Cleavage of the leptin receptor by matrix metalloproteinase-2 promotes leptin resistance and obesity in mice

Abstract

Obesity and related morbidities pose a major health threat. Obesity is associated with increased blood concentrations of the anorexigenic hormone leptin; however, obese individuals are resistant to its anorexigenic effects. We examined the phenomenon of reduced leptin signaling in a high-fat diet-induced obesity model in mice. Obesity promoted matrix metalloproteinase-2 (Mmp-2) activation in the hypothalamus, which cleaved the leptin receptor's extracellular domain and impaired leptin-mediated signaling. Deletion of Mmp-2 restored leptin receptor expression and reduced circulating leptin concentrations in obese mice. Lentiviral delivery of short hairpin RNA to silence Mmp-2 in the hypothalamus of wild-type mice prevented leptin receptor cleavage and reduced fat accumulation. In contrast, lentiviral delivery of Mmp-2 in the hypothalamus of Mmp-2^-/- mice promoted leptin receptor cleavage and higher body weight. In a genetic mouse model of obesity, transduction of cleavage-resistant leptin receptor in the hypothalamus reduced the rate of weight gain compared to uninfected mice or mice infected with the wild-type receptor. Immunofluorescence analysis showed that astrocytes and agouti-related peptide neurons were responsible for Mmp-2 secretion in mice fed a high-fat diet. These results suggest a mechanism for leptin resistance through activation of Mmp-2 and subsequent cleavage of the extracellular domain of the leptin receptor.

Fig. 1.. Mmp-2 activity is increased in brains after HFD intake.

(A) Representative gel zymography showing the activity of Mmp-2 in the brain of rats fed a HFD and a CD. (B) Values of the band density in (A) are presented as density units (DU). Data are shown as means ± SD (n = 8 per group). (C) Representative sections of the hypothalamus of rats fed a CD and a HFD after immunolabeling with an antibody against the active form of Mmp-2. The insets show an overview, with black squares delineating the magnified fields. Arrows point to the Mmp-2–positive cells. (D) WB analysis of Mmp-2 in brain homogenates of rats fed a HFD and a CD. Equal amounts of protein were subjected to SDS–polyacrylamide gel electrophoresis, and Mmp-2 (active form) was detected. (E) Mmp-2 and β-actin band densities were determined, and measurements were presented as relative density units (RDU) of the Mmp-2/β-actin ratio (*P < 0.05, HFD versus CD; data are shown as means ± SD; n = 9 per group). (F) Representative WB showing ObR extracellular domain signal in brains of the CD and HFD groups. (G) Optical density measurements of the WB signal and data are presented as DU (*P < 0.05, CD versus HFD; data are shown as means ± SD; n = 8). (H) IF imaging of the ObR extracellular domain in HFD and CD. White arrows point to positive staining in CD and HFD. (I) Silver stain of ObR (left), Mmp-2 (middle), and ObR incubated with Mmp-2 (right). (J) Extracellular domain of the leptin receptor. Cytokine receptor homology (CRH-2) is the main binding site for leptin. The immunoglobulin (Ig)–like and fibronectin type 3 (FN3) domains are essential for receptor signaling. Stars in the extracellular domain represent the cleavage sites determined by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Fig. 2.. ObR is cleaved and has reduced signaling capabilities after incubation with Mmp-2.

(A) Representative immunolabeling of the ObR extracellular domain (green) in mouse hypothalamic cells exposed to Mmp-2. DAPI (4′,6-diamidino-2-phenylindole; blue) shows cell nuclei. (B) Measurement of the ObR extracellular domain density on cells treated with/without Mmp-2 is presented as mean fluorescent intensity (MFI) ± SD [*P < 0.05, cells treated with Mmp-2 (+Mmp-2) versus without Mmp-2 (−Mmp-2); n = 6]. (C) The incubation media of cells treated with/without MMP-2 analyzed for ObR fragments by WB. (D and E) Activation of ADAM-17 (D) and ADAM-10 (E) by Mmp-2 in hypothalamic cells. Activity was recorded for 30 min and presented as the difference in optical density (dOD ± SD; n = 6). *P < 0.05. TNF, tumor necrosis factor; LPS, lipopolysaccharide. (F) Representative WB of p-ObR, p-STAT-3, p-ERK, and corresponding total protein in cells treated with Mmp-2 (last four lanes) and their respective controls (first four lanes). Leptin was added to the cells after Mmp-2 removal for selected time points (indicated in the figure). (G) p-ERK/ERK ratio in control and Mmp-2–treated cells after leptin stimulation. *P < 0.05, untreated versus Mmp-2–treated cells at t = 5 min (n = 3 in each group). (H) p-STAT-3/STAT-3 ratio in untreated and Mmp-2–treated cells after leptin stimulation. *P < 0.05, untreated versus Mmp-2–treated cells at t = 5 min; **P < 0.005, untreated versus Mmp-2–treated cells at t = 15 min (n = 3 in each group). (I) p-ObR/β-actin ratio in untreated and Mmp-2–treated cells after leptin stimulation. *P < 0.05, untreated versus Mmp-2–treated cells at t = 15 min (n = 3 in each group).

