Figure 1.
Npy induction within visceral fat and SVF with diet-induced obesity.
Npy gene expression by quantitative RT-PCR in (A) epididymal fat (EWAT) and (B) inguinal fat depots (IWAT) of normal diet (ND), 8 week high fat diet (HFD), and 20 week HFD C57Bl6 mice. N = 4 mice per group. (C) Npy expression in various fat depots of ND and 16 week HFD fed animals. N = 4 mice per group. (D) Npy expression in stratified adipose tissue. EWAT from HFD C57Bl6 mice was separated into adipocyte (Ad) and stromal vascular fraction (SVF) cells after collagenase digestion. Npy expression was assessed by quantitative RT-PCR. N = 4 per group. (E) Npy1r and Npy2r expression in adipocytes and SVF cell fractions from lean mice. N = 4 per group. (F) NPY protein expression in cultured SVF cells from HFD C57Bl6 mouse EWAT and IWAT. Media was removed from cells after 18 hours of culture and assessed for NPY by ELISA (N = 3 wells per group). *p<0.05, **p<0.01,***p<0.005 by t-test.
Figure 2.
Npy gene expression in fat is primarily derived from ATMs in obese animals.
(A) Flow cytometry analysis of SVF cells from EWAT of obese C57Bl/6 mice 18–20 wk HFD. SVF cells were stained with F4/80 and CD11b prior to analysis by flow cytometry. Fluorescent activated cell sorting (FACS) isolated purified ATM and non-ATM fractions (middle and right panels show validation plots). (B) Npy expression in ATM and non-ATM fractions by quantitative RT-PCR (N = 5 mice per group) (C) Comparative analysis of Npy expression in F4/80+ CD11b+ ATMs from ND, HFD, and db/db mice (N = 3 per group). (D) Npy expression in FACS purified M2 (CD206+) and M1 (CD11c+) ATMs (F4/80+ CD11b+) *p<0.05, **p<0.01,***p<0.005 by t-test.
Figure 3.
NPY production by bone marrow derived dendritic cells (BMDC) and macrophages (BMMP).
(A) Quantitative RT-PCR analysis of Npy expression in BMMPs and BMDCs from C57Bl/6 mice with or without LPS (100 ng/ml) for 24 hours. (B) NPY protein expression by ELISA on media from BMDC or BMMP cultured for 24 hours with and without LPS (100 ng/ml) or IL-4 (20 ng/ml) treatment. N = 3 per group. *p<0.05 **p<0.1 #p<0.05 between BMMP and BMDC, by t-test.
Figure 4.
NPY1R is expressed in pre-adipocytes.
Analysis of FACS purified ATM and Non-ATM populations from EWAT from 5 lean (ND) and 5 obese (16 wk HFD) C57Bl/6 mice for (A) Npy1r and (B) Npy2r expression by quantitative RT-PCR. No significant differences between groups by ANOVA. (C) Npy1r expression in 3T3-L1 cells during adipocyte differentiation (N = 5). (D) Npy1r expression in BMDC from lean and obese (HFD) mice (N = 3 per group) *p<0.05, **p<0.01, ***p<0.005, by t-test.
Figure 5.
NPY receptor blockade increases M1 cytokine expression and MHCII expression in dendritic cells.
Bone marrow dendritic cells (BMDCs) were differentiated for 7 days in the presence of antagonists against the Y1 receptor (BIBO-3304, 10 uM), Y2 receptor (BIIE-0246, 1 uM), and Y5 receptor (L-152,804, 10 uM) and then stimulated with LPS (10 ng/ml) for 18 hours. (A) Analysis of M1 cytokine gene expression by quantitative RT-PCR. (B) TNFα and IL-6 protein in media from cultured BMDC with and without antagonist treatment after stimulation with LPS (10 ng/ml) for 18 hours (N = 6). (C) Analysis of dendritic cell maturation genes in antagonist and vehicle treated BMDCs. (D) Flow cytometry analysis of DC maturation markers in BMDCs with and without antagonist. Right panel shows quantitation of CD11c+ MHCII+ cells in the BMDC groups. (N = 3). *p<0.05, by t-test.
Figure 6.
NPY treatment decreases circulating Ly6c+ monocyte and CD11c+ ATM content.
Lean C57Bl/6 mice were treated with NPY or NPY scramble control peptide (60 µg/kg/day) IP for 10 days. N = 4 mice per group. (A) Quantitiation of ATMs by flow cytometry. EWAT SVF cells were stained for ATM markers (F4/80, CD11b, CD11c) and analyzed by flow cytometry. ATM content normalized to fat pad weight. Gene expression analysis of EWAT for (B) inflammatory genes by quantitative RT-PCR. (C) light microscopy image showing larger adipocytes with NPY injection (left panel) compared to scramble control peptide injection (right panel) (D) Gene expression analysis of EWAT for adipocyte genes by quantitative RT-PCR. *p<0.05. (E) Quantitation of Ly6chi and Ly6clo CD115+ blood monocytes by flow cytometry (N = 4 per group). Data is representative data of one of 3 replicate experiments.
Figure 7.
Lack of NPY expression in hematopoietic cells increases M1 ATMs with diet-induced obesity.
Donor marrow from S129 wildtype and S129 Npy−/− mice were transplanted into lethally irradiated wild-type S129 mice. After reconstitution, both groups were placed on ND or HFD chow for 8 weeks (N = 5 in WT donor groups, N = 4 in KO donor groups). (A) Body Weight and (B) EWAT weight assessed at the end of the diet exposure. (C) Flow cytometry quantitation of ATMs (F4/80, CD11b) expressed as number for total per EWAT fat pad (D) ATM as percent of leukocytes in EWAT by flow cytometry. (E) Flow cytometry quantitation of F4/80+ CD11b+CD11c+ ATMs (M1) and F4/80+ CD11b+CD11c+ MHCII+ ATMs. (F) Npy expression in EWAT of BM chimeras by RT-PCR. (G) Tnfa EWAT gene expression by RT-PCR. (H) Fasting insulin levels from serum after 6 hour fast. *p<0.05, groups compared by two-way ANOVA and signficance values shown by individual t-tests.