Download Perspective: emerging evidence for signaling roles of mitochondrial

Am J Physiol Endocrinol Metab 288: E1–E15, 2005;
doi:10.1152/ajpendo.00218.2004.
Invited Review
Perspective: emerging evidence for signaling roles of mitochondrial
anaplerotic products in insulin secretion
Michael J. MacDonald, Leonard A. Fahien, Laura J. Brown,
Noaman M. Hasan, Julian D. Buss, and Mindy A. Kendrick
Childrens Diabetes Center, University of Wisconsin Medical School, Madison, Wisconsin
citrate oscillations; anaplerosis; insulin release; NADPH; pyruvate carboxylase;
cataplerosis; succinate mechanism
to indicate that fuel secretagogues stimulate insulin secretion primarily via their metabolism in mitochondria. However, only the initial stages of the
metabolism of these fuels are known. In addition to fuel
metabolism providing energy, metabolism of all physiological
insulin secretagogues results in the net synthesis of citric acid
cycle intermediates (anaplerosis). Mounting evidence indicates
that these intermediates act directly as, or as precursors of,
important signals in insulin secretion. It is assumed that products of anaplerosis are exported from the mitochondria
(cataplerosis) and have extramitochondrial signaling actions.
Even less is known about the extramitochondrial actions of
potential anaplerotic products than is known about the path-
THERE IS A GREAT DEAL OF EVIDENCE
Address for reprint requests and other correspondence: M. J. MacDonald,
Rm. 3459 Medical Science Center, 1300 University Ave., Madison, WI 53706
(E-mail: [email protected]).
http://www.ajpendo.org
ways of their formation. This article discusses some of the
potential products of anaplerosis and their possible extramitochondrial mechanisms of action. Numerous excellent
articles have addressed what is believed to be well known
about anaplerosis in fuel-induced insulin secretion (18, 19,
23, 83, 88, 92, 96, 108, 117). Much of the current review
will take a different approach and will explore less thoroughly studied and therefore less well understood processes.
Although a discussion of emerging evidence automatically
makes an article somewhat speculative, we believe that many
of the general processes mentioned here will ultimately be
substantiated, but concepts of their mechanistic details may
need some revision. We propose that most citric acid cycle
intermediates are exported to the cytosol, where their extramitochondrial actions trigger, potentiate, or support insulin secretion. Because so little is known about the direct signaling
mechanisms of the metabolites, and there is likely overlap
0193-1849/05 $8.00 Copyright © 2005 the American Physiological Society
E1
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
MacDonald, Michael J., Leonard A. Fahien, Laura J. Brown, Noaman M.
Hasan, Julian D. Buss, and Mindy A. Kendrick. Perspective: emerging evidence
for signaling roles of mitochondrial anaplerotic products in insulin secretion. Am J
Physiol Endocrinol Metab 288: E1–E15, 2005; doi:10.1152/ajpendo.00218.
2004.—The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing ␤-cell probably equals that of ATP production. In
glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high
and correlates more strongly with the glucose concentration the ␤-cell is exposed
to (and thus with insulin release) than does pyruvate decarboxylation, which
produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The
carboxylation pathway can increase the levels of citric acid cycle intermediates, and
this indicates that anaplerosis, the net synthesis of cycle intermediates, is important
for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates
have roles in signaling insulin secretion. Although evidence is quite good that all
physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric
acid cycle intermediates. This article speculates on their potential roles as signaling
molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and
malonyl-CoA, as well as ␣-ketoglutarate as a substrate for hydroxylases. We also
discuss the “succinate mechanism,” which hypothesizes that insulin secretagogues
produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria
in causing oscillations in ␤-cell citrate levels. These parallel oscillations in ATP and
NAD(P)H. Oscillations in ␤-cell plasma membrane electrical potential, ATP/ADP
and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with
pulsatile insulin release. Citrate oscillations might synchronize oscillations of
individual mitochondria with one another and mitochondrial oscillations with
oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria.
Thus citrate oscillations may synchronize mitochondrial ATP production and
anaplerosis with other cellular oscillations.
Invited Review
E2
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
between their possible direct initiating vs. supporting roles, we
will not attempt to distinguish between the two concepts.
EVIDENCE FOR MITOCHONDRIAL FACTORS IN ADDITION
TO ATP
PYRUVATE CARBOXYLATION DRIVES THE HIGH RATE OF
ANAPLEROSIS IN THE GLUCOSE-STIMULATED ␤-CELL AND
DEMONSTRATES THAT MITOCHONDRIA PRODUCE MORE
THAN ATP
The ␤-cell possesses a high level of the anaplerotic mitochondrial enzyme pyruvate carboxylase (13, 21, 54, 61, 62, 69,
76, 80, 81, 103), about equal to that in the gluconeogenic
organs, liver and kidney (61). However, unlike these tissues,
the ␤-cell cannot carry out glyconeogenesis from the oxaloacetate generated by pyruvate carboxylase because it lacks the
glyconeogenic enzymes phosphoenolpyruvate carboxykinase
(38, 68, 80) and fructose bisphosphatase (M. J. MacDonald,
unpublished data). This suggests a novel and interesting purpose for anaplerosis in the ␤-cell. We know that anaplerosis is
important for insulin secretion for several reasons. It has been
demonstrated by using the 14CO2 ratios method that ⬃50% of
glucose-derived pyruvate enters mitochondrial metabolism via
carboxylation catalyzed by pyruvate carboxylase (46, 58, 59,
60, 61). The other one-half of the glucose-derived pyruvate
enters mitochondrial metabolism via decarboxylation to acetylCoA catalyzed by the pyruvate dehydrogenase complex (46,
58, 59, 60, 61). We have shown that the rate of pyruvate
carboxylation correlates better than the rate of its decarboxylation with the concentration of glucose that the ␤-cell is
exposed to and thus also with the rate of insulin secretion (59).
Fig. 1. Mitochondrial metabolic signals in addition to ATP stimulate insulin secretion in the ␤-cell. Depicted are mitochondria producing ATP, which acts on
the sulfonylurea receptor (SUR) and the ATP-sensitive potassium (KATP) channel concomitant with mitochondrial anaplerosis and cataplerosis, producing
proximal metabolic signals. The latter signals, which we believe are rather specific to the ␤-cell, have direct actions of their own and also act on known and
unknown distal messengers that are present in many cells. Known distal messengers include G proteins (G), phospholipase C (PLC), generation of inositol
phosphates [phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate (IP3)], diacylglycerol (DG), and generation of nicotinic acid adenine
dinucleotide phosphate (NAADP). Depolarization of the plasma membrane, caused by the increase in intracellular K⫹ via activation of the KATP channel,
influences Ca2⫹ movements and, along with the cataplerotic and distal processes, mobilizes insulin granules (small shaded circles, as depicted in diagram between
“K⫹” and “Insulin”) to be extruded into the circulation. ATP also energizes contractile proteins that move insulin granules. Emerging evidence suggests that
numerous mitochondrial-generated messengers have extramitochondrial actions that work in parallel with ATP generation to signal and support insulin secretion.
ER, endoplasmic reticulum.
AJP-Endocrinol Metab • VOL
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
ATP is the only mitochondrial factor known with certainty
to couple metabolism to insulin exocytosis. An increase in the
ATP-to-ADP (ATP/ADP) ratio acting on the sulfonylurea
receptor (1) and the ATP-dependent potassium channel (KATP)
in the ␤-cell plasma membrane causes membrane depolarization. This opens a voltage-sensitive calcium channel in the
plasma membrane, and the resulting influx of calcium increases cytosolic calcium, which promotes the exocytosis of
insulin granules (5) (Fig. 1). However, as will be discussed, it
is clear that more than the KATP channel is involved in insulin
secretion. The work of Henquin et al. (39) and others (reviewed
in Ref. 108) showed that fuel secretagogues can stimulate
insulin release independently of the KATP channel. Although
ATP may participate in the KATP channel-independent pathway, there is evidence that mitochondrial factors in addition to
ATP (inter)act concomitantly with ATP to stimulate or enhance insulin secretion. The evidence for this idea comes from
the fact that the ␤-cell has a tremendous capacity for anaplerosis (58, 59, 61).
There are two phases of insulin secretion. The first phase
starts within seconds of an increased level of a fuel secretagogue making contact with the ␤-cell and has been called the
triggering phase. In this phase, a sharp peak of insulin is
released, probably from insulin granules stored immediately
next to the plasma membrane, and is believed to be due to an
increase in cellular calcium. In contrast, the second phase of
insulin release is of much longer duration and is quantitatively
the more important phase of insulin secretion. The second
phase also requires calcium and ATP and has been called the
amplification phase (39). Insulin secretagogues that do not
affect metabolism, such as high potassium or arginine, can
trigger the first phase of insulin release and raise intracellular
calcium but cannot cause the second phase of insulin secretion.
Because only fuel secretagogues can cause both the first and
second phases of insulin secretion, this has been taken as
evidence that the second phase is dependent on more products
of fuel metabolism than just ATP (39, 108).
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
In addition, it has been shown that a large amount of glucosederived pyruvate cycles through the pyruvate carboxylase reaction and that this cycling is proportional to the glucose
responsiveness of insulin secretion from various INS-1 cell
lines (55).
If ATP production alone is sufficient for stimulating insulin
secretion, pyruvate carboxylase would not need to be present in
the ␤-cell, because the decarboxylation pathway alone should
be sufficient to stimulate insulin secretion. (Many metabolically active tissues, such as cardiac muscle, possess little or no
pyruvate carboxylase.) The metabolism of acetyl-CoA in the
citric acid cycle provides ⬎95% of the ATP that a cell
produces. Acetyl-CoA metabolism produces mitochondrial
E3
NADH, which is oxidized by the mitochondrial respiratory
chain to supply the energy for ATP formation (Figs. 2 and 3).
The fact that the carboxylation pathway is so active in the
␤-cell suggests that anaplerosis (the net synthesis of citric acid
cycle intermediates and not simply their formation and utilization in the citric acid cycle) is important for insulin secretion.