Fig. 3.. Mmp-2^−/− mice are protected from obesity and leptin resistance.

(A) Body weight in WT and Mmp-2^−/− mice fed a HFD (n = 8). (B) Weight gain in WT and Mmp-2^−/− mice after HFD intake (means ± SD; *P < 0.05, n = 8). (C) Representative WB for the detection of active Mmp-2 in brain homogenate of mice fed a CD or a HFD. (D) Concentrations of Mmp-2 were quantified and are presented as RDU ± SD normalized against actin. *P < 0.05, CD versus HFD. (E) Representative WB of the ObR extracellular domain in Mmp-2^−/− mice fed a HFD, WT mice fed a CD, and WT mice fed a HFD. (F) The density of the bands was determined relative to β-actin, and results are presented as RDU ± SD. *P < 0.05, WT (HFD) versus Mmp-2^−/− (HFD); **P < 0.05, WT (HFD) versus WT (CD); n = 8. (G) Immunolabeling using an antibody against the extracellular domain of the ObR in the hypothalamus of WT mice fed a HFD, WT mice fed a CD, and Mmp-2^−/− mice fed a HFD. White arrows point to ObR staining. Scale bar, 20 μm. (H) Serum leptin concentrations measured with enzyme-linked immunosorbent assay after HFD (WT and Mmp-2^−/−) and CD intake (WT) [*P < 0.001, WT (HFD) versus WT (CD); **P < 0.001, WT (HFD) versus Mmp-2^−/− (HFD); n = 9 per group].

Fig. 4.. Hypothalamic Mmp-2 mediates weight gain and plasma leptin.

(A) The ARC part of the hypothalamus of WT mice was infected with viruses carrying shRNA to inhibit MMP-2 (shMMP-2-ARC) or an irrelevant sequence (shIRR-ARC), and mice were subjected to either CD or HFD. The shMMP-2-ARC on HFD gained significantly less weight compared to WT on HFD (*P < 0.05; n = 8). (B) Weight increase as a fraction of body weight (before the start of HFD/CD) (*P < 0.05, WT fed HFD versus shMmp-2 ARC fed HFD; n = 8). (C) Plasma leptin values in the experimental groups (*P < 0.05 shMmp-2 versus WT fed HFD; n = 8). (D) IF images showing the expression pattern of Mmp-2 (red) and ObR (yellow) in the hypothalamus of mice infected with shMmp-2 (GFP-green); blue indicates nuclear DAPI labeling. White arrows point to ObR-positive cells (second row, right), leti-infected cells (third row, left and right), and Mmp-2–positive cells (fourth row, left). Note that, in the presence of Mmp-2, ObR is reduced. Scale bar, 20 μm. (E) IF labeling (high magnification) of the ObR extracellular domain (red) and Mmp-2 (green) in the hypothalamus of obese mouse. (F) MMP-2^−/− were reintroduced with MMP-2 (MMP-2^−/−/L-MMP-2) and were subjected to either CD or HFD (means ± SD; *P < 0.05 versus MMP-2^−/− fed a HFD; n = 7). (G) Total weight increase as percentage of body weight (before the start of HFD/CD versus 16 weeks of CD or HFD). (H) Concentrations of plasma leptin after HFD intake in mice reintroduced with Mmp-2 compared to Mmp-2^−/− on the same diet.

Fig. 5.. MMP-2 mediates weight gain and is controlled by NF-κB.

(A) Representative confocal images showing reduced expression of ObR in cells of Mmp-2^−/− mice after reintroduction of Mmp-2 to the ARC part of the hypothalamus. White arrows point to locations with high expression of Mmp-2 but reduced expression of ObR. Scale bar, 20 μm. (B) Weight gain in db/db mice infected with MMP-2–resistant ObR [means ± SD; *P < 0.05, db/db^L-ObR (L60;I266) versus db/db^L-ObR (WT); **P < 0.01, db/dbL-ObR (L60;I266) versus db/db; n = 6]. (C) Reverse transcription (RT) qPCR for NF-κB target genes and Mmp-2 in GT1–7 cells treated with TNFα to activate NF-κB and with the IKK2 inhibitor TPCA. Results are presented as relative to glyceraldehyde-3-phosphate dehydrogenase. (D) Changes in blood glucose concentrations in mice fed a CD or a HFD treated with doxycycline (Dox) or saline. Data are presented as percent increase at day 5 versus day 0 (means ± SD, n = 8). (E) IF imaging of hypothalamus from obese mice labeled with glial fibrillary acidic protein (GFAP), Iba1, pro-opiomelanocortin (POMC), and AgRP and colocalized with Mmp-2. Colocalization was found in astrocytes (GFAP-positive; white arrows, upper right) and AgRP neurons (white arrows, lower right).