Additive actions of the cannabinoid and neuropeptide Y systems on adiposity and lipid oxidation

Aims: Energy homeostasis is regulated by a complex interaction of molecules and pathways, and new antiobesity treatments are likely to require multiple pharmacological targeting of anorexigenic or orexigenic pathways to achieve effective loss of excess body weight and adiposity. Cannabinoids, acting via the cannabinoid‐1 (CB1) receptor, and neuropeptide Y (NPY) are important modulators of feeding behaviour, energy metabolism and body composition. We investigated the interaction of CB1 and NPY in the regulation of energy homeostasis, hypothesizing that dual blockade of CB1 and NPY signalling will induce greater weight and/or fat loss than that induced by single blockade of either system alone. Methods: We studied the effects of the CB1 antagonist Rimonabant on food intake, body weight, body composition, energy metabolism and bone physiology in wild‐type (WT) and NPY knockout (NPY^?/?) mice. Rimonabant was administered orally at 10 mg/kg body weight twice per day for 3 weeks. Oral Rimonabant was delivered voluntarily to mice via a novel method enabling studies to be carried out in the absence of gavage‐induced stress. Results: Mice with dual blockade of CB1 and NPY signalling (Rimonabant‐treated NPY^?/? mice) exhibited greater reductions in body weight and adiposity than mice with single blockade of either system alone (Rimonabant‐treated WT or vehicle‐treated NPY^?/? mice). These changes occurred without loss of lean tissue mass or bone mass. Furthermore, Rimonabant‐treated NPY^?/? mice showed a lower respiratory exchange ratio than that seen in Rimonabant‐treated WT or vehicle‐treated NPY^?/? mice, suggesting that this additive effect of dual blockade of CB1 and NPY involves promotion of lipid oxidation. On the other hand, energy expenditure and physical activity were comparable amongst all treatment groups. Interestingly, Rimonabant similarly and transiently reduced spontaneous and fasting‐induced food intake in WT and NPY^?/? mice in the first hour after administration only, suggesting independent regulation of feeding by CB1 and NPY signalling. In contrast, Rimonabant increased serum corticosterone levels in WT mice, but this effect was not seen in NPY^?/? mice, indicating that NPY signalling may be required for effects of CB1 on the hypothalamo‐pituitary‐adrenal axis. Conclusions: Dual blockade of CB1 and NPY signalling leads to additive reductions in body weight and adiposity without concomitant loss of lean body mass or bone mass. An additive increase in lipid oxidation in dual CB1 and NPY blockade may contribute to the effect on adiposity. These findings open new avenues for more effective treatment of obesity via dual pharmacological manipulations of the CB1 and NPY systems.     

Ghrelin and the differential regulation of des-acyl (DSG) and oct-anoyl ghrelin (OTG) in human adipose tissue (AT)

Objectives Ghrelin, an important central acting orexigenic hormone, is predominantly secreted in the gastrointestinal tract. However little is known about the action of ghrelin in human adipose tissue (AT). Aim To study the expression of ghrelin in AT, the effects of octanoyl–(OTG) and des‐acyl (DSG) ghrelin on lipolysis and lipogenesis, leptin release and potential peripheral signalling through the Y1 receptor. Methods Ex vivo human AT was obtained from women undergoing elective surgery (46 (mean ± SD) 6·8 years, body mass index (BMI): 25·6 ± 5·0 kg/m², n = 20). Abdominal‐subcutaneous (AbdSc) adipocytes were isolated and treated with recombinant human (rh) OTG and DSG to assess lipid metabolism leptin release and the influence of Y1‐receptor blocker. Results Ghrelin was expressed in AbdScAT and negatively correlated with BMI (lean: 3·6 ± 0·74 optical‐density‐units (OD), obese: 1·64 ± 0·45OD, *P < 0·05). Only DSG significantly suppressed glycerol release (Control (C): 286 ± 58 µl/l; DSG 1 nm : 224 ± 38 µl/l↓*; DSG 100 nm : 172 ± 13 µl/l↓*,*↓P < 0·05, n = 7) and reduced hormone sensitive lipase expression (C: 1·0 ± 0·3OD; DSG 1 nm : 0·8 ± 0·3OD↓*; DSG 100 nm : 0·6 ± 0·1OD↓*, n = 4). However, both isoforms increased lipoprotein lipase expression (C: 1·0 ± 0·3OD; DSG 100 nm : 0·2 ± 0·4OD↑*; OTG 100 nm : 2·5 ± 0·3OD↑*, n = 4), whilst blockade of Y1 eliminated this effect in both. Leptin was down‐regulated by DSG only (DSG 1 nm : 5·3 ± 0·7 ng/ml; DSG 100 nm : 4·1 ± 0·7 ng/ml*) and was significant after BMI adjustment (P = 0·029). Conclusion Ghrelin was expressed in human AbdSc AT. In vitro, both OGT and DSG appear to mediate fat deposition with the lipogenic effects in part mediated by the Y1 receptor, whilst the influence of DSG affected lipolysis, lipogenesis and leptin secretion. Taken together, these studies support a local action for ghrelin isoforms on lipid and adipokine metabolism that further supports a cross talk between organs.     