In each turn of the citric acid cycle, carbon entering the cycle
as the two carbon acetate units of acetyl-CoA is balanced by
the release of two molecules of CO2. Thus, with pyruvate
decarboxylation and oxidation of acetyl-CoA, the cycle intermediates act somewhat like catalysts for the oxidation of acetyl
units, and their levels should remain relatively constant. In
contrast, the carboxylation of pyruvate can increase the intraDownloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
Fig. 2. Mitochondrial schematic showing respiration, the citric acid cycle, and transporters of metabolites across the mitochondrial inner membrane. Respiration
via the oxidation of NADH and flavoproteins pumps protons (H⫹) out of mitochondria. The diffusion of protons back across the inner mitochondrial membrane
powers ATP synthesis. Metabolism of substrates in the TCA cycle reduces NAD to NADH and reduces flavoproteins, e.g., succinate dehydrogenase. The
mitochondrial inner membrane is impermeable to many metabolites, and, therefore, transporters have evolved to carry metabolites across the inner membrane,
frequently in exchange for another metabolite. Some carriers are capable of transporting more than one metabolite, and some metabolites are transported on more
than one carrier. The red Xs at top right indicate that oxaloacetate (OAA), NAD(P)(H), and acetyl-CoA (Ac-CoA) are not transported as such across the inner
membrane and must be transported either as parts of other molecules (oxaloacetate and acetyl-CoA) or as equivalents in the form of reduced or oxidized
metabolites [NAD(P)(H)].
AJP-Endocrinol Metab • VOL
288 • JANUARY 2005 •
www.ajpendo.org
Invited Review
E4
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
mitochondrial level of citric acid cycle intermediates. Because
most citric acid cycle intermediates directly inhibit or activate
various enzymes of the cycle (examples are discussed in Ref.
73), the potential for increases in their concentrations means
that cycle function would be altered if excess amounts of
intermediates were not exported to the cytosol (cataplerosis).
In addition, increased levels of many intermediates would alter
the mitochondrial NAD-to-NADH (NAD/NADH) ratio, and
this would also alter the flux through the cycle. Experimental
studies have confirmed that cycle intermediates are exported
from ␤-cell mitochondria (57, 61, 64, 65). Because a cell
cannot afford to waste energy by synthesizing unutilized intermediates, this suggests that the metabolites are exported
from mitochondria for specific purposes, such as signaling and
supporting insulin secretion.
All fuel insulin secretagogues are capable of anaplerosis
(Fig. 4). It seems that, in order for a fuel to be a secretagogue,
it must be capable of forming either pyruvate or ␣-ketoglutarate and, in addition, fulfill other requirements. Fuels that
cannot form pyruvate directly, such as methyl esters of succinate, can become anaplerotic via their conversion to malate in
the mitochondrion and the export of malate to the cytosol
where it is converted to pyruvate by malic enzyme. Pyruvate is
then taken up into the mitochondrion where it can be carboxylated to oxaloacetate or decarboxylated to acetyl-CoA (Fig. 4,
reactions 21, 8, and 9 followed by reactions 12, 1, and 2).
Oxaloacetate can be converted to malate, fumarate, succinate,
and succinyl-CoA in the reverse of the reactions of the citric
acid cycle (Fig. 4, reactions 10, 9, 8, and 7). Fuels that increase
␣-ketoglutarate directly (Fig. 4, reactions 19, 20, and 18) can
be converted to the other intermediates of the cycle in the
forward reactions of the cycle (Fig. 4, reactions 6 –10). In
addition, ␣-ketoglutarate can be converted to isocitrate because
the mitochondrial NADP-linked isocitrate dehydrogenase reaction is reversible in the ␤-cell (MacDonald MJ, unpublished
data), as in some other cells (17, 24). Isocitrate can then be
converted to citrate in the aconitase reaction, which is freely
reversible (Fig. 4, reactions 5 and 4). Thus all citric acid cycle
AJP-Endocrinol Metab • VOL
intermediates can be synthesized from ␣-ketoglutarate but
perhaps not as efficiently as from pyruvate. Leucine and
␣-ketoisocaproate (␣-ketoisocaproic acid; KIC) can be metabolized directly to acetyl-CoA through a series of steps without
conversion to pyruvate (Fig. 4, reaction 19 and the 5 reactions
indicated by no. 25). The acetyl-CoA can combine with oxaloacetate for the synthesis of citrate and any citric acid cycle
intermediate (Fig. 4, reactions 3–10).
INITIAL STAGES OF METABOLISM OF
FUEL SECRETAGOGUES
Surprisingly, there are only a few fuel secretagogues, and
only the initial reactions of fuel secretagogue metabolism in the
␤-cell are understood. Briefly, the current consensus combined
with some of our own thoughts about their metabolism is as
follows.
Glucose. Glucose is the most potent physiological insulin
secretagogue, and it stimulates insulin release by its metabolism via aerobic glycolysis. It is metabolized to pyruvate, the
terminal product of aerobic glycolysis, from which all citric
acid cycle intermediates can be formed. Both decarboxylation
and carboxylation of pyruvate are necessary for glucose-induced insulin secretion (Fig. 4, reactions 1 and 2), as was
discussed above.
Leucine. Leucine is about one-third as potent an insulin
secretagogue as glucose (28). Leucine allosterically activates
glutamate dehydrogenase, thus enhancing conversion of glutamate to ␣-ketoglutarate (Fig. 4, reaction 18), which can be
metabolized in part of the cycle (Fig. 4, reactions 6 –10) to
form ATP, but less than can be obtained with metabolism of
pyruvate-derived acetyl-CoA in a complete turn of the cycle.
As mentioned in the previous section, ␣-ketoglutarate can be
converted in the reverse direction of the cycle to isocitrate and
citrate (Fig. 4, reactions 4 and 5). In addition to enhancing
endogenous glutamate metabolism, leucine can be converted to
KIC, which can be metabolized to hydroxymethylglutaryl-CoA
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
Fig. 3. ␤-Cell mitochondria, because of the
presence of pyruvate carboxylase, can synthesize citric acid cycle intermediates. The
conversion of glucose-derived pyruvate to
oxaloacetate catalyzed by pyruvate carboxylase can increase the level of citric acid cycle
intermediates (anaplerosis). These intermediates can then be exported from the mitochondria (cataplerosis), where, we postulate,
they have roles in signaling insulin secretion.
Pyruvate carboxylase is not present in all
body tissues; however, its level in the ␤-cell
is among the highest of any tissue (61).
Acetyl-CoA derived from pyruvate or other
fuels and its metabolism in the TCA cycle
reduce NAD to NADH. Electrons are passed
from NADH to the mitochondrial respiratory
chain to form 95% of ATP produced by a
cell. In the ␤-cell, the glycerol-phosphate
shuttle is particularly active (56). This shuttle passes electrons directly to the mitochondrial respiratory chain to oxidize cytosolic
NADH to make extra ATP.
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
E5
and acetyl-CoA (Fig. 4, reaction 19 and the 5 reactions
indicated by no. 25) (31, 33, 79).
Glutamine. Glutamine by itself is not an insulin secretagogue. However, the combination of glutamine and leucine is
about as potent as glucose in stimulating insulin release (28,
72, 87). This is most likely due to the activation of glutamate
dehydrogenase by leucine in the presence of a surfeit of
glutamine-derived glutamate. In addition, it is possible that the
metabolism of leucine combines with that of the glutamate to
create a synergistic stimulus on insulin secretion.
2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid. Although
2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) is a
nonmetabolizable leucine analog that is as potent as leucine in
activating glutamate dehydrogenase and enhancing conversion
of endogenous glutamate to ␣-ketoglutarate (Fig. 4, reaction
18) (25, 28, 36, 53, 87, 104), it is not as potent as leucine as a
secretagogue and is about one-fourth as potent as glucose
(MacDonald MJ, unpublished data). We believe BCH is a
slightly less potent insulin secretagogue than leucine, probably
because unlike leucine, BCH cannot be metabolized to hydroxymethylglutaryl-CoA and acetyl-CoA.
KIC. Interestingly, KIC, the first metabolite of leucine, is
much more potent an insulin secretagogue than leucine and is
about as potent a secretagogue as glucose. KIC probably
stimulates insulin secretion via its transamination with endogAJP-Endocrinol Metab • VOL
enous glutamate to form leucine and ␣-ketoglutarate (31, 51)
(Fig. 4, reaction 20) and via ␣-ketoglutarate’s metabolism, as
well as by the conversion of KIC to acetyl-CoA, (Fig. 4,
reaction 19 plus the 5 reactions indicated by no. 25). The
leucine derived from transamination can enhance glutamate
metabolism by activation of glutamate dehydrogenase, as mentioned above. The reason KIC is a much more potent secretagogue than leucine might be because metabolism of KIC
supplies acetoacetate and acetyl-CoA at a faster rate than does
metabolism of leucine.
Methyl esters of succinate. Methyl esters of succinate are
about one-third as potent as glucose as insulin secretagogues
and are about equal to leucine in potency (28, 34, 58, 59, 70,
71, 74, 75, 85, 86, 121) in fresh rat pancreatic islets. Methyl
esters of succinate probably stimulate insulin release by producing both oxaloacetate and acetyl-CoA. After hydrolysis of
the ester to succinate and conversion of the succinate to
fumarate and then malate in the mitochondrion, malate can be
exported to the cytosol and converted to pyruvate in the malic
enzyme reaction [Fig. 4, reactions 21, 8, 9 (or 22), and 12].
Pyruvate can then be taken up into the mitochondrion and
carboxylated to oxaloacetate (Fig. 4, reaction 2) or decarboxylated to acetyl-CoA (Fig. 4, reaction 1). The carboxylation
route of conversion of malate to oxaloacetate (Fig. 4, reactions
12 and 2) generates cytosolic NADPH and, unlike the direct
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
Fig. 4. Pathways of secretagogue metabolism and anaplerosis/cataplerosis in the ␤-cell. Individual reactions are referred to by their nos. when mentioned in the
text. At bottom left, the 5 steps of ␣-ketoisocaproic acid (KIC) metabolism indicated by no. 25 are its conversion to isovaleryl-CoA, methylcrotonyl-CoA,
methylglutaconyl-CoA, hydroxymethylglutaryl-CoA, and acetyl-CoA plus acetoacetate catalyzed by, respectively, the branched-chain ketoacid dehydrogenase
complex, isovaleryl-CoA dehydrogenase, methylcrotonyl-CoA carboxylase, methylglutaconyl-CoA hydratase, and hydroxymethylglutaryl-CoA lyase.
Invited Review
E6
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
EXPORT OF CITRIC ACID CYCLE INTERMEDIATES
FROM MITOCHONDRIA
When pyruvate is supplied to suspensions of ␤-cell mitochondria, the export of malate is increased markedly (64, 65),
and the export of other citric acid cycle intermediates increases
to various extents. Although the export of citrate is not increased as much as malate, evidence from intact cell experiments suggests that anaplerosis of citrate is necessary for
insulin secretion (19, 29, 30, 97, 102). We observed that most
of the citrate and malate are extramitochondrial in suspensions
of ␤-cell mitochondria (61), and others have previously made
the same observation with mitochondria from other tissues (44,
113). The extramitochondrial location of citrate may enable it
to act as a communicator among individual mitochondria to
synchronize their metabolism with one another and to synchronize mitochondrial processes with other cellular processes
(73), as discussed in the last section of this perspective.
