Download Life and death decisions of the pancreatic β

Clinical Science (2007) 112, 27–42 (Printed in Great Britain) doi:10.1042/CS20060115
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Life and death decisions of the pancreatic
β-cell: the role of fatty acids
Philip NEWSHOLME*, Deirdre KEANE*, Hannah J. WELTERS†
and Noel G. MORGAN†
*School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland, and †Institute
of Biomedical and Clinical Science, Peninsula Medical School, Universities of Exeter and Plymouth, Plymouth, Devon PL6 8BU,
U.K.
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Both stimulatory and detrimental effects of NEFAs (non-esterified fatty acids) on pancreatic β-cells
have been recognized. Acute exposure of the pancreatic β-cell to high glucose concentrations
and/or saturated NEFAs results in a substantial increase in insulin release, whereas chronic
exposure results in desensitization and suppression of secretion, followed by induction of
apoptosis. Some unsaturated NEFAs also promote insulin release acutely, but they are less toxic
to β-cells during chronic exposure and can even exert positive protective effects. Therefore
changes in the levels of NEFAs are likely to be important for the regulation of β-cell function and
viability under physiological conditions. In addition, the switching between endogenous fatty acid
synthesis or oxidation in the β-cell, together with alterations in neutral lipid accumulation, may
have critical implications for β-cell function and integrity. Long-chain acyl-CoA (formed from either
endogenously synthesized or exogenous fatty acids) controls several aspects of β-cell function,
including activation of specific isoenzymes of PKC (protein kinase C), modulation of ion channels,
protein acylation, ceramide formation and/or NO-mediated apoptosis, and transcription factor
activity. In this review, we describe the effects of exogenous and endogenous fatty acids on β-cell
metabolism and gene and protein expression, and have explored the outcomes with respect to
insulin secretion and β-cell integrity.
METABOLISM OF GLUCOSE AND FATTY
ACIDS IN THE β-CELL
In writing this review, we are fully aware that most of
the studies cited have utilized rat-, mouse- or hamsterderived insulinoma β-cell lines to study function in
vitro. This is due to the inherent difficulty in maintaining
primary rodent islet β-cell mass and function for more
than a few days in vitro and, of course, the scarcity of
human islets for research purposes. There are as yet no
suitable human β-cell lines available for unrestricted
in vitro studies; however, the major rodent β-cell lines
have provided substantial data and insights into cell
function in normal or pathogenic situations. The most
widely used cell lines include INS-1, MIN-6, RINm5F,
and BRIN-BD11. To stimulate insulin secretion from
Key words: acetyl-CoA, apoptosis, fatty acid toxicity, glucose metabolism, insulin secretion, non-esterified fatty acid (NEFA),
pancreatic β-cell.
Abbreviations: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated kinase; CPT-1, carnitine palmitoyltransferase-1; DAG, diacylglycerol; ER, endoplasmic reticulum; GLP-1, glucagon-like peptide-1; GSIS, glucose-stimulated insulin secretion; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA reductase; HNF-4α, hepatocyte nuclear factor 4α; HSL, hormone-sensitive lipase; iNOS,
inducible form of NO synthase; IRS-2, insulin receptor substrate-2; MPTP, mitochondrial permeability transition pore; mTOR, mammalian target of rapamycin; NEFAs, non-esterified fatty acids; NF-κB, nuclear factor κB; OGG1, 8-oxoguanine DNA glycosylase/apurinic lyase; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; PPAR, peroxisome-proliferatoractivated receptor; SREBP1c, sterol-regulatory-element-binding protein 1c; TAG, triacylglycerol; UCP, uncoupling protein.
Correspondence: Dr Philip Newsholme (email [email protected]).
