Anette Sams

Adiponectin Promotes Macrophage Polarization toward an Anti-inflammatory Phenotype

It is established that the adipocyte-derived cytokine adiponectin protects against cardiovascular and metabolic diseases, but the effect of this adipokine on macrophage polarization, an important mediator of disease progression, has never been assessed. We hypothesized that adiponectin modulates macrophage polarization from that resembling a classically activated M1 phenotype to that resembling alternatively-activated M2 cells. Peritoneal macrophages and the stromal vascular fraction (SVF) cells of adipose tissue isolated from adiponectin knock-out mice displayed increased M1 markers, including tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 and decreased M2 markers, including arginase-1, macrophage galactose N-acetyl-galactosamine specific lectin-1, and interleukin-10. The systemic delivery of adenovirus expressing adiponectin significantly augmented arginase-1 expression in peritoneal macrophages and SVF cells in both wild-type and adiponectin knock-out mice. In culture, the treatment of macrophages with recombinant adiponectin protein led to an increase in the levels of M2 markers and a reduction of reactive oxygen species and reactive oxygen species-related gene expression. Adiponectin also stimulated the expression of M2 markers and attenuated the expression of M1 markers in human monocyte-derived macrophages and SVF cells isolated from human adipose tissue. These data show that adiponectin functions as a regulator of macrophage polarization, and they indicate that conditions of high adiponectin expression may deter metabolic and cardiovascular disease progression by favoring an anti-inflammatory phenotype in macrophages.

Characterisation of the effects of a non-peptide CGRP receptor antagonist in SK-N-MC cells and isolated human cerebral arteries.

The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8-37) and Compound 1, yielding pK(i) values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8-37) and Compound 1 with pA(2) values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8-37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8-37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect. The clinical studies of non-peptide CGRP antagonists are awaited with great interest.

Pro‐Inflammatory macrophages increase in skeletal muscle of high fat‐Fed mice and correlate with metabolic risk markers in humans

In obesity, immune cells infiltrate adipose tissue. Skeletal muscle is the major tissue of insulin‐dependent glucose disposal, and indices of muscle inflammation arise during obesity, but whether and which immune cells increase in muscle remain unclear.

Expression of anti-inflammatory macrophage genes within skeletal muscle correlates with insulin sensitivity in human obesity and type 2 diabetes

Aims/hypothesisLow-grade systemic inflammation and adipose tissue inflammatory macrophages are frequently detected in patients with obesity and type 2 diabetes. Whether inflammatory macrophages also increase in skeletal muscle of individuals with metabolic disorders remains controversial. Here, we assess whether macrophage polarisation markers in skeletal muscle of humans correlate with insulin sensitivity in obesity and type 2 diabetes.MethodsSkeletal muscle biopsies were obtained from individuals of normal weight and with normal glucose tolerance (NGT), and overweight/obese individuals with or without type 2 diabetes. Insulin sensitivity was determined by euglycaemic–hyperinsulinaemic clamps. Expression of macrophage genes was analysed by quantitative RT-PCR.ResultsGene expression of the inflammatory macrophage phenotype marker cluster of differentiation (CD)11c was higher in muscle of type 2 diabetes patients (p = 0.0069), and correlated with HbA1c (p = 0.0139, ρ = 0.48) and fasting plasma glucose (p = 0.0284, ρ = 0.43), but not after correction for age. Expression of TGFB1, encoding the anti-inflammatory marker TGF-β1, correlated inversely with HbA1c (p = 0.0095, ρ = −0.50; p = 0.0484, ρ = −0.50) and fasting plasma glucose (p = 0.0471, ρ = −0.39; p = 0.0374, ρ = −0.52) in two cohorts, as did HbA1c with gene expression of macrophage galactose-binding lectin (MGL) (p = 0.0425, ρ = −0.51). TGFB1 expression was higher in NGT individuals than in individuals with type 2 diabetes (p = 0.0303), and correlated with low fasting plasma insulin (p = 0.0310, ρ = −0.42). In exercised overweight/obese individuals, expression of genes for three anti-inflammatory macrophage markers, MGL (p = 0.0031, ρ = 0.71), CD163 (p = 0.0268, ρ = 0.57) and mannose receptor (p = 0.0125, ρ = 0.63), correlated with high glucose-disposal rate.Conclusions/interpretationMuscle expression of macrophage genes reveals a link between inflammatory macrophage markers, age and high glycaemia, whereas anti-inflammatory markers correlate with low glycaemia and high glucose-disposal rate.