The high rate of carboxylation of pyruvate in the ␤-cell
indicates that it is possible that each citric acid cycle intermediate is synthesized in amounts that exceed its consumption in
the citric acid cycle, and the excess is exported from the
mitochondria to the cytosol, where it has a role in stimulating
or supporting insulin secretion. [Obviously, some intermediates cannot cross the mitochondrial inner membrane and are
exported in the form of another intermediate and are converted
back to the same intermediate outside the mitochondria. For
example, oxaloacetate cannot cross the mitochondrial inner
membrane but can be exported as malate, fumarate, citrate, or
isocitrate (Figs. 2 and 4, reactions 10, 9, 22, 12, 13, and 26),
and acetyl-CoA is also not transported as such but can be
transported as citrate (Figs. 2 and 4, reaction 13).] The following sections discuss the possible extramitochondrial roles for
each cycle intermediate. Less will be said about well-studied
extramitochondrial mechanisms and more will be said about
possible actions for which evidence is emerging.
Malate. The conversion of oxaloacetate to malate and the
export of malate to the cytosol form a pyruvate-malate shuttle
(61), permitting the export of NADPH equivalents to the
cytosol. Malate and NADP are converted to NADPH and
AJP-Endocrinol Metab • VOL
pyruvate by malic enzyme. Pyruvate can then reenter mitochondrial pools (Fig. 4, reactions 10, 12, 1, and 2). The
potential roles of NADPH are depicted (see Fig. 6) and discussed below.
Citrate and isocitrate. The citrate-pyruvate shuttle (21, 29,
30, 102, 103) utilizes portions of the malate-pyruvate shuttle
and additional pathways to export both NAD equivalents and
NADPH equivalents from mitochondria. Exported citrate is
cleaved to acetyl-CoA and oxaloacetate by ATP citrate lyase in
the cytosol. Cytosolic malate dehydrogenase catalyzes the
conversion of exported oxaloacetate and cytosolic NADH to
malate and NAD, thus exporting NAD equivalents from the
mitochondria. The malate can be converted to pyruvate by
malic enzyme, thus exporting NADPH equivalents to the
cytosol (Fig. 4, reactions 13, 11, and 12). The acetyl-CoA can
be carboxylated to malonyl-CoA, which can be used in the
synthesis of lipids. Malonyl-CoA itself is believed also to have
a signaling role (12, 18, 23, 29, 30, 102, 103) because it inhibits
carnitine palmitoyl-CoA transferase-1, an enzyme that is required for the transport of long-chain acyl-CoAs into mitochondria, where they are metabolized. Inhibition of this enzyme should increase the level of long-chain acyl-CoAs in the
cytosol, where these molecules are believed to have numerous
signaling activities (Fig. 5). The proposed signaling roles of
long-chain acyl-CoAs include influences on the KATP channel,
glucokinase, ATPases, and vesicular trafficking (18). The malonyl-CoA hypothesis, originally championed by the late Dennis McGarry (Chen et al., Ref. 15) and by Corkey and coworkers (18, 19) and Prentki et al. (97), is one of the more popular
and well-studied hypotheses about the role of anaplerotic
products in insulin secretion. Evidence in support of this
hypothesis abounds, and, as with all popular hypotheses, there
is some evidence against it (18, 39, 90). Many excellent
reviews of this hypothesis have been published (18, 23, 96).
␣-Ketoglutarate. ␣-Ketoglutarate, when it is exported from
mitochondria, can act as a transporter of oxidizing equivalents
of NAD out of mitochondria in the malate-aspartate shuttle
(Fig. 4, reactions 10, 11, 23, and 16) and of NADP into
mitochondria in the isocitrate shuttle, which transports
NADPH out of mitochondria (Fig. 4, reactions 5, 15, 23, 11,
10, and 16). There are no fuel secretagogues that cannot
produce ␣-ketoglutarate, as described below. ␣-Ketoglutarate
almost certainly has a fuel function, but evidence is emerging
that ␣-ketoglutarate has signaling roles beyond its acting as a
metabolite in the citric acid cycle and as a precursor for
anaplerosis of other cycle intermediates.
␣-Ketoglutarate is a substrate for ␣-ketoglutarate hydroxylases (dioxygenases). These enzymes use ferrous ion, molecular oxygen, and a reducing agent such as ascorbate to catalyze
hydroxylation of prolyl, lysyl, or aspartyl (and asparagine)
residues in proteins. Phytanoyl-CoA hydroxylase is also a
member of this enzyme family.
By using RT-PCR, we have detected transcripts for proline
and lysine hydroxylases as well as phytanoyl-CoA hydroxylase
in human and rat pancreatic islets and INS-1 cells. We have
also found enzyme activity and immunoreactivity for prolyl
hydroxylases in human and rat pancreatic islets and INS-1
cells. On the basis of our own RT-PCR studies and from
histochemical studies of the pancreas performed by J. Dinchuk
(personal communication), very little or no aspartyl hydroxylase is present in the ␤-cell. Although the ␣-ketoglutarate
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
conversion of malate to oxaloacetate (Fig. 4, reaction 10), does
not generate mitochondrial NADH. Succinate-derived oxaloacetate can then condense with acetyl-CoA derived from the
pyruvate (Fig. 4, reaction 3) to form any citric acid cycle
intermediate (58, 63, 64, 86). Interestingly, esters of other citric
acid cycle intermediates do not stimulate insulin release (75).
A possible explanation for this fact is that succinate is ideally
situated among metabolic pathways to supply both NADPH
and mevalonate as discussed below (see SUCCINATE MECHANISM).
Fatty acids. The consensus about the effect of fatty acids
such as palmitate on insulin release is that they potentiate
glucose-induced insulin secretion when added acutely to islets,
but they cannot stimulate insulin release by themselves (18, 34,
96). The long-term effect of incubation of islets with palmitate
is a reduction in glucose-induced insulin release (the lipotoxicity hypothesis) (9, 34, 92, 114). Fatty acids are metabolized
to acetyl-CoA, (Fig. 2), which can be metabolized in the citric
acid cycle, but acetyl-CoA cannot be anaplerotic unless there is
a source of oxaloacetate to combine with it. Perhaps this is why
fatty acids do not, by themselves, stimulate insulin release.
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
E7
hydroxylases are known to participate in chronic processes,
such as modification of collagen, or nonacute regulatory functions, such as the modification by prolyl hydroxylases of the
transcription of a number of genes regulated by the hypoxiainducible factor (HIF) system, we have evidence that these
hydroxylases participate in acute processes in the ␤-cell. Inhibitors of these enzymes decrease insulin release from islets in
proportion to their ability to inhibit the hydroxylation of
endogenous substrates as well as of generic peptides by extracts of ␤-cells. These data suggest that hydroxylation of
␤-cell proteins is occurring during fuel-induced insulin secretion.
LESSONS LEARNED FROM MOUSE ISLETS
Differences between mouse and rat pancreatic islets in their
responses to esters of succinate provide additional evidence
that anaplerosis of ␣-ketoglutarate or citrate and isocitrate is
necessary for insulin secretion. Succinic acid methyl esters are
potent insulin secretagogues in rat pancreatic islets, but not in
mouse pancreatic islets, which lack malic enzyme and cannot
form pyruvate from the succinate hydrolyzed from these esters
(63). Thus, in the mouse islet, succinate metabolism can
produce fumarate, malate, and oxaloacetate in the forward
direction of the citric acid cycle but not acetyl-CoA, from
which citrate, isocitrate, and ␣-ketoglutarate can be formed
(reactions 8 and 9 in Fig. 4 can occur, but reaction 12 plus
reactions 1– 6 cannot occur). Because the succinate thiokinase
reaction is reversible, succinate can be converted to succinylCoA in the reverse direction of the citric acid cycle (Fig. 4,
reaction 6). However, the irreversibility of the ␣-ketoglutarate
dehydrogenase reaction (Fig. 4, reaction 6) prevents succinylCoA from being converted to ␣-ketoglutarate, isocitrate, or
citrate. Glucose and any secretagogue that augments ␣-ketoglutarate production are equally potent in mouse islets and rat
islets. Isocitrate and citrate can be formed from ␣-ketoglutarate
in the reverse of the isocitrate dehydrogenase and aconitase
reactions (Fig. 4, reactions 5 and 4 or 15 and 26). Therefore,
anaplerosis of one or all of the intermediates (citrate, isocitrate,
and ␣-ketoglutarate) must be necessary for insulin secretion.
AJP-Endocrinol Metab • VOL
IS GLUTAMATE A MESSENGER IN THE ␤-CELL?
It has been proposed that glutamate formed from the amination of ␣-ketoglutarate in the reverse direction of the glutamate dehydrogenase reaction signals insulin secretion, and
elegant imaging methods have shown that glutamate might
have stimulatory effects on insulin secretory granules (84).
However, almost all studies of glutamate indicate that glutamate is a fuel secretagogue from which other metabolites can
be formed and not an anaplerotic product in the ␤-cell. Our
opinion is that, if glutamate is a messenger in insulin secretion,
it must be recruited from preexisting intracellular stores because the preponderance of evidence indicates that ␤-cell
glutamate is not increased by insulin secretagogues. Several
laboratories have been unable to show that glucose, the most
potent insulin secretagogue, significantly increases glutamate
in islets or INS-1 cells (8, 64, 65, 72, 87). In addition, the basal
level of glutamate is very high in the islet (64, 72), as in many
tissues, and it seems unlikely that increasing it to even higher
levels would create a signal. Perhaps the strongest evidence
against glutamate being a signal is that glutamine by itself
cannot stimulate insulin release from islets, even though it
increases islet glutamate levels up to 10-fold (53, 64, 65, 72, 87).