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primary islets and β-cell lines, glucose is transported into
the pancreatic β-cell by the non-insulin-dependent glucose transporter GLUT2 in rodents and by both GLUT1
and GLUT2 in humans. Once inside the cells, glucose is
phosphorylated by the low-affinity (K m = 6–11 mmol/l)
hexokinase IV (glucokinase). The glycolytic flux in
pancreatic islet β-cells is therefore regulated by a combination of the rate of glucose uptake and the glucokinase
activity, although it is unlikely that glucose uptake is ratelimiting under most conditions. Therefore glucokinase
has been referred to as the β-cell’s glucose sensor
[1]. The flux of metabolites through the pentose
phosphate pathway is low and glycogen synthesis
accounts for approx. 1–7 % of the glucose utilized
by β-cells. Furthermore, the β-cell is metabolically
distinct from almost all other mammalian cell types in
several respects; namely because (i) it can utilize
glucose in the physiologically relevant range (2–
20 mmol/l); (ii) it displays low lactate dehydrogenase and
plasma membrane monocarboxylate pyruvate/lactate
transporter activity and correspondingly high activity
in the mitochondrial malate/aspartate shuttle, so
ensuring mitochondrial oxidation of NADH; (iii) it
has a high activity of both pyruvate dehydrogenase
and pyruvate carboxylase, ensuring that both anaplerotic and oxidative metabolism of glucose/pyruvate can
co-exist. Acetyl-CoA formed from pyruvate can condense with oxaloacetate forming citrate for metabolism
in the tricarboxylic acid cycle (Krebs cycle), leading to
NADH and FADH2 production [2]. All these specific metabolic adaptations are geared to enhancing
mitochondrial tricarboxylic acid cycle activity, oxidative
phosphorylation and efficient ATP production. An
enhancement of the ATP/ADP ratio results in closure
of ATP-sensitive K+ channels, depolarization of the
plasma membrane, opening of voltage-activated Ca2+
channels [1], influx of Ca2+ and, finally, fusion of insulincontaining granules with the plasma membrane [3,4].
In addition to this metabolic complexity, the β-cell can
metabolize a number of key amino acids, which, via mitochondrial metabolism, can generate further stimulus–
secretion ‘coupling’ factors [5]. Indeed, additional signals
which ‘amplify’ the initial glucose-dependent stimulation
of insulin secretion may be generated independently of
ATP-sensitive K+ channel activity. These, as yet, unidentified signals appear to increase the efficacy of Ca2+
on the exocytosis of insulin granules [6].
Pancreatic β-cell and islet glucose metabolism results
in increased levels of cytosolic long-chain acyl-CoA
compounds, TAG (triacylglycerol) and phospholipid
turnover as a consequence of increased citrate and
malonyl-CoA production and subsequent inhibition of
β-oxidation of NEFAs (non-esterified fatty acids) [3,7].
Malonyl-CoA produced from glucose plays a role in
this shift from NEFA to glucose as an oxidative fuel,
since it inhibits CPT-1 (carnitine palmitoyltransferase-1),
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found on the outer mitochondrial membrane, thereby
reducing long-chain CoA entry into the mitochondria [8].
Long-chain acyl-CoA, phosphatidate and DAG
(diacylglycerol) contents are elevated in response to
increased glucose metabolism, leading to a subsequent
elevation in intracellular Ca2+ , activation of PKC (protein
kinase C) isoforms [9] and changes in the acylation state of
key proteins involved in regulating ion channel activity
and exocytosis [9–11]. On the other hand, long-chain
CoA may also stimulate insulin release by a more direct
effect on exocytosis, as it facilitates the fusion of insulinsecretory granules with the β-cell plasma membrane
[10].
The ability of the pancreatic islets to convert glucose into fatty acids by de novo synthesis has been
demonstrated directly by determination of the content
and composition of fatty acids from rat pancreatic
islets and culture medium after incubation for 1 and 3 h
in the absence and presence of 5.6, 8.3 or 16.7 mmol/l
glucose using HPLC analysis [12]. The total lipid content
of pancreatic islets was substantially reduced after
incubation for 1 h in the absence of glucose; however, the
lipid content was restored by incubation in the presence
of 5.6 mmol/l glucose and was increased further by
incubation with 8.3 or 16.7 mmol/l glucose. Pancreatic
islets exported a substantial amount of fatty acids to the
medium either in the absence or presence of glucose. After
incubation for 1 h, the addition of 5.6 mmol/l glucose
raised the medium content of palmitate (3.4-fold) and
stearate (5.4-fold) compared with islets incubated in the
absence of glucose. Martins et al. [12] concluded that
the synthesis and release of saturated and, to a lesser extent, unsaturated fatty acids from glucose-exposed islets
represents a novel mechanism for modulating GSIS
(glucose-stimulated insulin secretion) from pancreatic
β-cells.
