects in AT in vitro. However, a similar reduction in AT and systemic inflammation cannot be obtained by oral treatment with vitamin D in obese subjects.

Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle.

During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.

The effect of weight loss on serum mannose-binding lectin levels.

BACKGROUND: Serum levels of the mannose-binding lectin (MBL), which is an activator of the complement system, have been considered as a pathogenic factor in a broad range of diseases, and means of modulating MBL are therefore being evaluated. In this study we examine the effects of weight loss on MBL levels, and in continuation of this if MBL is synthesized in human adipose tissue. METHODS: 36 nondiabetic obese subjects received a very low-calorie diet (VLCD) of 800 kcal/day for 8 weeks. Blood samples were collected at baseline and after VLCD. Furthermore, we measured MBL mRNA levels by the real-time RT-PCR on human adipose tissue compared to liver tissue. RESULTS: The mean body weight was reduced from 106.3 ± 2.6 kg to 92.8 ± 2.4 kg, P < 0.0001. Median MBL at baseline was 746 µg/L (IQR 316-1190) versus 892 µg/L (IQR 336-1511) after 8 weeks, P = 0.23. No correlations were found between weight loss and changes in MBL (r = -0.098, P = 0.57). MBL real-time RT-PCR showed no expression of mRNA in adipose tissue, but as expected a good expression in liver tissue was seen. CONCLUSIONS: MBL levels are not affected by weight loss and MBL is not synthesized in human adipose tissue.

Investigations of the human endocannabinoid system in two subcutaneous adipose tissue depots in lean subjects and in obese subjects before and after weight loss.

CONTEXT: Endocannabinoids (ECs) have a role in obesity by affecting appetite and through peripheral effects. Obesity is associated with a dysregulation of the endocannabinoid system (ECS). OBJECTIVE: We aimed to determine the ECS in subcutaneous adipose tissue (AT) in obese subject and investigate the influence of diet-induced weight loss on this system. DESIGN: The obese study participants underwent a 12 weeks diet regimen resulting in 10-12% weight loss. All study participants underwent fasting blood samples and AT biopsies from abdomen and gluteal region, the obese subjects both before and after weight loss. SETTING AND PARTICIPANTS: A total of 21 healthy obese individuals (10 men/11 women, age 39.5 ± 1.6 years, body mass index (BMI): 37.5 ± 0.8 kg m(-2)) and 21 age- and gender-matched lean subjects (BMI: 23.8 ± 0.4 kg m(-2)) were studied. MAIN OUTCOME MEASURES: The activity of ECS in AT was determined by measuring arachidonoyl glycerol (2-AG) and N-arachidonoylethanolamine/anandamide in AT by mass spectrometry and gene expressions of enzymes and receptors involved in the ECS. RESULTS: The EC, 2-AG was reduced in obese individuals in the gluteal AT depot (P<0.01). Moreover, 2-AG increased in both depots in the obese subjects following weight loss (P<0.05). The gene expression of the CB1 was either not affected by the obese state (in the gluteal AT depot) or reduced (in the abdominal depot, P<0.05) and significantly affected by weight loss. The expression of the degrading enzymes FAAH, FAAH2, MGL and MGL2 was differently affected by obesity, AT depot and weight loss. CONCLUSION: We found reduced levels of 2-AG in subcutaneous AT in obesity, which increased after weight loss. In abdominal AT, the low CB1 expression was normalised after weight loss, whereas in gluteal AT the CB1 expression was reduced after weight loss. These findings support the concept of a dysregulated ECS in AT in association with obesity.

The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture.

The humoral cross-talk between bone and fat is an area of increasing interest. We investigated the expression and regulation of the bone-acting cytokines; bone morphogenetic protein 2 (BMP2), connective tissue growth factor (CTGF), osteoprotegerin (OPG), and transforming growth factor beta (TGFB1). Subcutaneous adipose tissue was aspirated from lean, healthy women. Tissue samples were incubated with interleukin 1-ß (IL1-ß), tumor necrosis factor-α (TNF-α), cortisol, troglitazone, IL1-ß + troglitazone, or vehicle. Gene expression in the adipose tissue was analyzed using qPCR and protein levels in the incubation media were analyzed using ELISA. OPG expression and secretion was diminished by 40.8% and 43.1% respectively, by cortisol, and OPG expression was diminished by 67.5% by troglitazone (p<0.05). The proinflammatory cytokines IL1-ß and TNF-α significantly increased the expression of CTGF (p<0.05) by 65.1% and 101.3%, respectively, and the expression and secretion of OGP by 62.3-165.8% (p<0.05). This interleukin 1-ß mediated increase in CTGF- and OPG expression and secretion was ameliorated by troglitazone. Troglitazone and related drugs are known to have adverse effects on bone. We suggest that this could be mediated via altered cytokine production in adipose tissue. Moreover, obese individuals have a low-grade inflammation in their adipose tissue and have higher bone mineral density than lean individuals. We suggest that this inflammation may increase the expression and secretion of OPG and CTGF and thereby increase BMD. In conclusion, bone acting cytokines are produced in the adipose tissue and may affect bone through endocrine mechanisms.