Consideration of the kinetic properties of glutamate dehydrogenase also suggests that net synthesis of glutamate is
unlikely. ␣-Ketoglutarate is inhibitory to glutamate dehydrogenase, indicating that flux through the reaction catalyzed by
the enzyme should be in the direction of ␣-ketoglutarate and its
removal by metabolism. In addition, several human patients
with hyperinsulinism, hyperammonemia, and hypoglycemia
have been reported to have a mutation in the GTP-binding
region of glutamate dehydrogenase (106, 107), which results in
severely decreased allosteric inhibition of glutamate dehydrogenase by GTP. This results in constitutive activation of islet
glutamate dehydrogenase and confirms the important role of
the enzyme in insulin secretion. Maintaining normal islets in
the presence of glucose inhibits leucine-induced insulin release
(31, 51, 74, 79), probably by increasing GTP (106, 107) and
inhibition of glutamate dehydrogenase. Glucose would not
stimulate insulin secretion via glutamate dehydrogenase if it
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
Fig. 5. Mitochondrially synthesized citrate supplies equivalents of acetyl-CoA, malonyl-CoA, NAD, and NADPH to the cytosol. Extramitochondrial citrate is
converted to acetyl-CoA and oxaloacetate by ATP-citrate lyase (ATP-CL). Citrate also activates acetyl-CoA carboxylase (ACC), the enzyme that forms
malonyl-CoA from acetyl-CoA. Malonyl-CoA itself has a signaling role by inhibiting carnitine palmitoyl-CoA transferase 1 (CPT-1), which is involved in the
transport of fatty acids into mitochondria, where they are metabolized. CPT-1 inhibition raises the level of long-chain (LC)-acyl-CoAs in the cytosol, where they
are believed to have signaling roles. In addition, lipids can be synthesized from malonyl-CoA. Lipids also have signaling roles, for example, by acylating proteins.
Oxaloacetate produced by ATP-CL can be converted to malate (thus exporting NAD equivalents from mitochondria), which can then be converted to pyruvate
(thus exporting NADPH equivalents from mitochondria). Pyruvate can reenter mitochondria pools (the pyruvate-malate shuttle; Ref. 63), as shown in Figure 4.
FFA, free fatty acid; SREBP-1c, sterol regulatory element-binding protein-1c.
Invited Review
E8
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
caused inhibition of the enzyme. This is consistent with evidence obtained from our studies of ␤-cell mitochondria that
indicates that flux through glutamate dehydrogenase is quiescent during glucose-induced insulin secretion (72).
NAD(H) SHUTTLES
NADPH
The production of NADPH equivalents in mitochondria
might be especially important in the ␤-cell because many
studies have shown that, in rat and mouse pancreatic islets,
very little glucose is metabolized through the pentose phosphate shunt (see Ref. 59 and references therein), the pathway
that forms a large amount of NADPH in the cytosol in many
cells. NADPH equivalents can be exported to the cytosol as
malate (Fig. 4, reactions 2, 10, and 12) and citrate (Fig. 4,
reactions 13, 11, 12, 1, 2, and 3) in human and rat islets and as
isocitrate (Fig. 4, reactions 1, 2, 3, 4, 15, 23, 11, and 10) in
islets of the mouse, rat, and human. In 1972, Ammon and
Steinke (3) showed that giving 6-aminonicotinamide (6-AN), a
compound that forms a metabolically inactive analog of
NADPH in vivo, caused hyperglycemia in rats. Furthermore,
glucose-induced (3) and amino acid-induced insulin releases
(67) were inhibited in islets isolated from the 6-AN-treated
rats. In addition, in 1977, Watkins and Moore (115) showed
that NADPH is taken up by insulin granules of the toadfish islet
and stimulates insulin release. Because of these findings, the
potential roles of NADPH in insulin secretion are summarized
(Fig. 6).
NADPH is a substrate for glutathione reductase, which is
plentiful in the pancreatic islet (4, 48). Whether this enzyme
plays a role in signaling insulin secretion or is present in the
␤-cell only to preserve the redox state of cellular thiols is
unknown. In most cells, the enzyme is present in the cytosol,
mitochondria, and endoplasmic reticulum, where its products
very likely interact with the thioredoxin and thioredoxin reductase reactions also using NADPH as a cofactor. Numerous
enzymes are likely regulated by the redox state of protein thiol
groups. These include many of the glycolytic enzymes, which
are more active when the ratio of reduced glutathione to
oxidized glutathione is high (42, 45, 66).
N-ethylmaleimide (NEM) inactivates thiol groups. The fact
that NEM-sensitive factor (NSF), a regulator of soluble NSF
attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells, including ␤-cells,
suggests that thiol status could regulate movement of insulin
granules. Glutathione also interacts with glutaredoxin, and the
ratio of reduced to oxidized glutathione (GSH/GSSG) influences the ratio of reduced to oxidized glutaredoxin. Evidence is
AJP-Endocrinol Metab • VOL
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
The activities of the mitochondrial NAD(H) shuttles are
particularly high in the ␤-cell, indicating they are important for
insulin secretion, and have been studied extensively. The
glycerol-phosphate shuttle (Fig. 3) and the malate-aspartate
shuttle (Fig. 4, reactions 10, 16, 11, and 23) oxidize NADH
formed in the cytosol by glycolysis and export NAD equivalents back to the cytosol (10, 11, 26, 56, 57, 77, 78, 101). The
malate-aspartate shuttle partially overlaps or intersects with
several anaplerotic pathways (Fig. 4), such as glutamate metabolism and the citrate and isocitrate shuttles, but the glycerolphosphate shuttle does not.
emerging that glutathionylation reversibly influences activities
of enzymes. For example, glutalthionylation inactivates ␣-ketoglutarate dehydrogenase, and glutaredoxin facilitates the
GSH-dependent recovery of the enzyme activity (94). The
GSH/GSSG ratio also influences the activity of protein disulfide isomerase (PDI) (Fig. 6). PDI is known to be plentiful in
rat ␤-cells, and we have detected transcripts for thioredoxin,
thioredoxin reductase, and glutaredoxin in mouse islets with
DNA microarrays (Brown LJ, unpublished data).
PDI has a number of functions, including influencing protein
folding and acting as a chaperone for proteins synthesized in
the endoplasmic reticulum. Protein folding in the endoplasmic
reticulum is necessary for packaging of insulin into secretory
granules. PDI is also the ␤-subunit for the prolyl hydroxylase
family of enzymes that use ␣-ketoglutarate as a substrate, as
mentioned above.
Interestingly, indirect evidence is emerging to suggest a role
for thioredoxin in insulin secretion. High glucose is known to
blunt the responsiveness of the ␤-cell to fuel secretagogues.
Shalev et al. (105) found that transcripts for thioredoxininteracting protein (Txnip), a protein that binds thioredoxin and
lowers its activity, are induced 11-fold in human islets after
prolonged incubation in the presence of high glucose. In
addition, Hui et al. (40) have shown that mice deficient in
Txnip have increased plasma insulin levels due to increased
insulin secretion and low blood glucose levels during brief
starvation. These inverse correlations between Txnip and insulin levels are consistent with the idea that thioredoxin activates or enhances insulin secretion.
NADPH is required for elongation of fatty acids, and evidence is emerging that fatty acid synthesis occurs in ␤-cells, as
mentioned below. NADPH is a substrate for stearoyl-CoA
desaturases, and mRNA transcripts for these enzymes have
been found in islets (30). Our laboratory (48) has found their
enzyme activity in islets, and Ramanadham et al. (99) have
identified glycerolphospholipid products of these desaturases
in INS-1 cells and islets (Fig. 6).
NADPH is a substrate for 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase, an enzyme in the pathway of mevalonate formation that is needed for protein isoprenylation.
Kowluru and coworkers (2, 47, 89) have found evidence for
protein isoprenylation in islets. Mevalonate is believed to have
additional unknown signaling properties, as discussed in SUCCINATE MECHANISM (Fig. 7).
NADPH is also a substrate for quinone reductases and
aldose/aldehyde reductases. These enzyme activities are very
high in the islet, but they are not likely directly involved in
insulin secretion, because potent inhibitors of these enzymes
do not inhibit insulin release (48). It is possible these enzymes
are present in islets to protect the cell against damaging
peroxides. However, interestingly, voltage-gated K⫹ channel
␤-subunits belong to the aldehyde reductase family of proteins
and are also present in the ␤-cell. Like the aldose and aldehyde
reductases, these K⫹ channel ␤-subunits are ⬃30,000 Da in
size and possess an oxidoreductase-binding site similar to that
for NADPH in aldehyde reductases. We have found transcripts
and immunoreactivity for these proteins in both human and rat
pancreatic islets and in INS-1 cells (16, 48). Gulbis et al. (35)
have crystallized these ␤-subunits and studied them by X-ray
diffraction and observed NADPH to be present within the
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
E9
native protein. This suggests the pyridine dinucleotide phosphate has the potential to act as a redox switch to regulate
channel activity. Although the ␤-subunits have never been
shown to possess aldehyde reductase enzyme activity per se,
Bahring et al. (7) have mutated the substrate-binding site of the
protein and shown that this alters the gating activity of the
␣-subunit (pore) of the channel. In the ␤-cell, these channels
repolarize the plasma membrane after it has been depolarized
by closure of the KATP channel. Therefore, inactivating the
voltage-gated channels should prolong the repolarization phase
of the plasma membrane current and potentiate insulin secretion. Interestingly, it was recently shown that increasing the
NADPH/NADP ratio in pancreatic ␤-cells prolongs the inactivation phase of these channels (82) (Fig. 6).
Finally, nitric oxide synthases use NADPH and are present
in islets. Recent evidence suggests that nitric oxide synthases
are directly involved in insulin secretion (91).
AJP-Endocrinol Metab • VOL
LIPID SYNTHESIS
As mentioned above, citrate exported from mitochondria is
a source of acetyl-CoA and malonyl-CoA used in lipid synthesis (Fig. 4, reactions 13 and 14, and Fig. 5). Until recently,
traditional dogma held that lipid synthesis does not occur to a
great extent in the islet. However, indirect evidence is emerging to indicate that it does occur (9, 30, 102, 114). Flamez et al.
(30), using microarrays, found that glucose increases the level
of many mRNAs, such as those for the sterol regulatory
element-binding protein-1c (SREBP-1c), and multiple lipogenic enzymes, including those for the mevalonate pathway.
␤-cell SREBP-1c is increased in type 2 diabetes, and overexpression of it increases lipid synthesis, implicating the protein
in the pathogenesis of ␤-cell lipotoxicity that occurs in type 2
diabetes (114). Others have found evidence for acylation of
proteins involved with exocytosis, such as G proteins (47, 89,
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
Fig. 6. Possible signaling mechanisms of NADPH in the ␤-cell. NADPH equivalents can be exported from mitochondria as reduced metabolites (such as malate,
which can form NADPH from NADP in the malic enzyme reaction in the cytosol, or as isocitrate via the NADP isocitrate dehydrogenase reaction in the cytosol).