REGULATION OF FATTY ACID METABOLISM
IN β-CELLS
Hyperlipidaemia is frequently associated with insulin
resistance, a condition that is associated with hyperglycaemia and hyperinsulinaemia, as found in Type 2
diabetes and obesity [13–15]. A direct effect of the fatty
acids on insulin secretion in vivo may explain the
co-existence of hyperlipidaemia and hyperinsulinaemia.
Although it has been accepted that fatty acid metabolism
is necessary for fatty acid stimulation of insulin secretion
[16,17], the precise mechanism(s) by which fatty acids
stimulate insulin secretion remain unknown.
Long-chain fatty acids may be transported into the cell
by free diffusion with no requirement for active transport
[18]. Extracellular NEFAs can be rapidly distributed
in lipid bilayers and move rapidly from cell to cell,
Role of non-esterified fatty acids in β-cell function and integrity
Figure 1 Potential pathways of fatty acid metabolism in the pancreatic β-cell
Fatty acids are ‘activated’ on entry into the β-cell to become acyl-CoA. This acyl-CoA may be oxidized in the mitochondrial matrix, or may be esterified in the cytosol
to become TAG. TAG/NEFA cycling may occur in the β-cell. Acyl-CoA may also give rise to lipid-based signalling molecules such as DAG. Glucose metabolism may
also give rise to cytosolic citrate via cataplerosis, which subsequently can be converted into acetyl-CoA and oxaloacetate by ATP-citrate lyase, and then conversion of
acetyl-CoA into malonyl-CoA via the action of ACC, the ‘committed’ step for fatty acid synthesis. Endogenously synthesized fatty acids may be incorporated into TAG,
signalling lipids or may be exported. NEFAs may also bind to GPR40 receptors and stimulate unknown signal transduction pathway(s). G3P, glucose 3-phosphate.
potentially acting as paracrine mediators. Islets express
LDL (low-density lipoprotein) receptors [19] and LPL
(lipoprotein lipase) [20], and could also obtain fatty acids
from circulating lipoproteins. Fatty acid metabolism is
mainly controlled by substrate supply. When the glucose
level is low, fatty acids are converted into long-chain acylCoA by ACS (acyl-CoA synthetase) and enter the mitochondria, where they are oxidized via the β-oxidation
pathway for energy production [7]. The shift from
fatty acid to glucose as an oxidative fuel occurs through
glucose conversion into the ‘regulatory’ compound
malonyl-CoA, as mentioned above, and involves inhibition of AMPK (AMP-activated kinase) and activation
of ACC (acetyl-CoA carboxylase) [8] (Figure 1).
In addition, the presence and action of HSL (hormonesensitive lipase) in β-cells reinforces the concept that
endogenous lipolysis participates in the regulation of
insulin secretion through the generation of NEFAs or
other lipid signalling molecules [21]. Hence the islet TAG
store, via HSL-mediated lipolysis, may play an important
role in the stimulus–secretion coupling mechanism of
GSIS (Figure 1). As discussed above, fatty acids, their
CoA derivatives or complex lipids formed from fatty
acids, play a key role for nutrient signalling in the
β-cell. Lipolysis of β-cell TAG also plays an important
role in the action of incretins, such as GLP-1 (glucagonlike peptide-1), due to the mechanism of potentiation
of GSIS, which involves an increase in intracellular
cAMP content [22,23]. cAMP can activate PKA (protein
kinase A), which is a potent stimulator of HSL. In fact,
HSL-knockout mice have reduced GSIS both in vivo
and in isolated islets [21]. Additionally an increase in glucose concentration induces HSL (LIPE) gene expression
in the β-cell, resulting in a 2-fold increase in HSL
protein and enzymatic activity [24]. This occurs concomitantly with an increase in basal insulin secretion [25], corroborating the proposition that lipid signalling molecules
are essential to GSIS [26,27].