Anti-inflammatory effect of resveratrol on adipokine expression and secretion in human adipose tissue explants.

OBJECTIVE: Human obesity is closely associated with a state of chronic low-grade inflammation, which also involves the adipose tissue with enhanced production of bioactive substances (adipokines). Calorie restriction (CR) reduces adipocytokine production and improves metabolic profile in rodents. Some of these effects are mediated through activation of the sirtuin 1 (Sirt1) enzyme, and in this study, we investigate whether the natural phytoalexin, resveratrol (RSV), which is a potent Sirt1 activator, has anti-inflammatory effects in human adipose tissue explants. DESIGN: The effect of RSV on interleukin 1ß (IL1ß)-induced change of adipokine mRNA gene expression and secretion were measured in human adipose tissue explants. RESULTS: Exposure of human adipose tissue in vitro to IL1ß for 24 h increased secretion of the proinflammatory adipokines IL6, IL8 and monocyte chemoattractant protein 1 (MCP-1) 3-7.7-fold (P<0.05) and increased IL6, IL8, MCP-1, IL1ß and PAI-1 mRNA expression 1.3-7.2-fold (P<0.05) accordingly. Concomitant incubations with RSV reversed the IL1ß-stimulated secretion (16-36%) and gene expression (25-48%) of these adipokines. IL1ß reduced adiponectin mRNA expression (40%), a decrement that was reversed by RSV treatment. Similar effects were observed in differentiated human preadipocytes in primary culture, indicating that human adipocytes are a potential target for RSV effects. Finally, the effects were neutralized by sirtinol, a Sirt1 inhibitor. CONCLUSION: This study is the first to show anti-inflammatory effects of RSV on adipokine expression and secretion in human adipose tissue in vitro through the SIRT1 pathway. Thus, RSV is hypothesized to possess beneficial effects and might improve the metabolic profile in human obesity.

Reduced cannabinoid receptor 1 protein in subcutaneous adipose tissue of obese.

BACKGROUND: Cannabinoid 1 receptors are identified in various tissues involved in the internal metabolism including adipose tissue and the endocannabinoid system is claimed to be overactive in the obese state. To study the potential involvement of cannabinoid receptor 1 in the endocannabinoid system over-activity in adipose tissue in the obese state, we investigated the cannabinoid receptor 1 levels in adipose tissue from different fat depots in lean and obese humans. MATERIALS AND METHODS: The adipose tissue samples were analysed by Western blot and by RT-PCR. RESULTS: Both the gene expression and the protein of cannabinoid receptor 1 were lower in subcutaneous abdominal adipose tissue from obese subjects as compared with lean subjects (P < 0.01 and P = 0.058). Moreover, in lean subjects, the level of cannabinoid receptor 1 was significantly higher in subcutaneous adipose tissue compared with visceral adipose tissue (P < 0.05) for both gene expression and protein. The level of cannabinoid receptor 1 was similar between the two depots in obese subjects. The expression of cannabinoid receptor 1 was higher in subcutaneous gluteal adipose tissue as compared with subcutaneous abdominal adipose tissue (P < 0.05). CONCLUSION: We found in lean subjects, a robust lower level of cannabinoid receptor 1 in visceral adipose tissue compared with subcutaneous adipose tissue (both RNA and protein levels), but similar levels of cannabinoid receptor 1 between the two depots in obese subjects. Our present findings do not indicate that cannabinoid receptor 1 is directly involved in the endocannabinoid system over-activity in adipose tissue in obesity.

Expression of 11beta-hydroxysteroid dehydrogenase 1 and 2 in subcutaneous adipose tissue of lean and obese women with and without polycystic ovary syndrome.

