Nuclear magnetic resonance spectroscopy-based metabolomics of the fatty pancreas: implicating fat in pancreatic pathology

Abstract

Background: Obesity is a worldwide epidemic and a significant risk factor for pancreatic diseases including pancreatitis and pancreatic cancer; the mechanisms underlying this association are unknown. Metabolomics is a powerful new analytical approach for describing the metabolome (compliment of small molecules) of cells, tissue or biofluids at any given time. Our aim was to analyze pancreatic fat content in lean and congenitally obese mice using both metabolomic analysis and conventional chromatography.

Methods: The pancreatic fat content of 12 lean (C57BL/6J), 12 obese leptin-deficient (Lep(ob)) and 12 obese hyperleptinemic (Lep(db)) mice was evaluated by metabolomic analysis, thin-layer and gas chromatography.

Results: Pancreata of congenitally obese mice had significantly more total pancreatic fat, triglycerides and free fatty acids, but significantly less phospholipids and cholesterol than those of lean mice. Metabolomic analysis showed excellent correlation with thin-layer and gas chromatography in measuring total fat, triglycerides and phospholipids.

Conclusions: Differences in pancreatic fat content and character may have important implications when considering the local pancreatic proinflammatory milieu in obesity. Metabolomic analysis is a valid, powerful tool with which to further define the mechanisms by which fat impacts pancreatic disease.

Copyright 2009 S. Karger AG, Basel.

Figures

Fig. 1.

a Typical 1 H spectra of intact pancreas tissue samples from lean, Lepob and Lepdb mice. All the spectra were obtained using magic angle sample spinning NMR techniques and plotted with identical scales for direct comparison of various lipids in different animal models. As can be seen from the spectra, triglycerides are higher in both Lepdb and Lepob compared with the lean mice by nearly an order of magnitude. On the other hand, phospholipids are higher in lean mice compared with Lepdb and Lepob mice (peak labeled as choline is a marker of phosphatidylcholine). b Score plot obtained from the PCA of the 1H NMR (Carr-Purcell-Meiboom-Gill) data of Lepdb, Lepob and lean mice. Both Lepdb and Lepob show distinctly separate clusters from the lean, indicating significant metabolic differences between obese and lean mice. The loading plot along PC1 direction indicates that lipids and triglycerides increase in obese mice (upward peaks), whereas the phospholipid content is higher in lean mice (downward peak).

Fig. 1.

a Typical 1 H spectra of intact pancreas tissue samples from lean, Lepob and Lepdb mice. All the spectra were obtained using magic angle sample spinning NMR techniques and plotted with identical scales for direct comparison of various lipids in different animal models. As can be seen from the spectra, triglycerides are higher in both Lepdb and Lepob compared with the lean mice by nearly an order of magnitude. On the other hand, phospholipids are higher in lean mice compared with Lepdb and Lepob mice (peak labeled as choline is a marker of phosphatidylcholine). b Score plot obtained from the PCA of the 1H NMR (Carr-Purcell-Meiboom-Gill) data of Lepdb, Lepob and lean mice. Both Lepdb and Lepob show distinctly separate clusters from the lean, indicating significant metabolic differences between obese and lean mice. The loading plot along PC1 direction indicates that lipids and triglycerides increase in obese mice (upward peaks), whereas the phospholipid content is higher in lean mice (downward peak).

Fig. 2.

a 31P NMR spectra of pancreas tissue from lean, Lepob and Lepdb mice. All spectra were obtained using magic angle sample spinning NMR techniques and plotted with identical scales for direct comparison of various phosphorus metabolites in different animal models. From the comparison of peak intensities, it can be seen that PME, phosphodiester (PDE) and Pi are higher in the lean than in Lepob and Lepdb. b Score plot obtained from the PCA of the 31P NMR data of Lepdb, Lepob and lean mice. Both Lepdb and Lepob show distinctly separate clusters from the lean, indicating significant metabolic differences between obese and lean mice. The loading plot along the PC1 direction indicates that the phosphorus metabolites are higher in lean compared with obese mice (upward peaks).

Fig. 2.

a 31P NMR spectra of pancreas tissue from lean, Lepob and Lepdb mice. All spectra were obtained using magic angle sample spinning NMR techniques and plotted with identical scales for direct comparison of various phosphorus metabolites in different animal models. From the comparison of peak intensities, it can be seen that PME, phosphodiester (PDE) and Pi are higher in the lean than in Lepob and Lepdb. b Score plot obtained from the PCA of the 31P NMR data of Lepdb, Lepob and lean mice. Both Lepdb and Lepob show distinctly separate clusters from the lean, indicating significant metabolic differences between obese and lean mice. The loading plot along the PC1 direction indicates that the phosphorus metabolites are higher in lean compared with obese mice (upward peaks).

Fig. 3.

a Pancreatic total fat (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly more fat than lean mice; good concordance between chromatographic and metabolomic analysis is apparent. b Pancreatic triglycerides (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly more triglycerides than lean mice; again, good concordance between chromatographic and metabolomic analysis is apparent. c Pancreatic phospholipids (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly lower phospholipid concentrations than lean mice. Again, chromatographic and metabolomic results are similar.

Fig. 3.

a Pancreatic total fat (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly more fat than lean mice; good concordance between chromatographic and metabolomic analysis is apparent. b Pancreatic triglycerides (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly more triglycerides than lean mice; again, good concordance between chromatographic and metabolomic analysis is apparent. c Pancreatic phospholipids (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly lower phospholipid concentrations than lean mice. Again, chromatographic and metabolomic results are similar.

Fig. 3.

a Pancreatic total fat (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly more fat than lean mice; good concordance between chromatographic and metabolomic analysis is apparent. b Pancreatic triglycerides (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly more triglycerides than lean mice; again, good concordance between chromatographic and metabolomic analysis is apparent. c Pancreatic phospholipids (μg/mg pancreatic tissue) as measured by conventional chromatography and metabolomic (NMR) analysis. Both congenitally obese strains of mice have significantly lower phospholipid concentrations than lean mice. Again, chromatographic and metabolomic results are similar.