α-KG, alpha-ketoglutarate; ACS, acetyl-CoA acetyltransfera-
se; ALT, alanine aminotransferase; AST, aspartate aminotran-
sferase; LDH, lactate dehydrogenase; ME, malic enzyme;
NMR, nuclear magnetic resonance; OAA, oxaloacetate; PBS,
phosphate buffered saline; PC,pyruvate carboxylase; PDH,py-
ruvate dehydrogenase; PEP, phosphoenolpyruvate carboxylase;
PK, pyruvate kinase; TCA, tricarboxylic acid.
There are many aspects of neuroendocrinology
that need to be addressed.One of these is to better un-
derstand the relationship between cell function and
bioenergetics (i.e., the link between the cell’s energe-
tic machinery and the ability to secrete hormones).
This may be particularly important in pituitary ade-
nomas, whose metabolic requirements and bioenerge-
tic machinery could differ from those of normal tissue,
and impact the function of the tissue. Presently, there
are methods readily available to study many aspects of
cellular metabolism on in vitro systems. For example,
oxygen consumption rates, glucose and lactate pro-
duction rates, hexokinase/glucokinase enzymatic cha-
racteristics,and Ca++flux can all be studied and related
to secretion under a variety of physiological condi-
tions. However, the addition of 13C NMR spectrosco-
pic techniques (on cellular extracts) to this analytical
arsenal provides a powerful means by which substan-
tial metabolic information concerning cellular energe-
tics can be obtained, particularly information regar-
ding that occurring in the mitochondria. Properly
analyzed NMR spectroscopic data may be related to
information gleaned from other assays to give a more
complete view of intracellular bioenergetics and its re-
lationship to the function of the cell.
13C NMR isotopomeric analysis and its application in the
study of endocrine cell metabolism and function
Nicholas E. Simpson, Ioannis Constantinidis
Division of Endocrinology, Department of Medicine,The University of Florida, FL, USA
Abstract. Defining mechanisms and enzymatic paths critical to cellular function (e.g., secretion) of en-
docrine cells is a key research goal that can lead toward novel avenues of therapeutic intervention for a vari-
ety of disorders.13C NMR spectroscopy and isotopomer analysis of cell extracts are excellent tools to quan-
titatively assess metabolism through intermediate labeling and estimate carbon entry to the TCA cycle. Dis-
cussed are: cell lines and in vitro culturing; extraction of intracellular material; NMR spectroscopy of the ex-
tract; isotopomeric analysis and modeling to obtain relative metabolic fluxes to the TCA cycle. This paper
describes issues related to the application of NMR spectroscopic techniques on cell line extracts. Included
are results of two studies that illustrate considerations that must be taken when performing analogous stud-
ies on neuroendocrine tissue: one involving the effect of media composition on cell behavior and isotopomer
labeling; the second looking at effects of applying different metabolic models to 13C data and inferences that
may be drawn. NMR isotopomeric analysis is a powerful technique that may be applied to better understand
endocrine cell function. (www.actabiomedica.it)
Key words:Isotopomer analysis, metabolism, NMR spectroscopy, secretion,TCA cycle
L E C T U R E
ACTA BIOMED 2007; 78; Suppl 1: 99-112
© Mattioli 1885
N.E. Simpson, I. Constantinidis
This NMR technique has been used in a variety
of cell systems to tease out biochemical information
(1-10).The bulk of these studies have been performed
in model insulin-secreting cell lines (i.e., insulinomas)
and attempt to determine critical links between cellu-
lar energetics and cellular function (e.g., insulin secre-
tion). However, the study of other model cell systems
has been enhanced by the application of isotopomer
analysis; specifically, the metabolic consequences of an
enzyme deficiency and its treatment in fibroblasts
(11).In theory,there is no reason why one cannot pur-
sue similar studies with endocrine tissues. For exam-
ple, one could study the pathogenesis of disease, or
identify novel molecular targets. Armed with a basic
metabolic knowledge of critical steps of metabolism
important to cellular function (e.g., secretion), new
avenues of therapeutic intervention may be tested to
up- or down-regulate these critical metabolic steps.To
aid the reader in appreciating the NMR technique,
following is a simplistic description of how the com-
plex NMR signals arise.