OBJECTIVE: To investigate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and 2 and hexose-6-phosphate dehydrogenase (H6PDH) mRNA in subcutaneous abdominal tissue from lean and obese women with and without polycystic ovary syndrome (PCOS), and to investigate the association between these enzymes and different measures of insulin sensitivity. DESIGN: Cross-sectional study. SUBJECTS: A total of 60 women, 36 women with PCOS, 17 lean (lean PCOS, LP) and 19 obese (obese PCOS, OP) and 24 age- and weight-matched control women, 8 lean (lean controls, LC) and 16 obese (obese controls, OC). Subcutaneous adipose tissue was collected from the abdomen. Peripheral insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp and determined as glucose disposal rate and insulin sensitivity index. Whole-body insulin sensitivity was calculated using homeostasis model assessment insulin resistance index. Body composition was evaluated by dual X-ray absorptiometry. Adipose mRNA expression of leptin and adiponectin were determined by real-time PCR. RESULTS: Polycystic ovary syndrome (P<0.05) and obesity (P<0.05) were independently associated with increased expression of 11beta-HSD1 mRNA. The subgroups LP and OC had increased 11beta-HSD1 and 11beta-HSD2 mRNA expression compared with LC (P<0.05, P<0.05). There were no effects of PCOS or obesity on11beta-HSD2 or H6PDH mRNA expression. Decreased peripheral insulin sensitivity (P<0.001) and increased upper body fat distribution (P<0.01) were associated with increased expression of 11beta-HSD1, but neither 11beta-HSD2 nor H6PDH. CONCLUSION: Polycystic ovary syndrome and obesity are independently associated with increased expression of 11beta-HSD1. This may lead to increased conversion of cortisone to cortisol in the peripheral adipose tissue and subsequently increased glucocorticoid activity. Decreased peripheral insulin sensitivity and central obesity was associated with increased expression of 11beta-HSD1.

Low Sirt1 expression, which is upregulated by fasting, in human adipose tissue from obese women.

OBJECTIVE: Calorie restriction increases the life span in a number of different organisms. This effect is dependent upon activation of the Sirt1 enzyme, and many of the beneficial effects of calorie restriction can be mimicked using resveratrol, which activates the Sirt1 enzyme. Nothing is known about this system in human adipose tissue; therefore, we investigated this system in human adipose tissue. DESIGN: Sirt1 mRNA was measured in adipose tissue biopsies from human volunteers before and after 6 days of total fasting. In addition, adipose tissue from lean and obese individuals was compared and in vitro investigations were performed. RESULTS: Long-term total fasting (6 days) of nine human volunteers increased Sirt1 mRNA expression in subcutaneous adipose tissue more than twofold (0.197-0.454 arbitrary units, P<0.05). Likewise, lean women (n=12) had more than twofold higher Sirt1 expression in subcutaneous adipose tissue compared to obese women (n=12; 0.33-0.73 arbitrary units, P<0.05). Sirt1 was equally expressed in the stroma-vascular fraction and the isolated adipocyte fraction. Finally, in vitro, we demonstrated that resveratrol (a Sirt1 activator) significantly enhanced the lipolytic effect of epinephrine in human adipose tissue (P<0.05). CONCLUSION: Human adipose tissue contains Sirt1 and the expression of Sirt1 can be regulated by calorie restriction as in other species. Furthermore, we demonstrated that resveratrol affects human fat-cell metabolism similar to the effects in rodents (that is, increased epinephrine induced lipolysis). These findings indicated that the beneficial effects of calorie restriction in humans might involve the activation of Sirt1. Thus, based on these findings, we propose that Sirt1 might play important roles for the beneficial effects of calorie restriction in humans.

Resveratrol displays anti-inflammatory properties in an ex vivo model of immune mediated inflammatory arthritis.

BACKGROUND: Resveratrol is a natural polyphenol found in berries, roots and wine that is well known to have anti-inflammatory and anti-oxidative properties. The anti-inflammatory effect has been reported for both immune cells and connective tissues, but only few studies have investigated effects on immune mediated inflammatory arthritis. None of which have studied this effect when combining resveratrol with methotrexate or adalimumab, two major drugs in the treatment of immune mediated inflammatory arthritis.We therefore aimed to investigate the anti-inflammatory effect of resveratrol alone and in combination with methotrexate or adalimumab in ex vivo models of immune mediated inflammatory arthritis. We furthermore aimed to describe any variations in this effect based on disease activity and cellular composition of the synovial fluid infiltrate. METHODS: Synovial fluid mononuclear cells from patients with rheumatoid arthritis (n = 7) and spondyloarthritis (n = 7) were cultured for either 48 h or 21 days. In both models, synovial fluid mononuclear cells were treated with resveratrol alone or in combination with methotrexate or adalimumab. Monocyte chemoattractant protein 1, matrix metalloproteinase 3 and tartrate resistant acidic phosphatase were measured to quantify inflammation, enzymatic degradation and osteoclast differentiation, respectively. RESULTS: Resveratrol reduced monocyte chemoattractant protein 1 production by synovial fluid mononuclear cells significantly (p = 0.005) compared to untreated controls. The effect of resveratrol was greatest in cultures from patients with low disease activity, i.e. DAS28CRP ≤ 3.2 (p = 0.022), and in cultures dominated by lymphocytes (p = 0.03). Further, the combination of methotrexate and resveratrol significantly reduced monocyte chemoattractant protein 1 levels compared with methotrexate alone in cultures from patients with low disease activity (p = 0.016), and in cultures with high lymphocyte count (p = 0.011). Resveratrol did not significantly affect matrix metalloproteinase 3 and tartrate resistant acidic phosphatase production. CONCLUSION: Resveratrol has anti-inflammatory properties in our ex vivo model of immune mediated inflammatory arthritis. Results show an additive effect of resveratrol, when combined with methotrexate in samples dominated by lymphocytes and samples from patients with low disease activity. This suggests further investigations in vitro and whether this effect may also be present in a clinical setting.