Chronic administration of DSP‐7238, a novel,potent, specific and substrate‐selective DPP IV inhibitor,improves glycaemic control and β‐cell damage in diabetic mice

Aims: The purpose of this study is to assess the in vitro enzyme inhibition profile of DSP‐7238, a novel non‐cyanopyrrolidine dipeptidyl peptidase (DPP) IV inhibitor and to evaluate the acute and chronic effects of this compound on glucose metabolism in two different mouse models of type 2 diabetes. Methods: The in vitro enzyme inhibition profile of DSP‐7238 was assessed using plasma and recombinant enzymes including DPP IV, DPP II, DPP8, DPP9 and fibroblast activation protein α (FAPα) with fluorogenic substrates. The inhibition type was evaluated based on the Lineweaver–Burk plot. Substrate selectivity of DSP‐7238 and comparator DPP IV inhibitors (vildagliptin, sitagliptin, saxagliptin and linagliptin) was evaluated by mass spectrometry based on the changes in molecular weight of peptide substrates caused by release of N‐terminal dipeptides. In the in vivo experiments, high‐fat diet‐induced obese (DIO) mice were subjected to oral glucose tolerance test (OGTT) following a single oral administration of DSP‐7238. To assess the chronic effects of DSP‐7238 on glycaemic control and pancreatic β‐cell damage, DSP‐7238 was administered for 11 weeks to mice made diabetic by a combination of high‐fat diet (HFD) and a low‐dose of streptozotocin (STZ). After the dosing period, HbA1c was measured and pancreatic damage was evaluated by biological and histological analyses. Results: DSP‐7238 and sitagliptin both competitively inhibited recombinant human DPP IV (rhDPP IV) with K_i values of 0.60 and 2.1 nM respectively. Neither vildagliptin nor saxagliptin exhibited competitive inhibition of rhDPP IV. DSP‐7238 did not inhibit DPP IV‐related enzymes including DPP8, DPP9, DPP II and FAPα, whereas vildagliptin and saxagliptin showed inhibition of DPP8 and DPP9. Inhibition of glucagon‐like peptide‐1 (GLP‐1) degradation by DSP‐7238 was apparently more potent than its inhibition of chemokine (C‐X‐C motif) ligand 10 (IP‐10) or chemokine (C‐X‐C motif) ligand 12 (SDF‐1α) degradation. In contrast, vildagliptin and saxagliptin showed similar degree of inhibition of degradation for all the substrates tested. Compared to treatment with the vehicle, single oral administration of DSP‐7238 dose‐dependently decreased plasma DPP IV activity and improved glucose tolerance in DIO mice. In addition, DSP‐7238 significantly decreased HbA1c and ameliorated pancreatic damage following 11 weeks of chronic treatment in HFD/STZ mice. Conclusions: We have shown in this study that DSP‐7238 is a potent DPP IV inhibitor that has high specificity for DPP IV and substrate selectivity against GLP‐1. We have also found that chronic treatment with DSP‐7238 improves glycaemic control and ameliorates β‐cell damage in a mouse model with impaired insulin sensitivity and secretion. These findings indicate that DSP‐7238 may be a new therapeutic agent for the treatment of type 2 diabetes.     

NPY modulates PYY function in the regulation of energy balance and glucose homeostasis

Aims: Both the neuronal‐derived neuropeptide Y (NPY) and the gut hormone peptide YY (PYY) have been implicated in the regulation of energy balance and glucose homeostasis. However, despite similar affinities for the same Y receptors, the co‐ordinated actions of these two peptides in energy and glucose homeostasis remain largely unknown. Methods: To investigate the mechanisms and possible interactions between PYY with NPY in the regulation of these processes, we utilized NPY/PYY single and double mutant mouse models and examined parameters of energy balance and glucose homeostasis. Results: PYY^?/? mice exhibited increased fasting‐induced food intake, enhanced fasting and oral glucose‐induced serum insulin levels, and an impaired insulin tolerance, ? changes not observed in NPY^?/? mice. Interestingly, whereas PYY deficiency‐induced impairment in insulin tolerance remained in NPY^?/?PYY^?/? mice, effects of PYY deficiency on fasting‐induced food intake and serum insulin concentrations at baseline and after the oral glucose bolus were absent in NPY^?/?PYY^?/? mice, suggesting that NPY signalling may be required for PYY's action on insulin secretion and fasting‐induced hyperphagia. Moreover, NPY^?/?PYY^?/?, but not NPY^?/? or PYY^?/? mice had significantly decreased daily food intake, indicating interactive control by NPY and PYY on spontaneous food intake. Furthermore, both NPY^?/? and PYY^?/? mice showed significantly reduced respiratory exchange ratio during the light phase, with no additive effects observed in NPY^?/?PYY^?/? mice, indicating that NPY and PYY may regulate oxidative fuel selection via partly shared mechanisms. Overall, physical activity and energy expenditure, however, are not significantly altered by NPY and PYY single or double deficiencies. Conclusions: These findings show significant and diverse interactions between NPY and PYY signalling in the regulation of different aspects of energy balance and glucose homeostasis.     