NADPH has been shown to be taken up by insulin granules and stimulate insulin release from toadfish islets (115). NADPH can partake in many enzyme reactions
in the cytosol, including thioredoxin (TRX) reductase and glutathione (GSSG) reductase, that influence processes such as protein folding. Activities of many
enzymes are also regulated via their sulfhydryl/disulfide status directly by the GSH/GSSG ratio and via protein disulfide isomerase (PDI). PDI also influences
protein folding. Glutathionation of proteins can regulate their activity and is influenced by glutaredoxin (GRX), as described in the text (see NADPH). NADPH
is also a substrate for fatty acid synthesis and for nitric oxide (NO) synthases. 6-Aminonicotinamide can block normal NADPH formation and inhibit insulin
secretion, as shown in Refs. 3 and 67. ERO1-L is a human protein that shares extensive homology with a yeast protein required for formation of disulfide bonds
in proteins including secretory proteins (14). NADPH is a substrate for stearoyl-CoA desaturases, which may increase unsaturated fatty acids in membranes and
facilitate insulin granule fusion with the plasma membrane. NADPH can act as a substrate for 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase to form
mevalonate, which is believed to have signaling roles according to the “succinate mechanism.” It can also act as a redox switch in ␤-subunits of voltage-gated
K⫹ channels, thus enabling an increased NADPH/NADP ratio or a reduced redox potential to potentiate insulin secretion.
Invited Review
E10
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
119) and other proteins in the ␤-cell (109, 118). Recently, Itoh
et al. (41) showed that free fatty acids, acting as a ligand and
without covalent attachment, can alter G protein activity.
Exactly how protein acylation affects insulin secretion is
largely unknown (Fig. 5).
Fuel-derived synthesis of cholesterol (95) and phospholipids
(100), including sphingolipids (50), may promote insulin exocytosis by stimulating insulin secretory granule packaging,
assembly, and movements as well as fusion of the granules
with the plasma membrane and extrusion of granular contents
into the circulation. Although the apparent necessity for
anaplerosis in the ␤-cell might suggest that synthesis of these
molecules is directly from the added fuel, it is currently not
known whether their formation is direct or whether the fuel
stimulates their synthesis and release from preexisting stores of
precursors. Membrane fluidity is regulated by the ratio of
cholesterol to phospholipids and the ratio of saturated to
unsaturated fatty acids incorporated into phospholipids (110).
Alteration of this ratio has been implicated in various diseases,
including type 2 diabetes (98). Stearoyl-CoA desaturases may
make membranes more fluid by increasing their content of
unsaturated fatty acids (98, 99), thus facilitating granule movement and fusion with the plasma membrane (Fig. 6).
AJP-Endocrinol Metab • VOL
SUCCINATE MECHANISM
We recently proposed the “succinate mechanism” of insulin
release (27). According to this mechanism, when mevalonate is
synthesized in islets, it or one of its metabolic products plays a
major role in triggering and/or supporting insulin release. This
concept is supported by experiments demonstrating that metabolites that are insulin secretagogues can also readily supply
HMG-CoA reductase with its substrate NADPH and precursors
for its other substrate, HMG-CoA (27, 28, 48, 51, 52, 61, 63,
64, 72) (Fig. 7).
Methyl esters of succinate are insulinotropic because they
enter the cell and are converted into succinate. The succinate
mechanism postulates that succinate is insulinotropic in rat
islets, because succinate is readily utilized for the production of
NADPH and pyruvate (Fig. 4, reactions 8, 9, and 12) (58, 63,
86). Acetyl-CoA produced from pyruvate by pyruvate dehydrogenase (Fig. 4, reaction 1) is then used by HMG-CoA
synthetase to generate HMG-CoA, which is reduced to mevalonate by NADPH in the HMG-CoA reductase reaction (Fig.
7, reactions 6, 7, 8, 10, 3, 11, and 4). Thus succinate is a source
of the two substrates of the HMG-CoA reductase reaction,
NADPH and HMG-CoA. This may explain why esters of
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
Fig. 7. The succinate mechanism of insulin release hypothesizes that all fuel secretagogues form NADPH and mevalonate. Individual reactions are referred to
by their nos. when mentioned in the text.
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
CITRATE OSCILLATIONS AS A SYNCHRONIZER OF
MITOCHONDRIA AND A COORDINATOR OF
MITOCHONDRIAL AND CELLULAR METABOLISM
It is well known that ␤-cell glycolysis, plasma membrane
activity, and ATP/ADP ratios oscillate in the ␤-cell and that
these processes are likely in synchrony with oscillations of
insulin release (20, 22, 32, 43, 93). If mitochondrial factors
influence cellular metabolism, such as the ATP/ADP ratio
modifying the activity of the KATP channel and the activities of
contractile proteins that propel insulin granules to the plasma
AJP-Endocrinol Metab • VOL
membrane, then ATP production and other signaling processes,
including anaplerosis, need to be synchronized.
Recent work in our laboratory (73) has shown that citrate
oscillates in suspensions of mitochondria from liver, pancreatic
islets, and INS-1 cells supplied with pyruvate and in intact
INS-1 cells supplied with glucose. Citrate oscillations were
synchronous with oscillations in ATP and NAD (73). Interestingly, in suspensions of mitochondria from tissues as diverse as
liver (113), pancreatic islets (61), and heart (49), most of the
citrate is extramitochondrial. The extramitochondrial location
of citrate suggests that citrate itself can establish communication among mitochondria within a cell (Fig. 8). Because citrate
inhibits its own synthesis by inhibiting citrate synthase (Fig. 4,
Fig. 8. Oscillations in citrate and ATP permit mitochondrial metabolism to be
interlocked with oscillations in glycolysis. When pyruvate is added to ␤-cell or
liver mitochondria, or glucose is added to INS-1 cells, citrate levels oscillate.
The fact that citrate levels oscillate in suspensions of ␤-cell and liver mitochondria and in INS-1 cells (73), and that the majority of citrate is extramitochondrial in suspensions of mitochondria from many tissues (49, 61, 113),
suggests that citrate itself can establish communication among mitochondria in
a cell. A mitochondrion receiving added pyruvate or pyruvate generated from
added glucose will increase citrate production and its export out of the
mitochondria. Citrate inhibits its own synthesis so that, when the citrate level
is high, its uptake and metabolism in mitochondria, combined with inhibition
of its own synthesis, will begin to lower its level. When its level is low enough
to relieve inhibition of its synthesis, its level will begin to increase again,
permitting another oscillation in its level. In an intact cell, high citrate and ATP
inhibit a cytosolic enzyme phosphofructokinase (PFK), believed to be the
major pacemaker of glycolytic oscillations. Inhibited glycolysis will decrease
the supply of pyruvate to mitochondria. Thus the decreased supply of pyruvate
in the cytosol is coordinated with the transition from high citrate and ATP to
lower citrate and ATP. Metabolism of citrate in the citric acid cycle will also
lower the mitochondrial NAD/NADH ratio by increasing the reduction of
NAD to NADH, and this will raise the ATP/ADP ratio. Because fructose
bisphosphate (FDP) is an activator of pyruvate kinase, which catalyzes the
terminal step in glycolysis, the FDP formed in the PFK reaction further
contributes to oscillations in pyruvate formation and thus glycolysis (66).
Overall, these reactions and probably additional reactions (44) link oscillations
of glycolysis in the cytosol with oscillations in mitochondrial anaplerosis and
energy production.
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
succinate, unlike other esters of citric acid cycle intermediates
or compounds structurally similar to succinate, are insulinotropic (28, 70, 71).
As was mentioned in previous sections, ␣-ketoglutarate can
be formed from all fuel secretagogues. However, most metabolites that increase ␣-ketoglutarate production are not insulinotropic. The succinate mechanism proposes that, to be insulinotropic, the product metabolite must be capable of increasing both HMG-CoA and NADPH production. This may
explain why enhancing the oxidative deamination of glutamate
by glutamate dehydrogenase results in enhanced insulin release
(28, 31, 36, 72, 89, 105), because both NADPH and ␣-ketoglutarate are produced in the glutamate dehydrogenase reaction. Thus leucine or leucine plus glutamine are insulinotropic
because leucine allosterically activates glutamate dehydrogenase, and glutamine provides glutamate, a substrate of glutamate dehydrogenase. On the other hand, increasing the rate of
conversion of glutamate to ␣-ketoglutarate by mitochondrial
aspartate aminotransferase is not necessarily accompanied by
enhanced insulin release, because this reaction does not directly increase NADPH production.
Leucine and KIC are the only amino acid and ketoacid,
respectively, that stimulate insulin release. According to the
succinate mechanism, this could be because both secretagogues can be metabolized to HMG-CoA and then mevalonate.
In addition, KIC can be transaminated to leucine, which can
activate glutamate dehydrogenase. Glutamate dehydrogenase
is the only known dehydrogenase that, when activated, is
accompanied by an increase in insulin release.
The succinate mechanism hypothesis might also explain
why glucose is the most potent insulin secretagogue. Glucose
metabolism forms pyruvate, which can then be utilized for the
synthesis of HMG-CoA and NADPH. HMG-CoA production
can be increased, because roughly one-half of glucose-derived
pyruvate is decarboxylated in the pyruvate dehydrogenase
reaction to acetyl-CoA, which is converted to substrate for
HMG-CoA synthetase. The other 50% of glucose-derived
pyruvate is converted to oxaloacetate by pyruvate carboxylation (59, 61), and this can increase the supply of acetoacetylCoA to HMG-CoA synthetase as a result of the combined
mitochondrial aspartate aminotransferase, ␣-ketoglutarate dehydrogenase, and succinyl-CoA acetoacetate transferase reactions (Fig. 7, reactions 18, 13, 2, and 3). In addition, acetoacetyl-CoA and oxaloacetate produced by decarboxylation and
carboxylation, respectively, of pyruvate would be utilized to
produce NADPH plus ␣-ketoglutarate in the combined citrate
synthase, aconitase, and NADPH isocitrate dehydrogenase
reactions (Fig. 4, reactions 3 and 4 or 26 plus 15).
E11
Invited Review
E12
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
ACKNOWLEDGMENTS
We thank Robert J. Gordon for excellent graphics work.
AJP-Endocrinol Metab • VOL
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-28348 and the Oscar C. Rennebohm Foundation.
REFERENCES
1. Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JP 4th, Boyd
AE 3rd, Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J, and Nelson
DA. Cloning of the beta cell high-affinity sulfonylurea receptor: a
regulator of insulin secretion. Science 268: 423– 426, 1995.
