(Received for publication, November 3, 1995; and in revised form, February 7, 1996)
From the
Long term exposure to elevated levels of long chain free fatty
acids decreases glucose-induced insulin secretion from pancreatic
islets and clonal pancreatic -cells. The mechanism for this loss
of glucose sensitivity is at present not known. In this study, we
evaluated the possibility that increases in long chain acyl-CoA esters
(LC-CoA), the metabolically active form of free fatty acids, might
mediate the loss of glucose sensitivity. We observed that cellular
levels of LC-CoA increased more than 100% in response to overnight
incubation with 0.5 mM palmitic acid complexed to albumin. In
the same studies, the total CoA pool increased by about 40%.
Patch-clamp studies demonstrated that saturated and unsaturated LC-CoA,
but not malonyl-CoA or free CoASH, induced a rapid and slowly
reversible opening of ATP-sensitive K
channels. The
effect was concentration-dependent between 10 nM and 1
µM. These findings indicate that the ATP-regulated
K
channel is a sensitive target for LC-CoA and suggest
that high levels of LC-CoA, which accumulate in response to
hyperglycemia or prolonged exposure to free fatty acids, may prevent
channel closure and contribute to the development of
-cell glucose
insensitivity.
Exposure to elevations in free fatty acids (FFA) ()decreases glucose-induced insulin secretion. The concept
of gluco-lipo-toxicity is increasingly being invoked to explain such
loss of glucose-induced insulin secretion, but the identity of putative
effector molecules that might mediate these effects is unknown. Normal
glucose-induced insulin secretion is associated with inhibition of FFA
oxidation and increased lipid synthesis in pancreatic
-cells(1, 2, 3, 4) . In
addition, exogenous FFA acutely potentiate glucose-stimulated insulin
secretion(4, 5) . We have demonstrated that glucose
causes marked alterations in the acyl-CoA profile of clonal pancreatic
-cells, with the largest (5-fold) and earliest (by 2 min) change
occurring in malonyl-CoA(1, 5) . However, we did not
obtain a good correlation between secretion and malonyl-CoA levels but
rather between secretion and decreases in long chain acyl-CoA (LC-CoA)
levels(5) .
The total CoA pool is fixed over short intervals and distributed between mitochondrial and cytosolic compartments that are not interchangeable(6, 7) . Thus, during an acute response, the maximum LC-CoA concentration is limited by the total CoA pool and its distribution between cytosol and mitochondria. In some cell types, high fat and certain drugs or steroids have the potential to increase the total CoA pool over a period of hours to days and also lead to increases in the LC-CoA pool(7) .
Our studies were
undertaken to determine whether increased levels of LC-CoA occur in
response to exposure of -cells to FFA and to assess the effect of
physiological concentrations of LC-CoA on the ATP-sensitive
K
channel (K
channel). We demonstrate,
for the first time, that both total CoA and LC-CoA levels increase in
cells cultured in elevated FFA and document dramatic increases in
K
channel activity in response to LC-CoA.
where n is the number of samples, i is the current registered in sample j and I
is the value of a user-defined base line. Traces
represent experiments that were performed at least five times using
cells from separate animals. Mean currents (i
)
were determined during selected intervals designated by time lines in
the figures. Statistical differences in concentrations of acyl-CoA
esters were compared using Student's t test for unpaired
two-tailed data.
Culture of isolated rat islets or clonal -cells in
elevated concentrations of FFA decreases the ability of glucose to
stimulate insulin secretion. We performed studies in which clonal
-cells (HIT-T15), incubated overnight in medium containing 0.5
mM palmitate, exhibited a 50% decrease in the ability of
glucose to stimulate insulin secretion (data not shown). This
observation is consistent with previously published findings in islets
from starved animals or animals exposed to high circulating levels of
FFA in which glucose-induced insulin release is
diminished(12, 13) . Analysis of soluble and insoluble
pools of acyl-CoA esters showed that overnight exposure to 0.5 mM palmitic acid increased the pool of LC-CoA, the metabolically
active form of FFA, as well as the total CoA pool without affecting the
short chain acyl-CoA ester pool (Table 1). The 100% increase in
LC-CoA was opposite in direction and greater in magnitude than the
change in secretion. These findings suggest that the accumulation of
LC-CoA could be causally related to a loss of responsiveness to
glucose.