N-butyldeoxynojirimycin causes weight loss as a result of appetite suppression in lean and obese mice

Aim: To determine the mechanism of weight loss caused by high doses of N‐butyldeoxynojirimycin (NB‐DNJ) in healthy lean and leptin–deficient obese (ob/ob) mice. Methods: Healthy lean and obese mice were treated with NB‐DNJ by the following methods: admixed with their diet, delivered by subcutaneously implanted mini‐pumps or by intraperitoneal or intracerebroventricular (ICV) injection. Daily changes in body weight and food intake were recorded during the experimental period. The effect of NB‐DNJ treatment on subcutaneous adipose tissue and on epididymal fat pads was measured. Results: Lean mice treated with NB‐DNJ, admixed with their diet, lost weight in the form of adipose tissue. This resulted in a 40% reduction in skin thickness (control, 358 ± 11 μm; NB‐DNJ treated 203 ± 6 μm) and a reduction in epididymal fat pad weights after 5 weeks of treatment at 2400 mg/kg/day (control, 0.0154 ± 0.001; NB‐DNJ treated, 0.0026 ± 0.0005 as ratios of fat pad weight to total body weight). Following the depletion of adipose tissue mass, the mice grew normally and did not have any reduction in lean mass. Obese mice treated with NB‐DNJ also lost weight or gained weight at a greatly reduced rate compared with non‐treated controls. Body weights at 6 months of age were: lean control, 29.10 ± 1.15 g; lean NB‐DNJ treated, 22.73 ± 0.29 g; obese control, 63.25 ± 1.5 g; obese NB‐DNJ treated from 5 weeks of age, 35.30 ± 1.68 g; obese NB‐DNJ treated from 12 weeks of age, 38.84 ± 1.26 g. Both the lean and obese groups of mice treated with NB‐DNJ ate up to 30% less than untreated controls. Daily food intake (powder diet) were: lean control, 4.15 ± 0.54 g; obese control, 4.14 ± 0.2 g; lean NB‐DNJ treated 2.9 ± 0.37 g; obese NB‐DNJ treated, 2.88 ± 0.47 g. Mice treated with the N‐substituted galactose imino sugar analogue, N‐butyldeoxygalactonojirimycin (NB‐DGJ) did not lose weight. Mice experienced similar weight loss or lack of weight gain when fed a restricted diet that mimics the drug‐induced level of food consumption. Delivery of 2 nmol NB‐DNJ by ICV injection into lean mice also caused similar reductions in food intake. Food intake: saline vehicle, 4.30 ± 0.12 g; NB‐DNJ, 3.37 ± 0.19 g; NB‐DGJ, 4.03 ± 0.16 g; 2‐deoxyglucose, 4.7 ± 0.15 g. Conclusion: NB‐DNJ causes weight loss as a result of reduced food consumption due to central appetite suppression.     

Levels of dipeptidyl peptidase IV/CD26 substrates neuropeptide Y and vasoactive intestinal peptide in rheumatoid arthritis patients

Neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) have their biological half-lives controlled by dipeptidyl peptidase IV (DPP IV/CD26). Several lines of evidence suggest the involvement of NPY in the regulation of rheumatoid arthritis (RA), and VIP has already been identified as a potent anti-inflammatory factor that reduces joint inflammation. The role of DPP IV/CD26 in the pathogenesis of RA has been indicated, but its mediator actions involving NPY and VIP have not been well investigated, so the aim of this study was to find an association between NPY, VIP, and DPP IV/CD26 in RA patients. Assessment of NPY, VIP, DPP IV/CD26 as well as some other inflammatory markers was carried out in 20 RA patients being treated with different types of drugs. Control group consisted of 18 osteoarthritis patients. Synovial fluid and serum content of investigated molecules was determined by ELISA and DPP IV/CD26 activity was measured spectrophotometrically. Immunodetection showed elevated levels of NPY and VIP in RA patients, with a significant increase in synovial fluid, while concentration and activity of DPP IV/CD26 were significantly decreased in both synovial fluid and serum. Positive correlations between serum DPP IV/CD26 concentration and activity (R = 0.6961), as well as between serum and synovial fluid concentration of VIP (R = 0.7029) were found. In RA group, NPY, VIP, and DPP IV/CD26 concentrations were not affected by the administration of drugs. The results of this study indicate a connection between elevated concentration of NPY and VIP and decreased DPP IV/CD26 activity and concentration, suggesting a potential role of these molecules in the immunomodulation of RA.     

Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

Objective: This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla. Methods: Equilibrium binding assays were conducted on intact cells with ¹²⁵I Bolton-Hunter labeled NPY (¹²⁵I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu³¹, Pro³⁴)NPY (a Y₁ receptor ligand, Y₁RL), NPY_13–36 (a Y₂ receptor ligand, Y₂RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation. Results: Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (K_d) value of 39.00 ± 12.84 nM and a maximal binding (B_max) of 489.89 ± 155.49 fmol/10⁶ cells (mean ± SE, n = 6). NPY, Y₁RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC₅₀ (nM) values of 4.06 ± 1.66 (n = 4), 2.94 ± 0.75 (n = 5), and 18.36 ± 10.36 (n = 3), respectively. APP and Y₂RL were unable to compete with ¹²⁵I-NPY in the concentration range 0.001–1 μM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3′,5′-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 ± 6.28%, n = 5 and an IC₅₀ of 5.96 ± 1.87 nM. The Y₁RL produced a comparable response (IC₅₀ = 5.35 ± 1.39 nM, n = 4; maximal inhibition of 61.05 ± 13.03%) and Y₂LR had no detectable effect at a similar concentration range. Conclusions: The results demonstrate the existence of a Y₁ receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.     

Neuropeptide Y receptor genes are associated with alcohol dependence, alcohol withdrawal phenotypes, and cocaine dependence

Background: Several lines of evidence in both human and animal studies suggest that variation in neuropeptide Y (NPY) or its receptor genes (NPY1R, NPY2R and NPY5R) is associated with alcohol dependence as well as alcohol withdrawal symptoms. Additional studies suggest that cocaine may affect NPY expression. Methods: A total of 39 single nucleotide polymorphisms (SNPs) were genotyped across NPY and its 3 receptor genes in a sample of 1,923 subjects from 219 multiplex alcoholic families of European American descent recruited as part of the Collaborative Studies on the Genetics of Alcoholism (COGA) study. Family‐based association analysis was performed to test the primary hypothesis that variation in these genes is associated with alcohol dependence. Secondary analyses evaluated whether there was an association of these SNPs with symptoms of alcohol withdrawal, cocaine dependence, or comorbid alcohol and cocaine dependence. Results: Although variations in NPY itself were not associated with these phenotypes, variations in 2 NPY‐receptor genes were. SNPs in NPY2R provided significant evidence of association with alcohol dependence, alcohol withdrawal symptoms, comorbid alcohol and cocaine dependence, and cocaine dependence (all p < 0.03). Haplotype analyses strengthened the evidence for these phenotypes (global 0.0004 < p < 0.005). SNPs in NPY5R demonstrated significant association with alcohol withdrawal characterized by seizures (p < 0.05). Conclusion: These results indicate that sequence variations in NPY receptor genes are associated with alcohol dependence, particularly a severe subtype of alcohol dependence characterized by withdrawal symptoms, comorbid alcohol and cocaine dependence, and cocaine dependence.     

Human RBP4 adipose tissue expression is gender specific and influenced by leptin

Objective The role of retinol‐binding protein‐4 (RBP4) in human insulin resistance remains controversial, which may in part be explained by a gender‐specific secretion of RBP4 in adipose tissue (AT). The aim of the study was to determine gender‐specific depot expression of RBP4 and to identify metabolic parameters and cytokines/adipokines associated with RBP4. Research Design and Methods The study is an ex ‐ vivo prospective analysis of paired AT‐samples from 22 men and 26 women of similar age [men: 43·4 ± 13 (mean ± SD)years, women: 44·1 ± 12 years], BMI (men: 41·9 ± 18kg/m², women: 38·4 ± 11kg/m²) and homeostasis model assessment of insulin resistance taken during elective surgery and ex‐vivo culture using visceral‐AT (VAT)‐explants (n = 10). Plasma RBP4 and cytokines were measured by ELISA and mRNA expression in AT by real‐time PCR. VAT‐explants were cultured with recombinant leptin and insulin and RBP4 determined by western blot analyses. Results Overall subcutaneous AT (SCAT)‐RBP4 mRNA expression was higher than VAT‐expression [3·1 ± 0·26 signal units (SU; mean ± SE) vs 1·79 ± 0·18 SU, n = 48, P < 0·0001], but neither correlated with circulating RBP4. SCAT‐RBP4 expression was higher in women and correlated with BMI (r = ?0·5, P = 0·009) and fat mass (r = ?0·5, P = 0·002). VAT‐RBP4 correlated positively with GLUT‐4 expression and adiponectin in men only (r = 0·54, P = 0·03 and r = 0·64, P < 0·002, respectively) when correcting for age and fat mass. Multiple regression determined leptin AT‐expression as a positive predictor of AT‐RBP4 in women (SCAT: β = 0·50, P = 0·002; VAT: β = 0·58, P = 0·003) and adiponectin for VAT‐RBP4 in men (β = 0·69; P = 0·001). AT‐RBP4 mRNA expression showed no relation with insulin resistance. Leptin stimulated RBP‐4 secretion ex‐vivo, whilst insulin did not affect RBP4. Conclusion AT‐derived RBP4‐mRNA expression is gender specific and regulated by leptin. Circulating RBP4 levels appear to be independent of AT‐RBP4 secretion.     

Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines

Summary In the human Ewing's sarcoma cell line WE-68, saturation analysis using³H-labelled neuropeptide Y ([³H]NPY) as the radioligand disclosed a homogeneous population of binding sites with a dissociation constant (K _d ) of 4.5 nM and maximal binding capacity (B _max) of 712 fmol/mg cell protein. Besides the WE-68 cell line, ten other human Ewing's sarcoma cell lines (FM-62, HS-80, HT-78, HT-M1-78, NT-68, RM-82, RS-63, VH-64, WE-M1-68, WE-M2-68) were also found to display NPY receptors withK _d varying from 3.5 nM to 10.7 nM andB _max=247–3744 fmol/mg cell protein. NPY, its natural analogues and the Y₁-receptor-specific peptide ligand [Leu³¹, Pro³⁴]NPY inhibited [³H]NPY binding in the potency order: [Leu³¹,Pro³⁴]NPYhuman NPYpeptide YY (PYY)>> salmon pancreatic polypeptide (PP) > human PP>porcine NPY_13–36NPY_22–36. In the Ewing's sarcoma cell lines NPY provoked inhibition of forskolin-stimulated cyclic AMP formation by up to 98%. Pertussis toxin alleviated the cyclic-AMP-inhibitory response to NPY. In isolated Ewing's sarcoma plasma membranes pertussis toxin [³²P]ADP-ribosylated a 41-kDa protein. The ability of NPY and analogues to inhibit cyclic AMP accumulation paralleled their potencies in displacing radioligand binding. By contrast, a cell line derived from an atypical form of Ewing's sarcoma did not express specific and functional NPY receptors. These results demonstrate that conventional Ewing's sarcoma cells possess G_i-potein-coupled NPY receptors of the Y₁ type, which upon interaction with NPY, PYY, and PP mediate inhibition of cyclic AMP generation.Abbreviations NPY neuropeptide Y - PP pancreatic polypeptide - PYY peptide YY - VIP vasoactive intestinal peptide     

The novel sodium glucose transporter 2 inhibitor dapagliflozin sustains pancreatic function and preserves islet morphology in obese, diabetic rats

Aims: To investigate whether glucose lowering with the selective sodium glucose transporter 2 (SGLT2) inhibitor dapagliflozin would prevent or reduce the decline of pancreatic function and disruption of normal islet morphology. Methods: Female Zucker diabetic fatty (ZDF) rats, 7–8 weeks old, were placed on high‐fat diet. Dapagliflozin (1 mg/kg/day, p.o.) was administered for ～33 days either from initiation of high‐fat diet or when rats were moderately hyperglycaemic. Insulin sensitivity and pancreatic function were evaluated using a hyperglycaemic clamp in anaesthetized animals (n = 5–6); β‐cell function was quantified using the disposition index (DI) to account for insulin resistance compensation. Pancreata from a matched subgroup (n = 7–8) were fixed and β‐cell mass and islet morphology investigated using immunohistochemical methods. Results: Dapagliflozin, administered from initiation of high‐fat feeding, reduced the development of hyperglycaemia; after 24 days, blood glucose was 8.6 ± 0.5 vs. 13.3 ± 1.3 mmol/l (p < 0.005 vs. vehicle) and glycated haemoglobin 3.6 ± 0.1 vs. 4.8 ± 0.26% (p < 0.003 vs. vehicle). Dapagliflozin improved insulin sensitivity index: 0.08 ± 0.01 vs. 0.02 ± 0.01 in obese controls (p < 0.03). DI was improved to the level of lean control rats (dapagliflozin 0.29 ± 0.04; obese control 0.15 ± 0.01; lean 0.28 ± 0.01). In dapagliflozin‐treated rats, β‐cell mass was less variable and significant improvement in islet morphology was observed compared to vehicle‐treated rats, although there was no change in mean β‐cell mass with dapagliflozin. Results were similar when dapagliflozin treatment was initiated when animals were already moderately hyperglycaemic. Conclusion: Sustained glucose lowering with dapagliflozin in this model of type 2 diabetes prevented the continued decline in functional adaptation of pancreatic β‐cells.     

Comparison of the antilipolytic effects of an A1 adenosine receptor partial agonist in normal and diabetic rats