Long-chain acyl CoA may additionally be esterified to
TAG in the β-cell in the presence of glycerol 3-phosphate
provided by glucose metabolism, or alternatively may be
oxidized when rates of glucose uptake and metabolism
are low. The key metabolic ‘sensor’ AMPK (which is
activated by a fall in the ATP/AMP ratio) may provide
a unique switching mechanism to allow β-cells to oxidize fatty acid when glucose oxidation (and ATP
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synthesis) is low, while allowing fatty acid and cholesterol synthesis to proceed when glucose oxidation (and
ATP synthesis) is higher. This regulation is achieved
through phosphorylation of key regulatory enzymes:
ACC, HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) and the translational regulator
mTOR (mammalian target of rapamycin). In β-cells, as
glucose transport and oxidation increases after a rise in
plasma glucose concentration, AMPK activity is reduced
(due to a rise in the ATP/AMP ratio) leading to dephosphorylation and activation of key lipid synthesizing
enzymes (ACC) and HMG-CoA reductase and dephosphorylation and activation of mTOR. This will result
in increased fatty acid, cholesterol and protein synthesis
while, at the same time, reducing fatty acid oxidation.
Thus glucose becomes the prime oxidative fuel for the
β-cell, maintaining the elevated ATP concentration and
thus stimulating insulin secretion. The newly synthesized fatty acids may become incorporated into lipidderived signalling molecules (such as DAG), leading
to promotion of insulin secretion via PKC activation
(Figure 1). Chronic exposure of β-cells to high levels
of saturated fatty acids will impair glucose oxidation,
resulting in a fall in the ATP/AMP ratio and activation
of AMPK, phosphorylation and inhibition of ACC,
reduction in fatty acid synthesis and promotion of
fatty acid oxidation, thus impairing GSIS. AMPK may
additionally regulate β-cell function more chronically
by changing the levels of expression of key transcription
factors controlling lipogenic and glycolytic enzymes, for
example SREBP1c (sterol-regulatory-element-binding
protein 1c) and HNF-4α (hepatocyte nuclear factor 4α).
AMPK activation caused a marked decrease in expression
of SREBP1c and HNF-4α in liver thus reducing fatty
acid synthesis. Overexpression of a constitutively active
form of SREBP1c in β-cells resulted in elevated TAG
accumulation and impairment of insulin secretion [28].
IMPACT OF NEFAs ON β-CELL GLUCOSE
METABOLISM
In pancreatic β-cells, cytoplasmic NEFAs are converted
into long-chain acyl-CoA by acyl-CoA synthase. Under
basal conditions, the long-chain acyl-CoA molecules are
transported into the mitochondria via CPT-1, where βoxidation takes place. High levels of glucose inhibit this
process (via inhibition of AMPK and subsequent ACC
activation) and induce a marked rise in the cytoplasmic
content of long-chain acyl-CoA [29]. Malonyl-CoA
inhibits CPT-1 activity, allowing the accumulation of
long-chain acyl-CoA in the cytosol [30,31]. Thus
malonyl-CoA acts by switching β-cell metabolism from
fatty acid oxidation to glucose oxidation. An important
consequence of this switching is a marked increase in
cytosolic long-chain acyl-CoA, which acts as an effector
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molecule in the β-cell signalling, as discussed above [32]
(Figure 1).
Randle et al. [14] proposed the glucose/fatty acid cycle
in their studies with rat heart muscle and rat diaphragm
muscle. In principle, a rise in plasma NEFA levels
decreases glucose uptake and oxidation by many tissues,
but the mechanism of this inhibition was unknown before
the work of Randle, Garland, Hales and Newsholme
[14]. The key steps of this hypothesis are co-ordinated
in the following way. The rise in plasma NEFA induces
β-oxidation with increased production of acetyl-CoA,
inhibiting pyruvate dehydrogenase and thus oxidation
of pyruvate. At the same time, the production of
citrate and ATP inhibits phosphofructokinase activity
and thus glycolysis, resulting in accumulation of glucose
6-phosphate. The increased level of glucose 6-phosphate,
in turn, inhibits muscle hexokinase activity with a
reduction in the glucose transport/phosphorylation
activity. However, the β-cell, due to the necessity of
operating a high glycolytic rate (in the presence of elevated glucose concentrations) in the presence of longchain acyl-CoA, does not appear to be subjected to the
same regulatory principles. For example, as a consequence of elevated glucose metabolism, resulting in high
cytosolic ATP and citrate concentrations, the β-cell
glycolytic rate is not impeded. β-Cell-specific expression
of key regulatory enzymes, such as glucokinase (which
is not subjected to allosteric inhibition by the product
glucose 6-phosphate), allows high rates of glycolysis in
an ‘energy rich’ environment.