It is well established that one of the initial events in
the -cell stimulus secretion coupling is closure of the K
channel. The activity of this channel is also the main
determinant of the
-cell resting potential(14) . One
possible explanation for the inhibition of glucose responsiveness
following exposure to high FFA levels could therefore be modulation of
K
channel activity. It should also be noted that the
molecular structure of the CoA moiety in the LC-CoA molecule bears a
very close resemblance to ADP, a known stimulator of the K
channel(15, 16, 17) . To test the
possibility of a direct effect of LC-CoA on the K
channel, we assessed regulation using single channel recordings
of dispersed mouse
-cells with the inside-out configuration of the
patch-clamp technique (Fig. 1). Fig. 1A illustrates a reversible stimulatory effect of 1 µM oleoyl-CoA on K
channel activity. Mean current
increased 5-fold, from 0.8 to 4.4 pA in the presence of the LC-CoA. The
onset of the effect was considerably faster than the recovery. Channel
activity is increased by the number of open channels and also by an
increased mean open time (data not shown), similar to the effect
exerted by low concentrations of ADP(17, 18) . The
second trace (Fig. 1B) shows a slight stimulatory
effect of 100 nM oleoyl-CoA with a long lag and slow recovery
in which the mean current increased approximately 2-fold from 0.6 to
1.5 pA. The kinetics of the oleoyl-CoA effects are probably related to
the lipophilicity of the compound. Addition of 10 nM oleoyl-CoA (Fig. 1C) induced little effect except
to possibly diminish run down of channel activity. In a previous study,
we found cytosolic binding sites for LC-CoA with an estimated K
of approximately 1 µM(9) ,
suggesting that the concentrations used are physiologically relevant.
Figure 1:
Effects
of different concentrations of oleoyl-CoA on K channel
activity in an inside-out patch shortly after patch excision. A, mean K
channel current increased 5-fold from
0.8 pA (i
)to 4.4 pA (i
) in the presence of 1 µM oleoyl-CoA. The onset of the effect is considerably faster than
the recovery. B, a slight stimulatory effect is seen following
addition of 100 nM of the ester. The mean current increased
approximately 2-fold, from 0.6 pA (i
) to
1.5 pA (i
). C, the effect of 10
nM oleoyl-CoA on K
channel activity is shown.
The run down of channel activity is partly reversed in the presence of
10 nM oleoyl-CoA. Mean currents i
and i
were found to be 0.8 and
1.1 pA, respectively.
Interaction between ATP and oleoyl-CoA was observed (Fig. 2).
When 0.1 mM ATP was present continuously, a concentration that
fully blocks K channel activity, 1 µM oleoyl-CoA induced a pronounced increase in channel activity (Fig. 2A). When 0.1 mM ATP was added to the bath
solution one min after exposure of the patch to LC-CoA, ATP had a
considerably less pronounced inhibitory effect on channel activity ( Fig. 2B). This reduced blocking effect of ATP can be explained
by the slow recovery of channel activity following exposure to the
LC-CoA ester, due to its lipophilicity, since after approximately five
min further washing, the normal sensitivity to ATP was regained (Fig. 2B). These experiments provide compelling
evidence that LC-CoA esters have the ability to prevent ATP-induced
closure of the K
channel, in vitro.
Interestingly, they may also be involved in the suppression of
glucose-induced depolarization following exposure to high levels of
lipids.
Figure 2:
Interactions between oleoyl-CoA and ATP on
K channel activity. A, the effect of 1
µM oleoyl-CoA on K
channel activity in an
inside-out patch in the continuous presence of 0.1 mM ATP. B, time dependence of the effectiveness of ATP in inhibiting
K
channel activity following removal of
LC-CoA.
The specificity for oleoyl-CoA was tested by comparing the
responses with the FFA and with free CoASH. As seen in Fig. 3,
no effect was observed with 1 µM oleic acid in a patch
that responded to ATP and oleoyl-CoA (Fig. 3A).
Likewise, no effect on K channel activity was observed
when CoASH was administered, either at low (data not shown) or at high
concentrations, to a patch where oleoyl-CoA induced a potent increase
in mean current (Fig. 3B). Thus, the stimulatory effect
of the LC acyl-CoA is dependent on both the acyl group and the CoA
component. We also studied the effects of LC-CoA of different chain
lengths on channel activity (Fig. 4). Stimulatory effects were
seen with CoA esters of chain length 14 (Fig. 4B) and
16 carbons (Fig. 4C) but not with the 3-carbon
malonyl-CoA (Fig. 4A). The effect of myristoyl-CoA
(C14:0) was less pronounced than that obtained with palmitoyl-CoA
(C16:0) or oleoyl-CoA (C18:1), both with regard to stimulatory ability
and to recovery of the effect. These findings indicate that the
K
channel is a target for LC-CoA with a responsiveness to
both saturated and unsaturated LC-CoA esters from C-14 to C-18 and are
consistent with the suggestion that high levels of LC-CoA, such as
occur in response to elevated FFA, may prevent channel closure in
response to glucose. This may explain the impairment in K
channel closure that has been observed in islets from the
diabetic Goto-Kakizaki rat(19) .
Figure 3:
Specificity for oleoyl-CoA stimulation of
channel activity. A, addition of 1 µM free oleic
acid to an inside-out patch did not affect K channel
activity. Mean currents were estimated to be 0.9 pA (i
) in the absence and 0.7 pA (i
) in the presence of 1 µM oleic acid. Subsequent addition of oleoyl-CoA to the same patch
resulted in an increased mean current of 4.2 pA (i
). B, addition of 100
µM CoASH was without effect on K
channel
activity. The mean current in the absence of CoASH was estimated to be
0.4 pA (i
), compared with 0.3 pA (i
) in the presence of CoASH. As a
control 1 µM oleoyl-CoA was added, increasing mean channel
current to 3.1 pA (i
).
