Serotonin Improves High Fat Diet Induced Obesity in Mice

There are two independent serotonin (5-HT) systems of organization: one in the central nervous system and the other in the periphery. 5-HT affects feeding behavior and obesity in the central nervous system. On the other hand, peripheral 5-HT also may play an important role in obesity, as it has been reported that 5-HT regulates glucose and lipid metabolism. Here we show that the intraperitoneal injection of 5-HT to mice inhibits weight gain, hyperglycemia and insulin resistance and completely prevented the enlargement of intra-abdominal adipocytes without having any effect on food intake when on a high fat diet, but not on a chow diet. 5-HT increased energy expenditure, O2 consumption and CO2 production. This novel metabolic effect of peripheral 5-HT is critically related to a shift in the profile of muscle fiber type from fast/glycolytic to slow/oxidative in soleus muscle. Additionally, 5-HT dramatically induced an increase in the mRNA expression of peroxisome proliferator-activated receptor coactivator 1α (PGC-1α)-b and PGC-1α-c in soleus muscle. The elevation of these gene mRNA expressions by 5-HT injection was inhibited by treatment with 5-HT receptor (5HTR) 2A or 7 antagonists. Our results demonstrate that peripheral 5-HT may play an important role in the relief of obesity and other metabolic disorders by accelerating energy consumption in skeletal muscle

Annexin A6 regulates catabolic events in articular chondrocytes via the modulation of NF-κB and Wnt/ß-catenin signaling.

Annexin A6 (AnxA6) is expressed in articular chondrocytes at levels higher than in other mesenchymal cell types. However, the role of AnxA6 in articular chondrocytes is not known. Here we show that complete lack of AnxA6 functions resulted in increased ß-catenin activation in Wnt3a-treated murine articular chondrocytes, whereas AnxA6 expressing articular chondrocytes showed decreased ß-catenin activation. High expression of AnxA6 in human articular chondrocytes showed the highest inhibition of Wnt/ß-catenin signaling. Inhibition of Wnt/ß-catenin signaling activity by AnxA6 together with cytosolic Ca2+ was achieved by interfering with the plasma membrane association of the Wnt signaling complex. AnxA6 also affected the cross-talk between Wnt/ß-catenin signaling and NF-κB signaling by decreasing ß-catenin activity and increasing NF-κB activity in Wnt3a-, interleukin-1beta (IL-1ß)-, and combined Wnt3a/IL-1ß-treated cells. Wnt3a treatment increased the mRNA levels of catabolic markers (cyclooxygenase-2, interleukin-6, inducible nitric oxide synthase) to a much lesser degree than IL-1ß treatment in human articular chondrocytes, and decreased the mRNA levels of matrix metalloproteinase-13 (MMP-13) and articular cartilage markers (aggrecan, type II collagen). Furthermore, Wnt3a decreased the mRNA levels of catabolic markers and MMP-13 in IL-1ß-treated human articular chondrocytes. High expression of AnxA6 resulted in decreased mRNA levels of catabolic markers, and increased MMP-13 and articular cartilage marker mRNA levels in Wnt3a-treated human articular chondrocytes, whereas leading to increased mRNA levels of catabolic markers and MMP-13 in human articular chondrocytes treated with IL-1ß, or combined Wnt3a and IL-1ß. Our findings define a novel role for AnxA6 in articular chondrocytes via its modulation of Wnt/ß-catenin and NF-κB signaling activities and the cross-talk between these two signaling pathways

AnxA6 interferes with plasma membrane association of Dvl1 in a Ca²⁺-dependent manner.

<p><b>A:</b> Immunoblot analysis of the plasma membrane fraction isolated from WT and AnxA6-/- mouse articular chondrocytes treated with Wnt3a for the time periods indicated, or treated with the intracellular Ca²⁺ chelator, BAPTA-2AM followed by Wnt3a treatment, and analyzed with antibodies specific for Dvl1and AnxA6. Blots were also analyzed with antibodies specific for ATP1A1 (a plasma membrane protein) to control for equal loading. <b>B:</b> The optical densities of the Dvl1 and AnxA6 bands were quantitated by densitometry and normalized to the optical densities of the ATP1A1 bands. Results are expressed relative to the normalized optical densities of the Dvl1 or AnxA6 bands of vehicle-treated WT cells, which were set as 1. <b>C:</b> Immunoblot analysis of the nuclear fraction isolated from WT and AnxA6-/- mouse articular chondrocytes treated with Wnt3a for 30 min, or treated with the intracellular Ca²⁺ chelator, BAPTA-2AM followed by Wnt3a treatment, and analyzed with antibodies specific for ß-catenin. Blots were also analyzed with antibodies specific for lamin B (a nuclear protein) to control for equal loading. <b>D:</b> The optical densities of the ß-catenin bands were quantitated by densitometry and normalized to the optical densities of the lamin B bands. Results are expressed relative to the normalized optical densities of the ß-catenin bands of Wnt3a-treated WT cells, which were set as 1. The blots in <b>A</b> and <b>C</b> are representative of 3 separate experiments with similar results. The optical densities were analyzed on three different immunoblots, and values are mean ± SD. (*p < 0.05; **p < 0.01; ***p < 0.001).</p

AnxA6 modulates the crosstalk between NF-κB and ß-catenin signaling activities.