Introduction and Aims: Elevated plasma free fatty acid (FFA) concentrations play a role in the pathogenesis of type 2 diabetes (2DM). Antilipolytic agents that reduce FFA concentrations may be potentially useful in the treatment of 2DM. Our previous observation that CVT‐3619 lowered plasma FFA and triglyceride concentrations in rats and enhanced insulin sensitivity in rodents with dietary‐induced forms of insulin resistance suggested that it might be of use in the treatment of patients with 2DM. The present study was undertaken to compare the antilipolytic effects of CVT‐3619 in normal (Sprague Dawley, SD) and Zucker diabetic fatty (ZDF) rats. Results: ZDF rats had significantly higher fat pad weight, glucose, insulin and FFA concentrations than those of SD rats. EC₅₀ values for forskolin‐stimulated FFA release from isolated adipocytes from SD and ZDF rats were 750 and 53 nM, respectively (p < 0.05). Maximal forskolin stimulation of FFA release was significantly (p < 0.01) less in ZDF rats (133 ± 60 μM) compared with SD rats (332 ± 38 μM). EC₅₀ values for isoproterenol to increase lipolysis in adipocytes from SD and ZDF rats were 2 and 7 nM respectively. Maximal isoproterenol‐stimulated lipolysis was significantly (p < 0.01) lower in adipocytes from ZDF rats (179 ± 23 μM) compared with SD rats (343 ± 27 μM). Insulin inhibited lipolysis in adipocytes from SD rats with an IC₅₀ value of 30 pM, whereas adipocytes from ZDF rats were resistant to the antilipolytic actions of insulin. In contrast, IC₅₀ values for CVT‐3619 to inhibit the release of FFA from SD and ZDF adipocytes were essentially the same (63 and 123 nM respectively). CVT‐3619 inhibited lipolysis more than insulin in both SD (86 vs. 46%, p < 0.001) and ZDF (80 vs. 13%, p < 0.001) adipocytes. In in vivo experiments, CVT‐3619 (5 mg/kg, PO) lowered FFA to a similar extent in both groups. Plasma concentrations of CVT‐3619 were not different in SD and ZDF rats. There was no significant difference in the messenger RNA expression of the A₁ receptors relative to β‐actin expression in adipocytes from SD (0.98 ± 0.2) and ZDF rats (0.99 ± 0.3). Conclusion: The antilipolytic effects of CVT‐3619 appear to be independent of insulin resistance and animal model.     

Lean mass and fat mass predict bone mineral density in middle-aged individuals with noninsulin-requiring type 2 diabetes mellitus

Objective Despite high bone mineral density (BMD), persons with type 2 diabetes are at greater risk of fracture. The relationship between body composition and BMD in noninsulin‐requiring diabetes is unclear. The aim was to examine how fat and lean mass independently affect the skeleton in this population. Research design and methods Subjects for this cross‐sectional analysis were men (n = 78) and women (n = 56) aged 40–65 years (56 ± 6 years) with uncomplicated, noninsulin‐requiring type 2 diabetes. Total body fat and lean mass, total body, hip and lumbar spine BMD were measured with dual energy X‐ray absorptiometry. Magnetic resonance imaging measured total abdominal, visceral and subcutaneous (SQ) fat. Results Subjects had normal all‐site BMD and were obese to overweight (body mass index 29–41 kg/m²) with controlled diabetes (HbA1c women 6·6 ± 1·2%, men 6·7 ± 1·6%). Lean mass was positively associated with total body, hip, femoral neck and hip BMD in both sexes. Fat mass, abdominal total and SQ fat were associated with total body and hip BMD in women. In multivariate analyses adjusted for sex, lean mass significantly predicted total, hip and femoral neck BMD in men and women. In unadjusted models, lean mass continued to predict BMD at these sites in men; fat mass also predicted total body, femoral and hip BMD in women. Conclusions In men and women with uncomplicated, noninsulin‐requiring diabetes, lean mass significantly predicted BMD at the total body, hip and femoral neck. Further research is needed to determine whether acquisition or maintenance of lean mass in T2DM can prevent hip fracture in this at‐risk population.     

Effect of beta-adrenergic stimulation on whole-body and abdominal subcutaneous adipose tissue lipolysis in lean and obese men

Aims/hypothesis Obesity is characterised by increased triacylglycerol storage in adipose tissue. There is in vitro evidence for a blunted beta-adrenergically mediated lipolytic response in abdominal subcutaneous adipose tissue (SAT) of obese individuals and evidence for this at the whole-body level in vivo. We hypothesised that the beta-adrenergically mediated effect on lipolysis in abdominal SAT is also impaired in vivo in obese humans. Methods We investigated whole-body and abdominal SAT glycerol metabolism in vivo during 3 h and 6 h [²H₅]glycerol infusions. Arterio–venous concentration differences were measured in 13 lean and ten obese men after an overnight fast and during intravenous infusion of the non-selective beta-adrenergic agonist isoprenaline [20 ng (kg fat free mass)⁻¹ min⁻¹]. Results Lean and obese participants showed comparable fasting glycerol uptake by SAT (9.7 ± 3.4 vs 9.3 ± 2.5% of total release, p = 0.92). Furthermore, obese participants showed an increased whole-body beta-adrenergically mediated lipolytic response versus lean participants. However, their fasting lipolysis was blunted [glycerol rate of appearance: 7.3 ± 0.6 vs 13.1 ± 0.9 μmol (kg fat mass)⁻¹ min⁻¹, p < 0.01], as was the beta-adrenergically mediated lipolytic response per unit SAT [Δ total glycerol release: 140 ± 71 vs 394 ± 112 nmol (100 g tissue)⁻¹ min⁻¹, p < 0.05] compared with lean participants. Net triacylglycerol flux tended to increase in obese compared with lean participants during beta-adrenergic stimulation [Δ net triacylglycerol flux: 75 ± 32 vs 16 ± 11 nmol (100 g tissue)⁻¹ min⁻¹, p = 0.06]. Conclusions/interpretation We demonstrated in vivo that beta-adrenergically mediated lipolytic response is impaired systematically and in abdominal SAT of obese versus lean men. This may be important in the development or maintenance of increased triacylglycerol stores and obesity.     

Identification and functional studies of a specific peptide YY-preferring receptor in dog adipocytes.

