Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing
Summary
Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed 'loss of function' experiments measuring the effect of leptin on HVR after CB denervation and 'gain of function' experiments measuring HVR after overexpression of the leptin receptor in CB.
Abstract
An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The carotid bodies (CB), a key organ of peripheral hypoxic sensitivity, express the long functional isoform of leptin receptor (LepRb) but the role of leptin signaling in control of breathing has not been fully elucidated. We examined the hypoxic ventilatory response (HVR) (1) in C57BL/6J mice before and after leptin infusion at baseline and after CB denervation; (2) in LepRb-deficient obese db/db mice at baseline and after LepRb overexpression in CBs. In C57BL/6J mice, leptin increased HVR and effects of leptin on HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB augmented the HVR. Therefore, we conclude that leptin acts in CB to augment responses to hypoxia.
Introduction
An adipocyte produced hormone leptin acts in the hypothalamus to suppress food intake and increase metabolic rate. Studies performed in our laboratory1,2 and by other investigators3,4 showed that leptin increases the hypercapnic ventilatory response (HVR) preventing obesity hypoventilation in leptin deficient obesity. However, a majority of obese individuals have high plasma leptin levels and demonstrate resistance to the metabolic and respiratory effects of the hormone5,6,7,8. Resistance to leptin is multifactorial, but limited permeability of the blood-brain barrier (BBB) to leptin plays a major role. We propose that leptin acts below BBB in a key organ of peripheral hypoxic sensitivity, the carotid bodies (CB), to defend breathing in obese individuals. CBs express the long functional isoform of leptin receptor, LepRb, but the role of CB in respiratory effects of leptin has not been sufficiently elucidated9,10.
The goal of our method was to examine the effect of leptin signaling in the CB on HVR. Our rationale was to perform (a) loss of function experiments infusing leptin in mice with intact carotid bodies and denervated carotid bodies followed by HVR measurements; (b) gain of function experiments in db/db mice lacking LepRb, in which we measured the HVR at baseline and after expression of LepRb exclusively in CB. The advantage of our techniques was that we performed all our experiments in unrestrained unanesthetized mice during sleep and wakefulness. Previous investigators either performed their experiments under anesthesia9 or did not measure effects of leptin during sleep10. In addition, our study is the first to utilize a unique gain of function approach with selective LepRb expression in CB described above.
In the broad context, our approach can be generalized to other receptors expressed in CB and their role in hypoxic sensitivity. Investigators can infuse a ligand to a receptor of interest and measure the HVR at baseline and after CB denervation. As a complementary approach, a receptor of interest can be overexpressed in CB and HVR measurements can be performed before and after overexpression using our technology described in this manuscript.
Protocol
Representative Results
Discussion
The main focus of our study was to examine respiratory effects of leptin signaling in the CB. Several protocols have been developed to assess the role of leptin in a mechanistic fashion. First, specific contribution of CB to the HVR was analyzed by careful quantification of the HVR during the first 2 min of hypoxic exposure. Second, the relevance of CB in leptin-mediated up-regulation of control of breathing was examined by two complementary approaches. In lean wild-type mice with low leptin levels, the HVR was measured …
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
R01HL138932, RO1HL133100, RO1HL128970, AHACDA34700025
Materials
| 1ml Insulin Syringes | BD Biosciences | 309311 | |
| 1x PBS (pH 7.4) | Gibco | 10010-023 | 500 ml |
| Ad-Lacz | Dr. Christopher Rhodes (University of Chicago) | 1×1010 pfu/ml | |
| Ad-LepRb-GFP | Vector Biolabs | ADV-263380 | 2-5×1010 pfu/ml |
| Anesthetic cart | Atlantic Biomedical | ||
| Betadine | Purdue Products Ltd. | 12496-0757-5 | |
| Buprenorphine (Buprenex) | Reckitt Benckiser Healthcare Ltd. | 12496-0757-5 | 0.3mg/ml |
| C57Bl/6J | Jackson laboratory | 000664 | Mice Strain |
| Cotton Gauze Sponges | Fisherbrand | 22-362-178 | |
| db/db | Jackson laboratory | 000697 | Mice Strain |
| Ethanol | Pharmco-AAPER | 111000200 | |
| Isoflurane | Vetone | 502017 | |
| Lab Chart | Data Science International (DSI) | Software | |
| Matrigel Matrix | BD Biosciences | 356234 | |
| Micro Spring Scissors | World Precision Instruments (WPI) | 14124 | |
| Mouse Ox Plus | STARR Life Sciences Corp. | Software | |
| Mouse Ox Plus Collar Sensor | STARR Life Sciences Corp. | 015022-2 | Medium Collar Clip Special 7” |
| Mouse Whole Body Plethysmography Chamber | Data Science International (DSI) | PLY3211 | |
| Ohio Care Plus Incubator | Ohmeda | HCHD000173 | |
| Operating Scissors | World Precision Instruments (WPI) | 501753-G | Straight |
| Osmotic Pump | Alzet | 1003D | 1ul per hour, 3 days |
| Phenol | Sigma-Aldrich | P4557 | |
| Recombinant Mouse Leptin protein | R&D systems | 498-OB-05M | 5mg |
| Saline | RICCA Chemical | 7210-16 | 0.9% Sodium Chloride |
| Sterile Surgical Suture | DemeTech | DT-639-1 | Silk, size 6-0 |
| Thermometer | Innovative Calibration Solutions (INNOCAL) | EW 20250-91 |
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