Dual CCR2/CCR5 antagonist treatment attenuates adipose inflammation, but not microvascular complications in ob/ob mice

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138252/1/dom12950.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138252/2/dom12950_am.pd

Sweet Taste Receptor Deficient Mice Have Decreased Adiposity and Increased Bone Mass

Functional expression of sweet taste receptors (T1R2 and T1R3) has been reported in numerous metabolic tissues, including the gut, pancreas, and, more recently, in adipose tissue. It has been suggested that sweet taste receptors in these non-gustatory tissues may play a role in systemic energy balance and metabolism. Smaller adipose depots have been reported in T1R3 knockout mice on a high carbohydrate diet, and sweet taste receptors have been reported to regulate adipogenesis in vitro. To assess the potential contribution of sweet taste receptors to adipose tissue biology, we investigated the adipose tissue phenotypes of T1R2 and T1R3 knockout mice. Here we provide data to demonstrate that when fed an obesogenic diet, both T1R2 and T1R3 knockout mice have reduced adiposity and smaller adipocytes. Although a mild glucose intolerance was observed with T1R3 deficiency, other metabolic variables analyzed were similar between genotypes. In addition, food intake, respiratory quotient, oxygen consumption, and physical activity were unchanged in T1R2 knockout mice. Although T1R2 deficiency did not affect adipocyte number in peripheral adipose depots, the number of bone marrow adipocytes is significantly reduced in these knockout animals. Finally, we present data demonstrating that T1R2 and T1R3 knockout mice have increased cortical bone mass and trabecular remodeling. This report identifies novel functions for sweet taste receptors in the regulation of adipose and bone biology, and suggests that in these contexts, T1R2 and T1R3 are either dependent on each other for activity or have common independent effects in vivo

CMKLR1 activation ex vivo does not increase proportionally to serum total chemerin in obese humans

Prochemerin is the inactive precursor of the adipokine chemerin. Proteolytic processing is obligatory for the conversion of prochemerin into active chemerin and subsequent regulation of cellular processes via the chemokine-like receptor 1 (CMKLR1). Elevated plasma or serum chemerin concentrations and differential processing of prochemerin have been reported in obese humans. The impact of these changes on CMKLR1 signalling in humans is unknown. The objective of this pilot study was to develop a cellular bioassay to measure CMKLR1 activation by chemerin present in human serum and to characterise how obesity modifies serum activation of CMKLR1 under fasted and fed conditions. Blood samples were collected from control (N = 4, BMI 20–25) and obese (N = 4, BMI >30) female subjects after an overnight fast (n = 2) and at regular intervals (n = 7) following consumption of breakfast over a period of 6 h. A cellular CMKLR1-luminescent reporter assay and a pan-chemerin ELISA were used to determine CMKLR1 activation and total chemerin concentrations, respectively. Serum total chemerin concentration (averaged across all samples) was higher in obese vs control subjects (17.9 ± 1.8 vs 10.9 ± 0.5 nM, P < 0.05), but serum activation of CMKLR1 was similar in both groups. The CMKLR1 activation/total chemerin ratio was lower in obese vs control subjects (0.33 ± 0.04 vs 0.58 ± 0.05, P < 0.05). After breakfast, serum total chemerin or CMKLR1 activation did not differ from baseline values. In conclusion, the unexpected observation that obese serum activation of CMKLR1 did not match increased total chemerin concentrations suggests impaired processing to and/or enhanced degradation of active chemerin in serum of obese humans

Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

<div><p>Chemerin is a leukocyte chemoattractant and adipokine with important immune and metabolic roles. Chemerin, secreted in an inactive form prochemerin, undergoes C-terminal proteolytic cleavage to generate active chemerin, a ligand for the chemokine-like receptor-1 (CMKLR1). We previously identified that adipocytes secrete and activate chemerin. Following treatment with the obesity-associated inflammatory mediator TNFα, unknown adipocyte mechanisms are altered resulting in an increased ratio of active to total chemerin production. Based on these findings we hypothesized adipocytes produce proteases capable of modifying chemerin and its ability to activate CMKRL1. 3T3-L1 adipocytes expressed mRNA of immunocyte and fibrinolytic proteases known to activate chemerin <em>in vitro</em>. Following treatment with a general protease inhibitor cocktail (PIC), the TNFα-stimulated increase in apparent active chemerin concentration in adipocyte media was amplified 10-fold, as measured by CMKLR1 activation. When the components of the PIC were investigated individually, aprotinin, a serine protease inhibitor, blocked 90% of the TNFα-associated increase in active chemerin. The serine proteases, elastase and tryptase were elevated in adipocyte media following treatment with TNFα and their targeted neutralization recapitulated the aprotinin-mediated effects. In contrast, bestatin, an aminopeptidase inhibitor, further elevated the TNFα-associated increase in active chemerin. Our results support that adipocytes regulate chemerin by serine protease-mediated activation pathways and aminopeptidase deactivation pathways. Following TNFα treatment, increased elastase and tryptase modify the balance between activation and deactivation, elevating active chemerin concentration in adipocyte media and subsequent CMKLR1 activation.</p> </div