2. Amin R, Chen HQ, Tannous M, Gibbs R, and Kowluru A. Inhibition
of glucose- and calcium-induced insulin secretion from beta TC3 cells by
novel inhibitors of protein isoprenylation. J Pharmacol Exp Ther 303:
82– 88, 2002.
3. Ammon HP and Steinke J. 6-Aminonicotinamide (6-AN) as a diabetogenic agent. In vitro and in vivo studies in the rat. Diabetes 21:
143–148, 1972.
4. Anjaneyulu K, Anjaneyulu R, Sener A, and Malaisse WJ. The
stimulus-secretion coupling of glucose-induced insulin release. Thiol:
disulfide balance in pancreatic islets. Biochimie 64: 29 –36, 1982.
5. Ashcroft FM. Ca2⫹ channels and excitation-contraction coupling. Curr
Opin Cell Biol 3: 671– 675, 1991.
6. Atkinson DE. The control of citrate synthesis and breakdown. In: Citric
Acid Cycle: Control and Compartmentation, edited by Lowenstein JM.
New York: Marcel Dekker, 1969, p. 137–162.
7. Bahring R, Milligan CJ, Vardanyan V, Engeland B, Young BA,
Dannenberg J, Waldschutz R, Edwards JP, Wray D, and Pongs O.
Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kv beta subunits. J Biol Chem 276: 22923–
22929, 2001.
8. Bertrand G, Ishiyama N, Nenquin M, Ravier MA, and Henquin JC.
The elevation of glutamate content and the amplification of insulin
secretion in glucose-stimulated pancreatic islets are not causally related.
J Biol Chem 277: 32883–32891, 2002.
9. Briaud I, Harmon JS, Kelpe CL, Segu VB, and Poitout V. Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent
esterification of fatty acids into neutral lipids. Diabetes 50: 315–321,
2001.
10. Brown LJ, Koza RA, Everett C, Reitman ML, Marshall L, Fahien
LA, Kozak LP, and MacDonald MJ. Normal thyroid thermogenesis,
but reduced viability and adiposity in mice lacking the mitochondrial
glycerol phosphate dehydrogenase. J Biol Chem 277: 32892–32898,
2002.
11. Brown LJ, Koza RA, Marshall L, Kozak LP, and MacDonald MJ.
Lethal hypoglycemic ketosis and glyceroluria in mice lacking both the
mitochondrial and the cytosolic glycerol phosphate dehydrogenases.
J Biol Chem 277: 32899 –32904, 2002.
12. Brun T, Roche E, Assimacopoulos-Jeannet F, Corkey BE, Kiro KH,
and Prentki M. Evidence for an anaplerotic/malonyl-CoA pathway in
pancreatic beta-cell nutrient signaling. Diabetes 45: 190 –198, 1996.
13. Brun T, Roche E, Kim K, and Prentki M. Glucose regulates acetylCoA carboxylase gene expression in a pancreatic ␤-cell line (INS-1).
J Biol Chem 268: 18905–18911, 1993.
14. Cabibbo A, Pagani M, Fabbri M, Rocchi M, Farmery MR, Bulleid
NJ, and Sitia R. ERO1-L, a human protein that favors disulfide bond
formation in the endoplasmic reticulum. J Biol Chem 275: 4827– 4833,
2000.
15. Chen S, Ogawa A, Ohneda M, Unger RH, Foster DW, and McGarry
JD. More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic beta-cell signaling.
Diabetes 43: 878 – 883, 1994.
16. Chouinard SW, Lu F, Ganetzky B, and MacDonald MJ. Evidence for
voltage-gated potassium channel ␤-subunits with oxidoreductase motifs
in human and rodent pancreatic beta cells. Receptors Channels 7:
237–243, 2000.
17. Comte B, Vincent G, Bouchard B, Benderdour M, and Des Rosiers
C. Reverse flux through cardiac NADP⫹-isocitrate dehydrogenase under
normoxia and ischemia. Am J Physiol Heart Circ Physiol 283: H1505–
H1514, 2002.
18. Corkey BE, Denney JT, Yaney GC, Tornheim K, and Prentki M. The
role of long-chain fatty acyl-CoA esters in beta-cell signal transduction.
J Nutr 130: 299S–304S, 2000.
19. Corkey BE, Glennon MC, Chen KS, Deeney JT, Matschinsky FM,
and Prentki M. A role for malonyl-CoA in glucose-stimulated insulin
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
reaction 3), and because succinyl-CoA, the product of the
␣-ketoglutarate dehydrogenase reaction (Fig. 4, reaction 6),
also inhibits citrate synthase (6, 116), oscillations in citrate
could synchronize the activity of the TCA cycle and ATP
synthesis among individual mitochondria in a cell. There are
theoretical reasons and also experimental data from decades of
research on mitochondria to indicate that the NAD/NADH and
ATP/ADP ratios, which are tightly coupled to each other, can
regulate or are coregulated by the same factors that regulate
each phase of citrate oscillations (see references cited in Ref.
73 for a more detailed explanation). It seems logical that
cytosolic metabolism, i.e., glycolysis, should be coordinated
with mitochondrial metabolism so that both are oscillating in
synchrony to permit the most efficient utilization of substrates
provided by glycolysis and the resulting energy production and
anaplerotic messengers supplied by mitochondria. Phosphofructokinase is believed to be the major pacemaker of glycolysis in the ␤-cell (111, 112, 120), as in many cells. This
enzyme is inhibited by citrate and ATP, and a constant increased level of citrate would inhibit the enzyme and thus
inhibit glycolysis. Continuous inhibition of glycolysis would
be counterproductive for glucose-induced insulin secretion.
Therefore, it makes a great deal of sense from the viewpoint of
cellular metabolism for inhibition of phosphofructokinase to be
intermittent. It is conceivable that oscillations in citrate and
ATP could, by influencing phosphofructokinase activity, coordinate oscillations in glycolysis and regulate the supply of
pyruvate, the terminal metabolite of aerobic glycolysis, to
mitochondria (Fig. 8). In addition, oscillating levels of fructose
bisphosphate, the product of phosphofructokinase, can directly
control pyruvate formation because fructose bisphosphate is an
activator of pyruvate kinase, the terminal enzyme of the glycolytic pathway (66). These may be only a few of the ways in
which oscillations are coordinated. There is evidence that the
control of glucose metabolism in non-␤-cells is distributed
across many enzyme reactions (44) and that the levels of many
metabolites can influence oscillatory activities of many metabolic enzymes in the ␤-cell (66).
In conclusion, evidence overwhelmingly supports the idea
that anaplerosis is important for insulin secretion and that the
formation and export of anaplerotic products from mitochondria correlate more with insulin secretion than does energy
production (46, 55, 58, 59, 61, 64). However, very little is
known about the extramitochondrial actions of these products,
and this makes the study of cataplerosis in the ␤-cell an
exciting and fruitful area for future research. Recent exciting
work even demonstrates unexpected direct signaling roles for
two citric acid cycle intermediates, ␣-ketoglutarate and succinate, in the kidney. At ⬍100 ␮M concentrations, these two
metabolites act as ligands for kidney G protein-coupled receptors. In addition, infusions of succinate into mice raise their
blood pressure via the renin-angiotensin system (37). Clearly,
there remains a lot to be learned about the role of the citric acid
cycle intermediates as intracellular and possibly even intercellular messengers.
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
20.
21.
22.
23.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
AJP-Endocrinol Metab • VOL
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
Shinohara T, Hinuma S, Fujisawa Y, and Fujino M. Free fatty acids
regulate insulin secretion from pancreatic beta cells through GPR40.
Nature 422: 173–176, 2003.
Johnson GS, Kayne MS, and Deal WC Jr. Metabolic control and
structure of glycolytic enzymes. 8. Reversal of the dissociation of rabbit
muscle pyruvate kinase into unfolded subunits. Biochemistry 8: 2455–
2462, 1969.
Jung SK, Kauri LM, Qian WJ, and Kennedy RT. Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free
Ca2⫹ in single islets of Langerhans. J Biol Chem 275: 6642– 6650, 2000.
Kashiwaya Y, Sato K, Tauchiya N, Thomas S, Fell DA, Veech RL,
and Passonneau JV. Control of glucose utilization in working perfused
rat heart. J Biol Chem 269: 25502–25514, 1994.
Kemp RG. Allosteric properties of muscle phosphofructokinase. II.
Kinetics of native and thiol-modified enzyme. Biochemistry 8: 4490 –
4496, 1969.
Khan A, Ling ZC, and Landau BR. Quantifying the carboxylation of
pyruvate in pancreatic islets. J Biol Chem 271: 2539 –2542, 1996.
Kowluru A, Chen HQ, and Tannous M. Novel roles for the rho
subfamily of GTP-binding proteins in succinate-induced insulin secretion
from ␤TC3 cells: further evidence in support of the succinate mechanism
of insulin release. Endocr Res 29: 363–376, 2003.
Laclau M, Lu F, and MacDonald MJ. Enzymes in pancreatic islets that
use NADP(H) as a cofactor including evidence for a plasma membrane
aldehyde reductase. Mol Cell Biochem 225: 151–160, 2001.
LaNoue K, Nicklas WJ, and Williamson JR. Control of citric acid
cycle activity in rat heart mitochondria. J Biol Chem 245: 102–111, 1970.
Laychock SG, Tian Y, and Sessanna SM. Endothelial differentiation
gene receptors in pancreatic islets and INS-1 cells. Diabetes 52: 1986 –
1993, 2003.
Li C, Najafi H, Daikhin Y, Nissim IB, Collins HW, Yudkoff M,
Matschinsky FM, and Stanley CA. Regulation of leucine-stimulated
insulin secretion and glutamine metabolism in isolated rat islets. J Biol
Chem 278: 2853–2858, 2003.
Li G, Regazzi R, Roche E, and Wollheim CB. Blockade of mevalonate
production by lovastatin attenuates bombesin and vasopressin potentiation of nutrient-induced insulin secretion in HIT-T15 cells: probable
involvement of small GTP-binding proteins. Biochem J 289: 379 –385, 1993.
Liu YJ, Cheng H, Drought H, MacDonald MJ, Sharp GWG, and
Straub SG. Activation of the KATP channel-independent signaling pathway by the nonhydrolyzable analog of leucine, BCH. Am J Physiol
Endocrinol Metab 285: E380 –E389, 2003.