Comparison of bone turnover markers in peripheral blood and bone marrow aspirate.

BACKGROUND: Bone remodeling takes place in the bone marrow environment. We investigated if levels of bone turnover markers (BTMs) differ between bone marrow and peripheral blood, if the bone marrow is an independent compartment, and how well the measurements in bone marrow correlate with bone mineral density. METHODS: Sixty-six men participated in a placebo controlled study designed to evaluate the effect of 16 weeks supplementation with resveratrol on bone mineral density and BTM. Bone marrow aspirates and blood samples were drawn at baseline and at week 16. Procollagen I N-terminal propeptide (PINP), C-terminal telopeptide of type I collagen (CTx), osteocalcin, bone specific alkaline phosphatase (BAP), and osteoprogeterin (OPG) were analyzed. Bland-Altman analysis was used to compare measurements across compartment to detect possible systematic or proportional differences. Paired t-test was performed if no proportional difference was revealed at the difference vs concentration plot. RESULTS: Measurements of PINP, CTx, and BAP differed proportionally between compartments depending on concentration; at low concentrations absolute values were only slightly different, while at higher average concentrations the levels were much higher in bone marrow than blood. Osteocalcin measures in bone marrow were systematically and significantly lower than in blood (mean ±â€¯SD; 14.4 µg/L ±â€¯5.3 µg/L versus 21.7 µg/L ±â€¯6.0 µg/L respectively, p < 0.001). OPG measures were comparable between compartments (p = 0.69). CTx and OPG measured in blood were negatively associated with lumbar spine BMD (ß = -0.22, p = 0.05 and ß = -0.02, p = 0.02, respectively), whereas both markers measured in bone marrow were not (p = 0.60 and p = 0.50 respectively). None of the BTMs, bone marrow or blood, were associated with total hip BMD. DISCUSSION: The levels of most BTMs differed significantly between bone marrow and peripheral blood, while OPG was comparable. Levels of PINP, CTx, and BAP differed between compartments depending on concentration, suggesting bone marrow to represent a compartment separate from the general circulation. Unexpectedly, osteocalcin was lower in the marrow, a gradient that was independent of concentration. BTMs measured in bone marrow did not show any association with bone mineral density. Although further studies are needed to investigate potential explanatory causes of the differences, BTMs in bone marrow do not seem to contribute further to fracture risk assessment.

Characterization of nuclear corticosteroid receptors in rat adipocytes. Regional variations and modulatory effects of hormones.

The corticosteroid receptor was investigated in isolated rat adipocytes with a new technique which characterizes the corticosteroid receptors that can be activated and tightly bound to the nucleus. The binding reaction with [3H]triamcinolone was performed with intact isolated adipocytes and the radioactivity associated with nucleus was subsequently determined after cell lysis. Scatchard analysis revealed a homogeneous class of nuclear corticosteroid receptors in rat epididymal adipocytes with an apparent Kd of 4.93 +/- 1.5 nM and a Bmax of 21.8 +/- 6.6 fmol/10(6) cells corresponding to about 13,000 receptors per nucleus. The corticosteroid binding exhibited regional variations in isolated adipocytes. The highest receptor number was found in epididymal adipocytes (Bmax 25.8 +/- 3.9 fmol/10(6) cells) whereas there were significantly lower nuclear binding sites in perirenal adipocytes (16.5 +/- 5.5 fmol/10(6) cells) (P less than 0.05) and subcutaneous adipocytes (4.8 +/- 1.5 fmol/10(6) cells) (P less than 0.01). The apparent affinity in the three fat depots were similar with Kd values about 4 nM. The nuclear corticosteroid receptor in adipocytes was steroid specific, as neither unlabelled estradiol nor testosterone were able to displace the [3H]triamcinolone binding at concentrations up to 100 microM. However, unlabelled progesterone and promegestrone (R5020) were able to compete with triamcinolone-binding (by 50-80%). In order to investigate whether the nuclear corticosteroid binding in adipocytes were under influence of other hormones we examined the effects of lipolytic and antilipolytic compounds on the binding. Preincubation with isoproterenol and dibutryl-cAMP for 1 h was able to decrease the corticosteroid binding by 30-50%. However, the antilipolytic hormone insulin had no effect in preincubations performed for up to 2 h. In conclusion, high affinity nuclear corticosteroid receptors were found in rat adipocytes. These receptors exhibited regional variations and were modulated by lipolytic hormones.