Figure 4:
Effects of different acyl-CoA chain
lengths on K channel activity. A, no effect on
K
channel activity could be observed when 1 µM malonyl-CoA was added, mean current was estimated to be 1.2 (i
) compared to 0.9 (i
) pA. B, in the presence of
myristoyl-CoA, channel activity increased from 0.4 pA (i
) to 1.1 pA (i
). C, addition of
palmitoyl-CoA to the intracellular solution also affected channel
activity with an estimated 4-fold increase in mean current going from
1.2 (i
) to 5.1 (i
) pA.
A previous report of FFA
modulation of K channel activity in clonal
-cells
did not differentiate between the effects of the FFA and their
metabolically active LC-CoA esters(20) . Since added FFA can be
rapidly converted to LC-CoA, it is necessary to compare the FFA and
LC-CoA in an excised inside-out patch, as we have done. Such studies
may also reveal a more general regulatory function of LC-CoA on
K
channel activity in other cell types.
Fig. 5compares the effect of oleoyl-CoA on different
K channels present in the
-cell. The upper
trace shows that the big conductance K
channel
(K
), which is voltage- and Ca
-dependent,
is not influenced by 1 µM oleoyl-CoA in contrast to the
K
channel, which is activated (compare the expanded
regions in Fig. 5A). The addition of 100 µM CoASH had no effect on either the K
channel or the
K
channel. Fig. 5B shows that the
8-picosiemens K
channel, described previously in the
-cell(21) , is also unaffected by the addition of
oleoyl-CoA. Hence, the ability of LC-CoA to increase K
conductance seems to be specific for the K
channel.
These findings identify a potent new regulator of the K
channel which causes up to fivefold increases in mean channel
current in response to the most common LC-CoA esters(7) . This
is observed at concentrations that are physiologically relevant and
approximately equal to the K
for cytosolic binding
sites for LC-CoA(9) . On a molar basis, the LC-CoA esters are
approximately 100-1000 times more potent than ADP in stimulating
channel activity.
Figure 5:
Illustration of the specificity of
oleoyl-CoA to activate the K channel. A,
response to CoASH and oleoyl-CoA of K
and large
conductance K
channel (K
) currents.
Whereas the activity of the K
channel is clearly
increased, no effect on the K
channel was observed. Insets show the indicated regions of the upper trace on an expanded time scale. B, recording of the
8-picosiemens K
channel in the absence and presence of
1 µM oleoyl-CoA. No effect on the 8-picosiemens channel
was observed, whereas a clear effect on K
channel
activity is registered. Insets show the indicated regions on
an expanded time scale. Arrows indicate the number of channel
levels and c denotes closed.
LC-CoA esters and products formed from them have
also been shown to be potent regulators of a variety of enzymes (22) and channels(20, 23) . Thus, LC-CoA
inhibits glucokinase activity, stimulates endoplasmic reticulum
Ca-ATPase, prevents malonyl-CoA inhibition of
carnitine palmitoyl transferase-1, and inhibits acetyl-CoA carboxylase (9, 22, 24, 25, 26, 27) .
The adenine nucleotide translocase, which plays an important role in
controlling the cytosolic ATP/ADP ratio(26) , and the sodium
pump (28, 29, 30) are stimulated by LC-CoA in
some cells. Based on these opposite effects on different established
targets of LC-CoA and the ability of FFA to increase the total CoA pool
in
-cells, as in other tissues(7, 11) , the net
effect of LC-CoA on glucose-induced insulin secretion can be either
stimulatory or inhibitory and may depend on the LC-CoA level achieved.
We find it attractive to hypothesize that elevated circulating FFA,
leading to increased cellular levels of LC-CoA, play a role in the
development of glucose insensitivity and that elevated glucose levels,
as in tissue culture or non-insulin-dependent diabetes mellitus,
exacerbate the ``toxic'' action of lipids, because high
glucose inhibits fatty acid oxidation and elevates cytosolic LC-CoA
levels(1) . Thus, isolated islets from 48-h lipid-infused rats
display reduced glucose oxidation and insulin release in response to
glucose(12) , and the insulin secretory response of islets
isolated from fat-fed mice is similar to the defective secretory
pattern observed in human NIDDM (31) . Furthermore, perifusion
of islets for 3 h with palmitate causes impaired insulin release in
response to glucose(32) . This may also be compatible with the
loss of insulin responsiveness to glucose seen during starvation, a
condition that leads to increased levels of FFA (33) . How long
term increases in FFA impair glucose-induced insulin secretion is not
known. It may be that LC-CoA inhibits the action of glucose by
preventing the closure or promoting the opening of K channels as we have shown in this study.