Specific binding sites for peptide YY (PYY) and neuropeptide Y (NPY) as well as functional responses were identified in dog adipocytes. Studies were carried out using the radioligand [125I-Tyr1]monoiodo-PYY on crude adipocyte membranes. [125I]PYY bound to dog adipocyte membranes with a high affinity (156 +/- 24 pM) and binding capacity of 314 +/- 48 fmol/mg protein. Competition studies revealed a higher affinity of the binding sites for PYY than NPY (inhibition constants were 118 +/- 17 pM and 300 +/- 53 pM, respectively, P < or = 0.001). NPY analogs displaced [125I]PYY specific binding with the following order of potency: NPY-(13-36) > NPY-(18-36) > NPY-(22-36) > [Leu31-Pro34]NPY. Neither adrenergic nor adenosine agents (activating or inhibiting other antilipolytic systems) interacted with [125I]PYY binding sites. So [125I]PYY binding was specific, saturable, and reversible. Lipolysis experiments performed with PYY, NPY, and NPY analogs confirm the relative order of potency found in competition experiments. The data agree with the definition of PYY-preferring receptor which resembles a Y2 receptor subtype since NPY-(13-36), a specific Y2 receptor agonist, inhibited binding and lipolysis in a similar way to PYY, whereas [Leu31-Pro34]NPY did not. No difference was observed in the antilipolytic response between IC50 values measured on omental, perirenal, and subcutaneous fat deposits. Moreover, PYY and NPY (10(-6) M) significantly attenuated forskolin-stimulated cAMP levels, involving inhibition of adenylyl cyclase as a transmembrane signaling mechanism. Cross-linking of bound [125I]PYY to membranes indicated that the mol wt of the receptor was 62K. The relative importance of such a receptor on fat cells alongside another powerful antilipolytic receptor--the alpha 2-adrenoceptor--is discussed.     

Lower expression of adiponectin mRNA in visceral adipose tissue in lean and obese subjects

Adiponectin is an adipocyte-specific protein suggested to play a role in mediating the metabolic effects of obesity. In the present study, we investigated adiponectin mRNA levels in both visceral and subcutaneous abdominal adipose tissue (AT) from lean and obese subjects. Investigations on both "fresh" fat biopsies and incubations of AT fragments were performed. Regional differences in the effects of the cytokine interleukin-1beta (IL-1beta) were investigated. Adiponectin gene expression was 33% lower in visceral AT than in subcutaneous AT of lean subjects (P < 0.05), and 28% lower in obese subjects, albeit non-significant (P = 0.3). In both lean and obese subjects adiponectin mRNA expression in incubated AT fragments was significantly lower in visceral AT than in subcutaneous AT (lean: P < 0.01; obese: P < 0.05). No difference was found in adiponectin mRNA levels in gluteal compared to abdominal subcutaneous AT. IL-1beta suppressed adiponectin mRNA levels substantially in both subcutaneous and visceral AT. In conclusion, adiponectin gene expression is lower in visceral AT than in subcutaneous abdominal AT, suggesting subcutaneous AT to be more important for circulating adiponectin levels.     

Fatty acid binding protein expression in different adipose tissue depots from lean and obese individuals

Aims/hypothesis: This study investigated the expression of adipose tissue fatty acid binding proteins (FABPs) in subcutaneous and visceral human adipose tissue depots from lean and obese individuals. Methods: Adipocyte lipid binding protein (ALBP) and keratinocyte lipid binding protein (KLBP) expression was quantified by western blot in subcutaneous and omental adipose tissue from 20 obese and 9 lean individuals. RNA expression was quantified by Northern blot in the obese subjects. Results: In the obese subjects, ALBP protein and RNA expression was higher in subcutaneous compared with omental adipose tissue (increases of 31 ± 14 % and 40 ± 13 % respectively, both p < 0.05), whereas in the lean group, KLBP protein levels were 32 ± 9 % lower in subcutaneous fat (p < 0.03). However, the ALBP/KLBP ratio was greater in subcutaneous compared to omental adipose tissue from both lean and obese subjects: increases of 187 ± 71 % (p = 0.01) and 52 ± 23 % (p = 0.17) respectively for the protein ratio, and 21 ± 6 % for RNA (p = 0.01, obese individuals). In lean subjects, insulin concentrations correlated positively with the ALBP/KLBP protein ratio in both depots (both p≤ 0.03). Conclusion/interpretation: There are regional differences in adipose tissue FABP expression, which could be influenced by obesity. However, the ALBP/KLBP ratio is greater in subcutaneous than visceral adipose tissue in lean as well as in obese subjects. Investigation of adipose tissue FABPs could further our understanding of the role of fatty acids in the insulin resistance syndrome. [Diabetologia (2001) 44: 1268–1273] Received: 1 February 2001 and in revised form: 25 June 2001     

Effects of in vitro antagonism of endocannabinoid‐1 receptors on the glucose transport system in normal and insulin‐resistant rat skeletal muscle