<p><b>A:</b> Immunoblot analysis with antibodies specific for AnxA6 of a lysate from human articular chondrocytes transfected with empty pcDNA expression vector (EV) or pcDNA expression vector containing full-length <i>AnxA6</i> cDNA (<i>AnxA6</i>), or a lysate from late stage OA human articular cartilage tissue. Late stage OA cartilage was defined as deep fibrillated cartilage with loss of cartilage of the superficial and middle zones. Blots were also analyzed with antibodies specific for beta-actin to control for equal loading. <b>B:</b> The optical densities of the AnxA6 bands were quantitated by densitometry and normalized to the optical densities of the actin bands. Results are expressed relative to the normalized optical densities of the AnxA6 bands of human articular chondrocytes transfected with empty expression vector (EV), which were set as 1. The blot in <b>A</b> is representative of 3 separate experiments with similar results. The optical densities were analyzed on three different immunoblots, and values are mean ± SD. <b>C:</b> ß-catenin activity as determined by luciferase activity from the TOPFlash (TOP) or FOPFlash (FOP) luciferase reporter in vehicle-treated, Wnt3a-treated, IL-1ß-treated, Wnt3a/IL-1ß-treated human articular chondrocytes overexpressing AnxA6. After serum starvation, TOPFlash or FOPFlash reporter- and empty pcDNA (pcDNA)-transfected, or TOPFlash or FOPFlash reporter- and pcDNA containing full-length <i>AnxA6</i> (<i>AnxA6</i>)-transfected human articular chondrocytes were vehicle-treated, or treated with Wnt3a, IL-1ß, or Wnt3a and IL-1ß for 24h. Cell extracts were then analyzed for luciferase activity. Transfection efficiency was monitored by co-transfection with Renilla luciferase vector, which provides constitutive expression of the Renilla luciferase reporter. <b>D:</b> NF-κB activity as determined by luciferase activity from the pNFκB-Met-Luc2 reporter in vehicle-treated, Wnt3a-treated, IL-1ß-treated, Wnt3a/IL-1ß-treated human articular chondrocytes overexpressing AnxA6. After serum starvation, pNFκB-Met-Luc2 reporter- and empty pcDNA (pcDNA)-transfected, or pNFκB-Met-Luc2 reporter- and pcDNA containing full-length <i>AnxA6</i> (<i>AnxA6)</i>-transfected human articular chondrocytes were vehicle-treated, or treated with Wnt3a, IL-1ß, or Wnt3a and IL-1ß for 24h. Samples were then analyzed for luciferase activity. Transfection efficiency was monitored by co-transfection with pSEAP vector, which provides constitutive expression of human placental alkaline phosphatase (SEAP). Secreted SEAP activity was monitored with the chemiluminescence substrate CSPD. Values in <b>C</b> and <b>D</b> were normalized to Renilla or pSEAP luciferase activity. Data are expressed relative to the normalized luciferase activity levels of vehicle-treated WT cells transfected with empty expression vector, which was set as 1. Data in <b>C</b> and <b>D</b> (<i>n</i> = 4) are expressed as mean ± SD. (*p < 0.05; **p < 0.01; ***p < 0.001).</p

mRNA levels for articular cartilage markers (aggrecan, type II collagen (α1(II)) and catabolic markers (Cox-2, IL-6, iNOS, MMP-13) in vehicle-treated, Wnt3a-treated, IL-1ß-treated, or combined Wnt3a- and IL-1ß-treated human articular chondrocytes overexpressing AnxA6.

<p>Human articular chondrocytes were transfected with empty expression vector (EV) or expression vector containing full-length AnxA6 (<i>AnxA6</i>). Twelve hours after transfection cells were serum-starved for 24 h followed by treatment with IL-1ß, Wnt3a, IL-1ß, or combined IL-1ß and Wnt3a for 24h. Levels of mRNA were determined by real-time PCR using SYBR Green and normalized to the level of 18S RNA. The mRNA levels are expressed relative to the level of vehicle-treated cells transfected with empty expression vector, which was set as 1. Data were obtained from triplicate PCRs using RNA from 3 different cultures (<i>n</i> = 3). Values are the mean ± SD. (**p < 0.01; ***p < 0.001).</p

ß-catenin activity in mouse AnxA6-/- and WT articular chondrocytes.

<p><b>A:</b> ß-catenin activity was determined by luciferase activity from the TOPFlash (TOP) or FOPFlash (FOP) luciferase reporter in vehicle-treated, Wnt3a-treated, and BAPTA-2AM/Wnt3a-treated WT and AnxA6-/- mouse articular chondrocytes. <b>B:</b> ß-catenin activity was determined by luciferase activity from the TOPFlash (TOP) or FOPflash (FOP) luciferase reporter in vehicle-treated, and Wnt3a-treated WT or AnxA6-/- mouse articular chondrocytes that were transfected with empty pcDNA expression vector (pcDNA) or pcDNA expression vector containing full-length AnxA6 cDNA (AnxA6). Mouse articular chondrocytes after transfection in <b>A</b> and <b>B</b> were serum starved followed by treatment with Wnt3a or the intracellular Ca²⁺ chelator, BAPTA-2AM followed by Wnt3a. Cell extracts were then analyzed for luciferase activity 48 h post-transfection. Transfection efficiency was monitored by co-transfection with Renilla luciferase vector, which provides constitutive expression of the Renilla luciferase reporter. Values were normalized to Renilla luciferase activity. Data are expressed relative to the normalized luciferase activity levels from the TOPFlash reporter of vehicle-treated WT cells, which was set as 1. Data (<i>n</i> = 4) are expressed as mean ± SD. (*p < 0.05; **p < 0.01; ***p < 0.001).</p