Louis NA and Witters LA. Glucose regulation of acetyl-CoA carboxylase in hepatoma and islet cells. J Biol Chem 267: 2287–2293, 1992.
Lu D, Mulder H, Zhao P, Burgess SC, Jensen MV, Kamzolova S,
Newgard CB, and Sherry AD. 13C NMR isotopomer analysis reveals a
connection between pyruvate cycling and glucose-stimulated insulin
secretion (GSIS). Proc Natl Acad Sci USA 99: 2708 –2713, 2002.
MacDonald MJ. High content of mitochondrial glycerol 3-phosphate
dehydrogenase in pancreatic islets and its inhibition by diazoxide. J Biol
Chem 256: 8287– 8290, 1981.
MacDonald MJ. Evidence for the malate-asparate shuttle in pancreatic
islets. Arch Biochem Biophys 213: 643– 649, 1982.
MacDonald MJ. Metabolism of the insulin secretagogue methyl succinate by pancreatic islets. Arch Biochem Biophys 300: 201–205, 1993.
MacDonald MJ. Estimates of glycolysis, pyruvate (de)carboxylation,
pentose phosphate pathway and methyl succinate metabolism in incapacitated pancreatic islets. Arch Biochem Biophys 305: 205–214, 1993.
MacDonald MJ. Glucose enters mitochondrial metabolism via both
carboxylation and decarboxylation of pyruvate in pancreatic islets. Metabolism 42: 1229 –1231, 1993.
MacDonald MJ. Feasibility of a mitochondrial pyruvate malate shuttle
in pancreatic islets: further implication of cytosolic NADPH in insulin
secretion. J Biol Chem 270: 20051–20058, 1995.
MacDonald MJ. Influence of glucose on pyruvate carboxylase expression in pancreatic islets. Arch Biochem Biophys 319: 128 –132, 1995.
MacDonald MJ. Differences between mouse and rat pancreatic islets:
succinate responsiveness, malic enzyme, and anaplerosis. Am J Physiol
Endocrinol Metab 283: E302–E310, 2002.
MacDonald MJ. The export of metabolites from mitochondria and
anaplerosis in insulin secretion. Biochim Biophys Acta 1619: 77– 88, 2002.
MacDonald MJ. Export of metabolites from pancreatic islet mitochondria as a means to study anaplerosis in insulin secretion. Metabolism 52:
993–998, 2003.
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
24.
secretion from clonal pancreatic ␤-cells. J Biol Chem 264: 21608 –21612,
1989.
Corkey BE, Tornheim K, Deeney JT, Glennon MC, Parker JC,
Matschinsky FM, Ruderman NB, and Prentki M. Linked oscillations
of free Ca2⫹ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract.
J Biol Chem 263: 4254 – 4268, 1988.
Curi R, Carpinelli AR, and Malaisse WJ. Hexose metabolism in
pancreatic islets: pyruvate carboxylase activity. Biochimie 73: 583–586,
1991.
Deeney JT, Kohler M, Kubik K, Brown G, Schultz V, Tornheim K,
Corkey BE, and Berggren PO. Glucose-induced metabolic oscillations
parallel those of Ca2⫹ and insulin release in clonal insulin-secreting cells.
A multiwell approach to oscillatory cell behavior. J Biol Chem 276:
36946 –36950, 2001.
Deeney JT, Prentki M, and Corkey BE. Metabolic control of beta-cell
function. Semin Cell Dev Biol 11: 267–275, 2000.
Des Rosiers C, Fernandez CA, David F, and Brunengraber H.
Reversibility of the mitochondrial isocitrate dehydrogenase reaction in
the perfused rat liver. J Biol Chem 269: 27179 –27182, 1994.
Erecinska M and Nelson D. Activation of glutamate dehydrogenase by
leucine and its nonmetabolizable analogue in rat brain synaptosomes.
J Neurochem 54: 1335–1343, 1990.
Eto K, Tsubamoto Y, Terauchi Y, Sugiyama T, Kishimoto T, Takahashi N, Yamauchi N, Kubota N, Murayama S, Aizawa T, Akanuma
Y, Aizawa S, Kasai H, Yazaki Y, and Kadowaki T. Role of NADH
shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion. Science 12: 981–955, 1999.
Fahien LA and MacDonald MJ. The succinate mechanism of insulin
release. Diabetes 51: 2669 –2676, 2002.
Fahien LA, MacDonald MJ, Kmiotek EH, Mertz RJ, and Fahien
CM. Regulation of insulin release by factors which also modify glutamate dehydrogenase. J Biol Chem 263: 13610 –13614, 1988.
Farfari S, Schulz V, Corkey B, and Prentki M. Glucose-regulated
anaplerosis and cataplerosis in pancreatic beta-cells: possible implication
of a pyruvate/citrate shuttle in insulin secretion. Diabetes 49: 718 –726,
2000.
Flamez D, Berger V, Kruhoffer M, Orntoft T, Pipeleers D, and
Schuit FC. Critical role for cataplerosis via citrate in glucose-regulated
insulin release. Diabetes 51: 2018 –2024, 2002.
Gao Z, Young RA, Li G, Najafi H, Buettger C, Sukumvanich SS,
Wong RK, Wolf BA, and Matschinsky FM. Distinguishing features of
leucine and ␣-ketoisocaproate sensing in pancreatic beta-cells. Endocrinology 144: 1949 –1957, 2003.
Gilon P, Ravier MA, Jonas JC, and Henquin JC. Control mechanisms
of the oscillations of insulin secretion in vitro and in vivo. Diabetes 51:
S144 –S151, 2002.
Giroix MH, Saulnier C, and Portha B. Decreased pancreatic islet
response to L-leucine in the spontaneously diabetic GK rat: enzymatic,
metabolic and secretory data. Diabetologia 42: 965–977, 1999.
Grill V, Sako Y, Ostenson CG, and Jalkannen P. Multiple abnormalities in insulin response to nonglucose nutrients in neonatally streptozotocin diabetic rats. Endocrinology 128: 2195–2203, 1991.
Gulbis JM, Mann S, and MacKinnon R. Structure of a voltagedependent K⫹ channel beta subunit. Cell 97: 943–952, 1999.
Gylfe E. Comparison of the effects of leucines, non-metabolizable
leucine analogues and other insulin secretagogues on the activity of
glutamate dehydrogenase. Acta Diabetol 13: 20 –24, 1976.
He W, Miao FJP, Lin DCH, Schwandner RT, Wang Z, Gao J, Chen
JL, Tian H, and Ling L. Citric acid cycle intermediates as ligands for
orphan G-protein-coupled receptors. Nature 429: 188 –193, 2004.
Hedescov CJ, Capito K, and Thams P. Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2⫹ activation. Biochim Biophys Acta 791: 37– 44, 1984.
Henquin JC, Ravier MA, Nenquin M, Jonas JC, and Gilon P.
Hierarchy of the ␤-cell signals controlling insulin secretion. Eur J Clin
Invest 33: 742–750, 2003.
Hui TY, Sheth SS, Diffley JM, Potter DW, Lusis AJ, Attie AD, and
Davis RA. Mice lacking thioredoxin-interacting protein provide evidence linking cellular redox state to appropriate response to nutritional
signals. J Biol Chem 279: 24387–24393, 2004.
Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi
S, Ogi K, Hosoya M, Tanaka Y, Uejima H, Tanaka H, Maruyama M,
Satoh R, Okubo S, Kizawa H, Komatsu H, Matsumura F, Noguchi Y,
E13
Invited Review
E14
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
AJP-Endocrinol Metab • VOL
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
metabolism of glutamine in pancreatic islets. J Biol Chem 257: 3754 –
3758, 1982.
Meglasson MD and Matschinsky FM. Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev 2: 163–
214, 1986.
Metz SA, Rabaglia M, Stock TB, and Kowluru A. Modulation of
insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins. Biochem J 295: 31– 40, 1993.
Mulder H, Lu D, Finley IVJ, An J, Cohen J, Antinozzi PA, McGarry
JD, and Newgard CB. Overexpression of a modified human malonylCoA decarboxylase blocks the glucose-induced increase in malonyl-CoA
level but has no impact on insulin secretion in INS-1-derived (832/13)
␤-cells. J Biol Chem 276: 6479 – 6484, 2001.
Nakada S, Ishikawa T, Yamamoto Y, Kaneko Y, and Nakayama K.
Constitutive nitric oxide synthases in rat pancreatic islets: direct imaging
of glucose-induced nitric oxide production in beta-cells. Pflügers Arch
447: 305–311, 2003.
Newgard C and McGarry J. Metabolic coupling factors in pancreatic
␤-cell signal transduction. Annu Rev Biochem 64: 689 –719, 1995.
Nilsson T, Schultz V, Berggren PO, Corkey BE, and Tornheim K.
Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate
and cytoplasmic free Ca2⫹ in glucose-stimulated pancreatic ␤-cells.
Biochem J 314: 91–94, 1996.
Nulton-Persson AC, Starke DW, Mieyal JJ, and Szweda LI. Reversible inactivation of ␣-ketoglutarate dehydrogenase in response to alterations in the mitochondrial glutathione status. Biochemistry 42: 4235–
4242, 2003.
Orci L, Amherdt M, Montesano R, Vassalli P, and Perrelet A.
Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells. Philos Trans R Soc Lond B Biol Sci
296: 47–54, 1981.
Prentki M. New insights into pancreatic ␤-cell metabolic signaling in
insulin secretion. Eur J Endocrinol 134: 272–286, 1996.
Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, and
Corkey BE. Malonyl-CoA and long chain acyl-CoA esters as metabolic
coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:
5802–5803, 1992.
Rahman SM, Dobrzyn A, Dobrzyn P, Lee SH, Miyazaki M, and
Ntambi J. Stearoyl-CoA desaturase 1 deficiency elevates insulin-signaling components and down-regulates protein-tyrosine phosphatase 1B in
muscle. Proc Natl Acad Sci USA 100: 11110 –11115, 2002.
Ramanadham S, Zhang S, Ma Z, Wohltmann M, Bohrer A, Hsu FF,
and Turk J. Delta6-, Stearoyl CoA-, and Delta5-desaturase enzymes are
expressed in beta-cells and are altered by increases in exogenous PUFA
concentrations. Biochim Biophys Acta 1580: 40 –56, 2002.
Rana RS, Mertz RJ, Kowluru A, Dixon JF, Hokin LE, and MacDonald MJ. Evidence for glucose-responsive and -unresponsive pools of
phospholipid in pancreatic islets. J Biol Chem 260: 7861–7867, 1985.