Chronic Administration of the Glucagon-Like Peptide-1 Analog, Liraglutide, Delays the Onset of Diabetes and Lowers Triglycerides in UCD-T2DM Rats

OBJECTIVE The efficacy of liraglutide, a human glucagon-like peptide-1 (GLP-1) analog, to prevent or delay diabetes in UCD-T2DM rats, a model of polygenic obese type 2 diabetes, was investigated. RESEARCH DESIGN AND METHODS At 2 months of age, male rats were divided into three groups: control, food-restricted, and liraglutide. Animals received liraglutide (0.2 mg/kg s.c.) or vehicle injections twice daily. Restricted rats were food restricted to equalize body weights to liraglutide-treated rats. Half of the animals were followed until diabetes onset, whereas the other half of the animals were killed at 6.5 months of age for tissue collection. RESULTS Before diabetes onset energy intake, body weight, adiposity, and liver triglyceride content were higher in control animals compared with restricted and liraglutide-treated rats. Energy-restricted animals had lower food intake than liraglutide-treated animals to maintain the same body weights, suggesting that liraglutide increases energy expenditure. Liraglutide treatment delayed diabetes onset by 4.1 ± 0.8 months compared with control (P < 0.0001) and by 1.3 ± 0.8 months compared with restricted animals (P < 0.05). Up to 6 months of age, energy restriction and liraglutide treatment lowered fasting plasma glucose and A1C concentrations compared with control animals. In contrast, liraglutide-treated animals exhibited lower fasting plasma insulin, glucagon, and triglycerides compared with both control and restricted animals. Furthermore, energy-restricted and liraglutide-treated animals exhibited more normal islet morphology. CONCLUSIONS Liraglutide treatment delays the development of diabetes in UCD-T2DM rats by reducing energy intake and body weight, and by improving insulin sensitivity, improving lipid profiles, and maintaining islet morphology.

Expression of calcitonin receptor-like receptor and receptor-activity-modifying proteins in human cranial arteries.

Recently a new type of proteins modulating the pharmacological profile of the calcitonin receptor-like receptor (CRLR) were identified. The receptor-activity-modifying proteins (RAMPs) were shown to be essential for the expression of a functional CRLR and furthermore the RAMPs seemed to modify ligand selectivity of CRLR: coexpression of CRLR and RAMP1 resulted in a CGRP1 type of receptor while an adrenomedullin receptor resulted when CRLR and RAMP2 were coexpressed. In the present study significant molecular expression of CRLR concomitant with RAMP1, 2 and 3 were demonstrated in human meningeal, cerebral and temporal arteries by use of reverse transcriptase polymerase chain reactions (RT-PCR). These findings support previous studies demonstrating functional CGRP1 receptors in human cranial arteries. Furthermore the present study suggests the potential for functional adrenomedullin receptors in human cranial arteries.

An Ongoing Role of α-Calcitonin Gene–Related Peptide as Part of a Protective Network Against Hypertension, Vascular Hypertrophy, and Oxidative Stress

&agr;-Calcitonin gene–related peptide (&agr;CGRP) is a vasodilator, but there is limited knowledge of its long-term cardiovascular protective influence. We hypothesized that &agr;CGRP protects against the onset and development of angiotensin II–induced hypertension and have identified protective mechanisms at the vascular level. Wild-type and &agr;CGRP knockout mice that have similar baseline blood pressure were investigated in the angiotensin II hypertension model for 14 and 28 days. &agr;CGRP knockout mice exhibited enhanced hypertension and aortic hypertrophy. &agr;CGRP gene expression was increased in dorsal root ganglia and at the conduit and resistance vessel level of wild-type mice at both time points. &bgr;CGRP gene expression was also observed and shown to be linked to plasma levels of CGRP. Mesenteric artery contractile and relaxant responses in vitro and endothelial NO synthase expression were similar in all groups. The aorta exhibited vascular hypertrophy, increased collagen formation, and oxidant stress markers in response to angiotensin II, with highest effects observed in &agr;CGRP knockout mice. Gene and protein expression of endothelial NO synthase was lacking in the aortae after angiotensin II treatment, especially in &agr;CGRP knockout mice. These results demonstrate the ongoing upregulation of &agr;CGRP at the levels of both conduit and resistance vessels in vascular tissue in a model of hypertension and the direct association of this with protection against aortic vascular hypertrophy and fibrosis. This upregulation is maintained at a time when expression of aortic endothelial NO synthase and antioxidant defense genes have subsided, in keeping with the concept that the protective influence of &agr;CGRP in hypertension may have been previously underestimated.

CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with ‘Compound 1' (WO98/11128), a non-peptide antagonist

Calcitonin gene‐related peptide (CGRP), amylin and adrenomedullin (AM) belong to the same family of peptides. Accumulating evidence indicate that the calcitonin (CT) receptor, the CT receptor‐like receptor (CRLR) and receptor‐activity‐modifying proteins (RAMPs) form the basis of all the receptors in this family of peptides. Using reverse transcriptase–polymerase chain reaction the presence of mRNA sequences encoding the CRLR, RAMP1 and RAMP2 were demonstrated in porcine left anterior descending (LAD) coronary arteries, whereas porcine calcitonin (CT) receptor mRNA was not present. The partial porcine mRNA sequences shared 82–92% nucleotide identity with human sequences. The human peptides αCGRP, βCGRP, AM and amylin induced relaxation with pEC50 values of 8.1, 8.1, 6.7 and 6.1 M respectively. The antagonistic properties of a novel non‐peptide CGRP antagonist ‘Compound 1’ (WO98/11128), βCGRP8–37 and the proposed AM receptor antagonist AM22–52 were compared to the well‐known CGRP1 receptor antagonist αCGRP8–37. The αCGRP8–37 and βCGRP8–37 induced concentration‐dependent (10−7–10−5 M) rightward shift of both the αCGRP and βCGRP concentration‐response curves. βCGRP8–37 (10−6 M) had the same effect as αCGRP8–37 (10−6 M), but with less potent rightward shift of the concentration‐response curves for αCGRP, AM and amylin. Preincubation with ‘Compound 1’ (10−7–10−5 M) and AM22–52 (10−6 M) had no significant antagonistic effect. In conclusion, the building blocks forming CGRP and AM receptors were present in the porcine LAD, whereas those of the amylin receptor were not. αCGRP, βCGRP, AM and amylin mediated vasorelaxation via the CGRP receptors. No functional response was detected to adrenomedullin via the adrenomedullin receptor.

CGRP and adrenomedullin receptor populations in human cerebral arteries: in vitro pharmacological and molecular investigations in different artery sizes

The aim of the present study was to determine functional and molecular characteristics of receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin in three different diameter groups of lenticulostriate arteries. Furthermore, the presence of perivascular neuronal sources of CGRP was evaluated in these arteries. In the functional studies, in vitro pharmacological experiments demonstrated that both CGRP and adrenomedullin induce alpha-CGRP-(8-37) sensitive vasodilation in artery segments of various diameters. The maximal amounts of vasodilation induced by CGRP and adrenomedullin were not different, whereas the potency of CGRP exceeded that of adrenomedullin by 2 orders of magnitude. Significant negative correlations between artery diameters and maximal responses were demonstrated for CGRP and adrenomedullin. In addition, the potency of both peptides tended to increase in decreasing artery diameter. In the molecular experiments, levels of mRNAs encoding CGRP receptors and receptor subunits were compared using reverse transcriptase polymerase chain reactions (RT-PCR). The larger the artery, the more mRNA encoding receptor activity-modifying proteins 1 and 2 (RAMP1 and RAMP2) was detected relative to the amount of mRNA encoding the calcitonin receptor-like receptor. By immunohistochemistry, perivascular CGRP containing nerve fibres were demonstrated in all the investigated artery sizes. In conclusion, both CGRP and adrenomedullin induced vasodilation via CGRP receptors in human lenticulostriate artery of various diameter. The artery responsiveness to the CGRP receptor agonists increased with smaller artery diameter, whereas the receptor-phenotype determining mRNA ratios tended to decrease. No evidence for CGRP and adrenomedullin receptor heterogeneity was present in lenticulostriate arteries of different diameters.

Recombinant Adiponectin Does Not Lower Plasma Glucose in Animal Models of Type 2 Diabetes

Aims/Hypothesis Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats. Methods Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10–30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5–15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days. Results Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active. Conclusions/Interpretation Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.