With the recent successful completion of the human genome sequencing project, biomedical researchers are now struggling to identify the functions of the 30,000 genes that appear to be expressed, and the role played by individual genes in human disease.

 

Much effort is focused on comprehensive approaches for evaluating changes in gene expression, as represented by changes in mRNA and protein levels. While genomics and proteomics have the potential for providing dazzling insights, it can be argued that these technologies do not provide the ultimate view of the status of a living organism, because this resides at the level of chemistry and metabolism. Therefore, a major focus of the Sarah Stedman Nutrition and Metabolism Center is to become a world leader in comprehensive mass spectrometry (MS)- and nuclear magnetic resonance (NMR)-based analysis of small molecule metabolites and metabolic flux in biological fluids, cells, and tissues. Application of these tools for comprehensive metabolic analysis is sometimes called metabolic profiling or metabolomics.

The technology platform for metabolic profiling that has been developed in the Sarah W. Stedman Nutrition and Metabolism Center consists of three major components:

At the heart of this consortium is the analytic platform. A major advance for the Sarah Stedman Nutrition and Metabolism Center was the recruitment of Dr. David Millington and Dr. Robert Stevens of the Duke Mass Spectrometry Laboratory and the Department of Pediatrics. Drs. Millington and Stevens have directed a world-renowned reference laboratory that uses mass spectrometry to detect inborn errors of metabolism for many years. They have teamed with Dr. Newgard, Dr. Mark Butler, Dr. James Bain, and Dr. Olga Ilkayeva of the Stedman Center to create a research-dedicated MS facility in the Sarah Stedman Center for metabolic profiling of pre-clinical and clinical research samples. The ultimate goal of this endeavor is to make metabolic profiling available to all interested investigators at Duke and collaborating institutions. Currently, the Stedman Center laboratory deploys GC/MS and MS/MS for "targeted" quantitative measurements of intermediary metabolites of known identity in 4 classes:

The laboratory has also recently obtained the new Waters LCT Premier™ LC/MS system and associated MarkerLynx™ software. This instrument allows "unbiased" analysis of some 3000 individual metabolites in a single specimen. Although the identity of most of the analytes is unknown when using this system, statistical analysis can be used to define principal components (groups of analytes) that discriminate different samples. Once a set of metabolites has been shown to correlate with functional states, the identity of the individual analytes can be determined by traditional chemical and physical methods.

This comprehensive metabolic profiling is complemented by a new hormone and cytokine assay core laboratory directed by Dr. Mike Muehlbauer. This core laboratory measures 18 hormones that regulate metabolic fuel homeostasis and energy balance (eg, leptin, ghrelin, adiponectin) as well as 23 pro- and anti-inflammatory cytokines.

 

Finally, in collaboration with Drs. Bill Kraus and Cris Slentz, the Stedman Center conducts human physiologic profiling, including resting metabolic rate and Rq by indirect calorimetry, body composition by DEXA, CT and BodPod, and frequently-sampled intravenous glucose tolerance testing. Integration of these metabolic, endocrine, inflammatory marker, and physiologic profiling tools affords Stedman Center investigators a unique and comprehensive view of the metabolic status of cells, animal models of disease, and human subjects.

Another unique feature of the Stedman Center program is its long-standing collaboration with the Ralph and Mary Nell Rogers NMR Center at UT Southwestern Medical Center in Dallas (UTSWMC). Prior to coming to Duke, Dr. Newgard was a faculty member at UTSWMC for 15 years, and this allowed development of a strong collaboration with Drs. Dean Sherry and Craig Malloy, the leaders of the Rogers Center. Drs. Sherry and Malloy have developed elegant methods for NMR-based analysis of flux through specific metabolic pathways. This involves administration of metabolic fuels containing stable isotopes (eg, ¹³C glucose, ²H₂O) to cells, animals, or humans, and analysis of metabolic byproducts via NMR-based mass isotopomer analysis. Drs. Sherry and Newgard have collaborated to apply these tools to the study of pathways of glycogen synthesis in liver and mitochondrial pathways of glucose metabolism that are involved in glucose-stimulated insulin secretion. This provides Stedman Center investigators with a unique capacity to test hypotheses about regulation of metabolic pathways that may emanate from MS-based analysis of individual metabolites.

 

Examples of the successful use of these technologies are shown in the following publications from Stedman Center scientists.

1) ODoherty R, Jensen PB, Anderson P, Jones JG, Berman HK, Kearney D, Newgard CB. Overexpression of a Glycogen-Targeting Subunit of Protein Phosphatase-1 in Liver of Normal Rats Activates Direct and Indirect Pathways of Glycogen Synthesis. J Clin Invest. 2000;105:479-488.

2) Yang R, Cao L, Gasa G, Brady M, Sherry AD, Newgard CB. Glycogen targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes. J Biol Chem. 2002;277:1514-1523.

3) Lu D, Mulder H, Zhao P, Burgess SC, Jense MV, Kamzolova S, Newgard CB, Sherry AD. 13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion. PNAS. 2002;99:2708-2713.

4) Wu JY, Kao HJ, Li SC, Stevens R, Hillman S, Millington D, Chen YT. ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease. J Clin Invest. 2004;113:434-440.

5) An J, Muoio DM, Shiota M, Fujimoto Y, Cline GW, Shulman GI, Stevens R, Millington D. Newgard CB. Hepatic expression of malonyl CoA decarboxylase reverses muscle, liver, and whole-animal insulin resistance. Nat Med. 2004;10(3):268-274.

6) Boucher A, Lu D, Burgess S, Telemaque-Potts S, Jensen M, Mulder H, Wang M-Y, Unger RH, Sherry AD, Newgard CB. Mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and its reversal by an analogue of malate. J Biol Chem. 2004;279:27263-27271.