In contrast with the acute effects of glucose and
fatty acids, the chronic inhibitory effect of NEFAs
on GSIS may be due to a metabolic effect. Reduced
glucose oxidation may result from decreased conversion
of pyruvate into acetyl-CoA, a consequence of a decline
in islet pyruvate dehydrogenase activity, due to the inhibitory action of increased NADH production via NEFA
β-oxidation [33], an increase in pyruvate dehydrogenase
kinase activity [34] or via changes in expression of key
metabolic genes or transcription factors. Adiponectin, an
adipose-tissue-derived peptide hormone, may additionally regulate fatty acid metabolism in the β-cell, mainly
through activation of AMPK [35–41]. Adiponectin is
required for normal insulin action in the primary target
tissues and regulation of energy homoeostasis [42,43].
MODULATION OF INSULIN SECRETION
BY NEFAs
In vivo studies
Insulin secretion is influenced, at any given time, predominately by the blood glucose concentration and by the
prevailing fatty acids in the circulation [44]. Gut-derived
incretins, other hormones and neurotransmitters may
also exert an influence. Many groups have investigated the
Role of non-esterified fatty acids in β-cell function and integrity
effect of fatty acids on the process of GSIS (for example,
[7,45–49]). The rapid effect of NEFAs to potentiate GSIS
in vitro, while having little effect on secretion at nonstimulatory glucose concentrations, would suggest that
they act to amplify metabolic stimulus–secretion coupling mechanisms [50,51]. The potency of fatty acids to
promote glucose-induced insulin release increases with
the chain length and decreases with the degree of unsaturation [52,53]. Long-chain fatty acids (such as palmitate,
linoleate and α-linolenate) potentiate insulin secretion in
response to basal glucose concentrations [54].
Acute lowering of plasma fatty acids levels is associated
with a decreased insulin response to glucose [45,55], but
full secretory function can be restored by inclusion of
fatty acids during perfusion of pancreas from fasted rats
[48,49,56]. GSIS by perifused islets of rats fasted for 24 h is
reduced when compared with the response of islets from
fed rats [48,49,56,57]. In rats deprived of food for 18–
24 h, the ability of the β-cell to secrete insulin in response
to a glucose load is fully dependent upon the elevated
levels of circulating NEFAs characteristic of the fasted
state [48]. In fact, circulating fatty acids are essential for
an efficient glucose stimulation of insulin release after
prolonged fasting in humans and rats [58,59].
A recent advance in the understanding of the mechanism(s) by which NEFAs modulate insulin secretion
in vivo is the discovery of high levels of expression of the
membrane-bound G-protein-coupled receptor GPR40,
a putative NEFA receptor in human and animal islet
β-cell preparations [60]. The GPR40 mRNA level was
positively correlated with the insulinogenic index [60].
The potential signalling mechanism(s) by which GPR40
regulates insulin secretion are still under investigation,
but are likely to involve changes in intracellular Ca2+
mobilization [61–63].
In vitro studies
Pancreatic islets exposed to high concentrations of
NEFAs for periods of 24–48 h [64,65] display enhanced
basal insulin secretion, decreased insulin synthesis,
depletion of stored insulin and an impaired response of
the β-cell to stimulation by glucose; all of which are
characteristics seen in Type 2 diabetes. Other studies
using pancreatic islets have demonstrated that palmitate
increased cytosolic Ca2+ [50]. Rat and human islets
exposed to fatty acids for 48 h demonstrated increased
insulin release at basal glucose concentration (3 mmol/l),
but decreased release at an elevated (stimulatory) glucose
concentration (27 mmol/l) [33].
The effects of high concentrations of NEFA on isolated
islets and clonal pancreatic insulin-secreting cells are
dependent on the period of exposure during cell culture.