Objective: We determined the direct effects of modulating the endocannabinoid‐1 (CB1) receptor on the glucose transport system in isolated skeletal muscle from insulin‐sensitive lean Zucker and insulin‐resistant obese Zucker rats. Methods: Soleus strips were incubated in the absence or presence of insulin, without or with various concentrations of the CB1 receptor antagonist SR141716 or with the CB1 receptor agonist arachidonyl‐2‐chloroethylamide (ACEA). Results: CB1 receptor protein expression in visceral adipose (57%), soleus (40%) and myocardial (36%) tissue was significantly (p < 0.05) decreased in obese compared to lean animals, with a trend for a reduction (17%, p = 0.079) in the liver. In isolated soleus muscle from both lean and obese Zucker rats, CB1 receptor antagonism directly improved glucose transport activity in a dose‐dependent manner. Basal glucose transport activity was maximally enhanced between 100 and 200 nM SR141716 in lean (26–28%) and obese (22–31%) soleus. The maximal increase in insulin‐stimulated glucose transport for lean muscle (～30%) was achieved at 50 nM SR141716 and for obese muscle (～30%) at 100 nM SR141716. In contrast, CB1 receptor antagonism did not alter hypoxia‐stimulated glucose transport activity. CB1 receptor agonism (1 mM ACEA) significantly decreased both basal (15%) and insulin‐stimulated (22%) glucose transport activity in isolated lean soleus. This effect was reversed by 200 nM SR141716. In both lean and obese muscle, the functionality of key signalling proteins (insulin receptor β‐subunit, Akt, glycogen synthase kinase‐3β (GSK‐3β), AMP‐dependent protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38 MAPK)) was not altered by either CB1 receptor agonism or antagonism. Conclusion: These results indicate that the engagement of CB1 receptor can negatively modulate both basal and insulin‐dependent glucose transport activity in lean and obese skeletal muscles, and that these effects are not mediated by the engagement of elements of the canonical pathways regulating this process in mammalian skeletal muscle.     

Investigation of the effect of oral metformin on dipeptidylpeptidase‐4 (DPP‐4) activity in Type 2 diabetes1

Aims Glucagon‐like peptide‐1 (GLP‐1) is an insulinotropic hormone and major component of the enteroinsular axis. Its therapeutic potential in human diabetes is limited by rapid degradation and inactivation by the enzyme dipeptidylpeptidase‐4 (DPP‐4). We investigated the acute effects of metformin with and without food on DPP‐4 activity in Type 2 diabetes. Methods Ten subjects with Type 2 diabetes (6 male/4 female, age 65.8 ± 2.6 years, body mass index 30.0 ± 1.2 kg/m², glycated haemoglobin (HbA_1c) 6.3 ± 0.2%, mean ± sem ) received metformin 1 g orally or placebo together with a standard mixed meal (SMM) in a random crossover design. Six subjects re‐attended fasting and received metformin 1 g without a SMM. Results Following SMM (n = 10), DPP‐4 activity was not suppressed by metformin compared with placebo [area under curve (AUC)_{0–4 h} 1574 ± 4 vs. 1581 ± 8 μmol/ml/min, respectively]. Plasma glucose, insulin and active GLP‐1 were not different. However, DPP‐4 activity was suppressed with metformin following fasting compared with a SMM (n = 6) (AUC_{0–4 h} 1578 ± 4 vs. 1494 ± 9 μmol/min, P < 0.02). Metformin serum levels were significantly lower (P < 0.001) after SMM than fasting (AUC_{0–4 h} 350 ± 66 vs. 457 ± 55 mg/ml/min). Conclusion Metformin inhibits DPP‐4 activity in Type 2 diabetic patients in the fasting state but not when taken with a standard mixed meal. Metformin serum concentrations are lower if the drug is taken with food. These findings should be taken into account in establishing how to maximize efficacy of the drug.     

Adipose tissue oxygenation is associated with insulin sensitivity independently of adiposity in obese men and women

Adipose tissue (AT) dysfunction contributes to the pathophysiology of insulin resistance and type 2 diabetes. Previous studies have shown that altered AT oxygenation affects adipocyte functionality, but it remains to be elucidated whether altered AT oxygenation is more strongly related to obesity or insulin sensitivity. In the present study, we tested the hypothesis that AT oxygenation is associated with insulin sensitivity rather than adiposity in humans. Thirty‐five lean and obese individuals (21 men and 14 women, aged 40‐65 years) with either normal or impaired glucose metabolism participated in a cross‐sectional single‐centre study. We measured abdominal subcutaneous AT oxygenation, body composition and insulin sensitivity. AT oxygenation was higher in obese insulin resistant as compared to obese insulin sensitive (IS) individuals with similar age, body mass index and body fat percentage, both in men and women. No significant differences in AT oxygenation were found between obese IS and lean IS men. Moreover, AT oxygenation was positively associated with insulin resistance (r = 0.465; P = .005), even after adjustment for age, sex and body fat percentage (standardized β = 0.479; P = .005). In conclusion, abdominal subcutaneous AT oxygenation is associated with insulin sensitivity both in men and women, independently of adiposity. AT oxygenation may therefore be a promising target to improve insulin sensitivity.