Rocheleau JV, Head WS, Nicholson WE, Powers AC, and Piston
DW. Pancreatic islet ␤-cells transiently metabolize pyruvate. J Biol
Chem 23: 30914 –30920, 2002.
Roduit R, Nolan C, Alarcon C, Moore P, Barbeau A, DelghingaroAugusto V, Przybykowski E, Morin J, Masse F, Massie B, Ruderman
N, Rhodes C, Poitout V, and Prentki M. A role for the malonyl-CoA/
long-chain acyl-CoA pathway of lipid signaling in the regulation of
insulin secretion in response to both fuel and nonfuel stimuli. Diabetes
53: 1007–1019, 2004.
Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, and
Prentki M. Metabolic fate of glucose in purified islet cells. J Biol Chem
272: 18572–18579, 1997.
Sener A, Malaisse-Lagae F, and Malaisse WJ. Stimulation of pancreatic islet metabolism and insulin release by a nonmetabolizable amino
acid. Proc Natl Acad Sci USA 78: 5460 –5464, 1981.
Shalev A, Pise-Masison CA, Radonovich M, Hoffman SC, Hirshberg
B, Brady JN, and Harlan DM. Oligonucleotide microarray analysis of
intact human pancreatic islets: identification of glucose-responsive genes
and a highly regulated TGF beta signaling pathway. Endocrinology 148:
3695–3698, 2002.
Stanley CA, Fang J, Kutyna K, Hsu BYL, Ming JE, Glaser B, and
Poncz M. Molecular basis and characterization of the hyperinsulinism
(hyperammonemic) syndrome. Diabetes 49: 667– 673, 2000.
Stanley CA, Lieu YK, Hsu BYL, Burlina AB, Greenberg CR, Hopwood NJ, Perlmar K, Rich BR, Zammarchi E, and Poncz M.
Hyperinsulinism and hyperammonemia in infants with regulatory muta-
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
66. MacDonald MJ, Al-Masri H, Jumelle-Laclau M, and Cruz MO.
Oscillations in activities of enzymes in pancreatic islet subcellular fractions induced by physiological concentration of effectors. Diabetes 46:
1996 –2001, 1997.
67. MacDonald MJ, Ammon HPT, Patel TN, and Steinke J. Failure of
6-aminonicotinamide to inhibit the potentiating effect of leucine and
arginine on glucose-induced insulin release in vitro. Diabetologia 10:
761–765, 1974.
68. MacDonald MJ and Chang CM. Do pancreatic islets contain significant amounts of phosphoenolpyruvate carboxykinase or ferroactivator
activity? Diabetes 34: 246 –250, 1985.
69. MacDonald MJ, Efendic S, and Ostenson CG. Normalization by
insulin treatment of low mitochondrial glycerol phosphate dehydrogenase and pyruvate carboxylase in pancreatic islets of the GK rat. Diabetes
45: 886 – 890, 1996.
70. Macdonald MJ and Fahien LA. Glyceraldehyde phosphate and methyl
esters of succinic acid. Diabetes 37: 997–999, 1988.
71. MacDonald MJ and Fahien LA. Insulin release in pancreatic islets by
a glycolytic and a Krebs cycle intermediate: contrasting patterns of
glyceraldehyde phosphate and succinate. Arch Biochem Biophys 279:
104 –108, 1990.
72. MacDonald MJ and Fahien LA. Glutamate is not a messenger in
insulin secretion. J Biol Chem 275: 34025–34027, 2000.
73. MacDonald MJ, Fahien LA, Buss JD, Hasan NM, Fallon MJ, and
Kendrick MA. Citrate oscillates in liver and pancreatic beta cell mitochondria and INS-1 insulinoma cells. J Biol Chem 278: 51894 –51900, 2003.
74. MacDonald MJ, Fahien LA, McKenzie DI, and Moran SM. Novel
effects of insulin secretagogues on capacitation of insulin release and
survival of cultured pancreatic islets. Am J Physiol Endocrinol Metab
259: E548 –E554, 1990.
75. MacDonald MJ, Fahien LA, Mertz RJ, and Rana RS. Effects of esters
of succinic acid and other citric acid cycle intermediates on insulin
release and inositol phosphate formation by pancreatic islets. Arch
Biochem Biophys 269: 400 – 406, 1989.
76. MacDonald MJ, Kaysen JH, Moran SM, and Pomije CE. Pyruvate
dehydrogenase and pyruvate carboxylase. Sites of pretranslational regulation by glucose of glucose-induced insulin release in pancreatic islets.
J Biol Chem 266: 22392–22397, 1991.
77. MacDonald MJ and Marshall LK. Mouse lacking NAD⫹-linked glycerol phosphate dehydrogenase has normal pancreatic beta cell function
but abnormal metabolite pattern in skeletal muscle. Arch Biochem Biophys 384: 143–153, 2000.
78. MacDonald MJ and Marshall LK. Survey of normal appearing mouse
strain which lacks malic enzyme and NAD⫹-linked glycerol phosphate
dehydrogenase: normal pancreatic beta cell function, but abnormal metabolite pattern in skeletal muscle. Mol Cell Biochem 220: 117–125, 2001.
79. MacDonald MJ, McKenzie DI, Kaysen JH, Walker TM, Moran SM,
Fahien LA, and Towle HC. Glucose regulates leucine-induced insulin
release and the expression of the branched chain keto acid dehydrogenase
E1a subunit gene in pancreatic islets. J Biol Chem 266: 1335–1340, 1991.
80. MacDonald MJ, McKenzie DI, Walker TM, and Kaysen JH. Lack of
glyconeogenesis in pancreatic islets: expression of gluconeogenic enzyme genes in islets. Horm Metab Res 24: 158 –160, 1992.
81. MacDonald MJ, Tang J, and Polonsky KS. Low mitochondrial glycerol phosphate dehydrogenase and pyruvate carboxylase in pancreatic
islets of Zucker diabetic fatty rats. Diabetes 45: 1626 –1630, 1996.
82. MacDonald PE, Salapatek AM, and Wheeler MB. Temperature and
redox state dependence of native Kv2.1 currents in rat pancreatic ␤-cells.
J Physiol 546: 647– 653, 2003.
83. Maechler P. Mitochondria as the conductor of metabolic signals for
insulin exocytosis in pancreatic ␤-cells. Cell Mol Life Sci 59: 1803–18,
2002.
84. Maechler P and Wollheim CB. Mitochondrial glutamate acts as a
messenger in glucose-induced insulin exocytosis. Nature 402: 685– 689,
1999.
85. Malaisse WJ, Rasschaert J, Villanueva-Penacarrillo ML, and
Valverde I. Respiratory, ionic and functional effects of succinate esters
in pancreatic islets. Am J Physiol Endocrinol Metab 264: E440 –E446, 1993.
86. Malaisse WJ and Sener A. Metabolic effects and fate of succinate esters
in pancreatic islets. Am J Physiol Endocrinol Metab 264: E434 –E439, 1993.
87. Malaisse-Lagae F, Sener A, Garcia-Morales P, Valverde I, and
Malaisse WJ. The stimulus-secretion coupling of amino acid-induced
insulin release. Influence of a nonmetabolized analog of leucine on the
Invited Review
SIGNALING ROLES OF MITOCHONDRIAL ANAPLEROTIC PRODUCTS
108.
109.
110.
111.
112.
113.
AJP-Endocrinol Metab • VOL
115.
116.
117.
118.
119.
120.
121.
SREBP-1c is instrumental in the development of ␤-cell dysfunction.
J Biol Chem 278: 16622–16629, 2003.
Watkins DT and Moore M. Uptake of NADPH by islet secretion
granule membranes. Endocrinology 100: 1461–1467, 1977.
Williamson JR and Cooper RH. Regulation of the citric acid cycle in
mammalian systems. FEBS Lett 117, Suppl: K73–K85, 1980.
Wollheim CB and Maechler P. Beta-cell mitochondria and insulin
secretion: messenger role of nucleotides and metabolites. Diabetes 51:
S37–S42, 2002.
Yajima H, Komatsu M, Yamada S, Straub SG, Kaneko T, Sato Y,
Yamauchi K, Hashizume K, Sharp GW, and Aizawa T. Cerulenin, an
inhibitor of protein acylation, selectively attenuates nutrient stimulation of
insulin release: a study in rat pancreatic islets. Diabetes 49: 712–717, 2000.
Yamada S, Komatsu M, Sato Y, Yamauchi K, Aizawa T, and Kojima
I. Nutrient modulation of palmitoylated 24 kDa protein in rat pancreatic
islets. Endocrinology 144: 5232–5241, 2003.
Yaney GC, Schultz V, Cunningham BA, Dunaway GA, Corkey BE,
and Tornheim K. Phosphofructokinase isozymes in pancreatic islets and
clonal beta-cells (INS-1). Diabetes 44: 1285–1289, 1995.
Zawalich WS, Zawalich KC, Cline G, Shulman G, and Rasmussen H.
Comparative effects of monomethylsuccinate and glucose on insulin
secretion from perifused rat islets. Diabetes 42: 843– 850, 1993.
288 • JANUARY 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on May 12, 2017
114.
tions of the glutamate dehydrogenase gene. N Engl J Med 338: 1352–
1357, 1998.
Straub SG and Sharp GW. Glucose-stimulated signaling pathways in
biphasic insulin secretion. Diabetes Metab Res Rev 18: 451– 463, 2002.
Straub SG, Yajima H, Komatsu M, Aizawa T, and Sharp GW. The
effects of cerulenin, an inhibitor of protein acylation, on the two phases of
glucose-stimulated insulin secretion. Diabetes 51, Suppl 1: S91–S95, 2002.
Thewke D, Kramer M, and Sinensky MS. Transcriptional homeostatic
control of membrane lipid composition. Biochem Biophys Res Commun
278: 1– 4, 2000.
Tornheim K. Are metabolic oscillations responsible for normal oscillatory insulin secretion? Diabetes 46: 1375–1380, 1997.
Tornheim K, Andres V, and Schultz V. Modulation by citrate of
glycolytic oscillations in skeletal muscle extracts. J Biol Chem 266:
15675–15678, 1991.
Walter P, Paetkau V, and Lardy HA. Paths of carbon in gluconeogenesis and lipogenesis. 3. The role and regulation of mitochondrial processes involved in supplying precursors of phosphoenolpyruvate. J Biol
Chem 241: 2523–2532, 1966.
Wang H, Maechler P, Antinozzi PA, Herrero L, Hagenfeldt-Johansson KA, Bjorklund A, and Wollheim CJ. The transcription factor
E15