NMR spectroscopy and isotopomer analysis
Although it’s a vastly oversimplified statement,
atoms with nuclei that possess a nuclear spin (angular
momentum) can be manipulated within a magnetic
field to create the NMR phenomenon, and obtain si-
gnal. The growing discipline of magnetic resonance
(which includes both spectroscopy and imaging) takes
advantage of this phenomenon to obtain vast amounts
of diverse information; biochemical, structural (che-
mical), anatomical, etc., depending on the experiment
performed. For a complete description of the NMR
phenomenon,the following books are suggested to the
reader (12, 13).
As stated earlier, NMR spectroscopy is a power-
ful tool by which one can obtain bioenergetic infor-
mation. One method is through studying changes in
the carbon labeling of compounds involved in meta-
bolic pathways. Although the usual isotope of carbon
(12C) is not NMR-detectable (i.e., it has no nuclear
spin), the non-radioactive isotope 13C does have a nu-
clear spin, and is readily NMR-observable. Because
this isotope of carbon has an extremely low natural
abundance, feeding cells a metabolizable label that in-
cludes 13C is essential for detection and analysis. If a
13C label is given to cells (e.g.,13C-labeled glucose),the
carbon label will enter the cell, and be distributed
among various compounds reflective of the processes
of metabolism. A standard model of glucose metabo-
lism (14, 15), including glycolysis and pyruvate entry
to the TCA cycle, is shown in Figure 1. In this model,
following our example of stimulation with
uniformly-labeled glucose, glucose is metabolized to
pyruvate through glycolysis. Glycolysis turns molecu-
les of uniformly-labeled glucose into molecules of
uniformly-labeled pyruvate, which have a number of
potential metabolic fates. Among these are: 1) conver-
sion to lactate (via lactate dehydrogenase: LDH); 2)
conversion to alanine (via alanine aminotransferase,
ALT); 3) entrance to mitochondria and the TCA cy-
cle either by conversion to acetyl-CoA via the pyruva-
te dehydrogenase complex (PDH), or anaplerotic car-
bon entry via PC to form oxaloacetate (OAA). This
cytosolic pyruvate pool can also be replenished from
TCA cycle intermediates malate or OAA by malic
enzyme (ME) or phosphoenolpyruvate carboxylase
Figure 1. Standard model of glucose metabolism. In this mo-
del, glucose is metabolized to pyruvate through glycolysis, and
then has a number of potential fates. Important enzymes in-
clude: ALT (alanine transaminase); LDH (lactate dehydroge-
nase); PDH (pyruvate dehydrogenase complex); PC (pyruvate
carboxylase); ACS (acetyl-CoA acetyltransferase); AST
(aspartate transaminase); “ATF” (aminotransferase); ME (ma-
lic enzyme); PEP/PK (phosphoenolpyruvate carboxylase/pyru-
Applications of 13C isotopomer analysis
new avenues for exploration towards defining critical
metabolic pathways related to hormone release.
However, caution is offered to perform the experi-
ments with the most physiologically relevant media
and choose appropriate metabolic models to assure
that the results obtained are valid and relevant to the
actual physiology of the pituitary adenoma under
study. With proper application of powerful NMR
spectroscopic techniques, important information re-
garding cellular energetics of pituitary adenomas will
The authors are grateful for the technical assistance of Jo-
se Oca-Cossio, Chiab Simpson, Nata Khokhlova and Carol
Sweeney. This work was supported by grants from the NIH
(DK56890, DK47858). The program ‘tcaCALC’ was obtained
from the University of Texas Southwestern, and developed th-
rough H-47669-16 and RR-02584. NMR data were obtained
at the Advanced Magnetic Resonance Imaging and Spectro-
scopy (AMRIS) facility in the McKnight Brain Institute of the
University of Florida.
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Correspondence: Nicholas E. Simpson, PhD,
Division of Endocrinology
1600 SW Archer Rd., PO Box 100226,
Gainesville, FL 32610-0226
Tel. (352) 846-2723
Fax: (352) 846-2635