Bestatin heightens the apparent adipocyte media concentration of active chemerin.

<p>CMKLR1 bioassay and western blot analysis were used to identify the effect of bestatin, an inhibitor of aminopeptidases, alone and in combination with TNFα or a vehicle control on the apparent (<b>A</b>) and total (<b>B</b>) media chemerin concentration of 3T3-L1 adipocytes. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. Western blot using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. ^† P<0.05 compared to the TNFα/vehicle control, two-way ANOVA followed by Bonferroni’s <i>post hoc</i> test (<b>A</b>).</p

Serine and cysteine protease inhibitors attenuate TNFα-dependent increases in the apparent concentration of active chemerin in adipocyte media.

<p>3T3-L1 or BMSC adipocytes were treated with TNFα or 0.1% BSA/PBS vehicle control in combination with 0–30 µM of the serine protease inhibitor aprotinin or 0–100 µM of the cysteine protease inhibitor E-64 or an equivalent volume of their respective vehicle controls, water or 0.9% NaCl. The effect of these treatments on the apparent concentration of active chemerin in adipocyte media was measured by the CMKLR1 bioassay (A–C). The effect of these treatments on the immunodetectable levels of total chemerin in adipocyte media was measured by western blot and quantified by densitometry (D–F). For densitometry analysis, the dual vehicle treatment (i.e. 0.1% BSA/PBS with H₂0 or 0.9% NaCl) served as the reference control and was assigned a value of 100%. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. Western blot using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. * P<0.05 compared to the within group 0.1% BSA/PBS vehicle control, ^† P<0.05 compared to the TNFα/vehicle control groups, two-way ANOVA, followed by Bonferroni’s <i>post hoc</i> test (A–C).</p

3T3-L1 adipocytes express immunocyte and fibrinolytic associated enzymes.

<p>The mRNA expression of <i>neutrophil elastase</i> (<b>A</b>), <i>mast cell tryptase</i> (<b>B</b>), <i>angiotensin converting enzyme</i> (<b>C</b>), <i>tissue plasminogen activator (tPA)</i> (<b>D</b>), <i>tissue plasminogen activator urokinase (uPA)</i> (<b>E</b>) and <i>cathepsin K</i> (<b>F</b>) were analyzed in 3T3-L1 adipocytes throughout differentiation. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. * P<0.05 compared to the control (D0, preadipocytes), one-way ANOVA, followed by Tukey’s <i>post hoc</i> test.</p

Elastase and tryptase are responsible for the TNFα-mediated increase in the apparent concentration of active chemerin in adipocyte media.

<p>The concentrations of elastase, tryptase, and tPA in 24 hour conditioned media from 3T3-L1 and BMSC adipocytes treated with 20 ng mL⁻¹ of TNFα or equivalent volume of 0.1% BSA/PBS vehicle control were measured by western blot analysis (A). The apparent concentration of active chemerin in 24 h conditioned media from 3T3-L1 adipocytes treated with 20 ng mL⁻¹ of TNFα (B) or equivalent volume of 0.1% BSA/PBS (B, Inset) together with neutralizing antibodies for elastase and tryptase (alone or in combination) or IgG control was measured using the CMKLR1 bioassay. All bars represent the mean ± s.e.m. of 3 samples, and are representative of 2 independent experiments. Western blots are representative of 4 samples per group and 3 independent experiments. * P<0.05 compared to the TNFα/IgG or the 0.1% BSA/PBS/IgG (Inset) treated cells, ^† P<0.05 compared to the TNFα+anti-elastase or anti-tryptase treated cells, two-way ANOVA, followed by Bonferroni’s <i>post hoc</i> test.</p