Acute exposure (1–3 h) of pancreatic islets to NEFAs
enhances insulin secretion [66,67] and plays a critical role
in modulating the stimulatory effect of glucose on insulin
release [50,68]. Acute removal of exogenous NEFAs may,
however, result in excessively high rates of GSIS. After
incubation of rat pancreatic islets for 4 h with a high
concentration of fatty-acid-free BSA, they responded
to glucose with extraordinarily high rates of secretion,
without changing the typical biphasic pattern of the
response [69]. The latter results may argue in favour of a
buffering role for NEFAs, such that they siphon off some
glucose (in the form of a downstream metabolite, glycerol
3-phosphate) for formation of TAG (see later discussion)
so as to ensure that excessively high rates of insulin
secretion do not occur. The TAG stores may subsequently
release NEFAs upon appropriate stimulation via specific
TAG lipases. Expression of UCP-2 (uncoupling protein2) in β-cells is increased by NEFAs. UCPs are located
in the inner mitochondrial membrane and act as proton
channels. They uncouple the electrochemical gradient
produced by the respiratory chain from ATP synthesis
[70,71]. Up-regulation of UCP-2 or overactivity of UCP2 leads to inhibition of ATP synthesis. Fatty acids
increase UCP-2 mRNA and protein levels in several cells
including β-cells. As a result, ATP synthesis is reduced
and GSIS is blunted. UCP-2−/− mice have lower fasting
blood glucose and elevated insulin levels when fed a highfat diet compared with wild-type mice [72]. Exposure
to palmitate reduced GSIS in wild-type islets, whereas
UCP2−/− islets had enhanced GSIS.
Importantly, few studies have attempted to explore
the interplay between glucose, amino acids and fatty
acids with respect to β-cell mass and functional integrity
in vitro. In a key recent study [73], culture of clonal
BRIN BD11 β-cells for 24 h with the polyunsaturated
fatty acid arachidonate increased β-cell proliferation
and enhanced amino-acid (alanine)-stimulated insulin
secretion. Conversely exposure to the saturated fatty acid
palmitate for 24 h was found to decrease β-cell viability
(by increasing apoptosis) and increase the intracellular concentration of TAG, while inhibiting alaninestimulated insulin secretion.
INTRODUCTION TO THE EFFECT OF NEFAs
ON β-CELL APOPTOSIS
In addition to the acute effects of fatty acids on the
insulin secretory function of pancreatic β-cells described
above, it has become increasingly clear that fatty acids
can also exert complex effects on β-cell viability during
more chronic periods of exposure. This response is often
perceived as primarily a detrimental effect leading to
compromised viability (a condition that has often been
described as ‘lipotoxicity’) (reviewed in [74]). However,
this is an oversimplification, as it is increasingly evident
that both the chain length and the degree of saturation
of fatty acids can exert a dramatic influence on their
capacity to affect β-cell viability and that not all fatty acids
are intrinsically toxic. In addition, the ambient glucose
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concentration also plays a role, leading some authors
to suggest that the toxic effects should be described as
‘glucolipotoxicity’ rather than simply as lipotoxicity [75].
DOES LIPOTOXICITY (OR
GLUCOLIPOTOXICITY) OCCUR IN β-CELLS
IN VIVO ?
It is important to consider whether (gluco)lipotoxicity
proceeds in vivo and if it might contribute to β-cell loss
under conditions such as those found in Type 2 diabetes,
as some workers have proposed that fatty-acid-mediated
β-cell toxicity may be primarily an in vitro phenomenon
that is unrepresentative of the situation found in vivo [76].
Robertson et al. [77] have considered this matter in
some detail and have argued that the primary alteration
associated with Type 2 diabetes is increased oxidative
stress that is imposed on pancreatic β-cells by virtue of
the prevailing hyperglycaemia. However, when hyperlipidaemia is superimposed on top of hyperglycaemia,
this leads to a still more rapid demise of the cells due
to the attendant cytotoxic effects of fatty acids operating
in the context of elevated glucose.
It is known that elevated circulating NEFAs occur
both under fasting conditions and post-prandially in patients with Type 2 diabetes and, therefore, it must be accepted that the potential for lipotoxic effects at the level
of the β-cell clearly exists. However, it must also be
acknowledged that it is very difficult to obtain conclusive
proof to confirm the involvement of lipotoxicity in
the progression of Type 2 diabetes in man, although
evidence from animal studies is abundantly supportive
[78–80]. Moreover, there is strong evidence that net βcell apoptosis persists in patients with long-term Type 2
diabetes [81] and it is not imprudent, therefore, to deduce
that at least part of this is likely to be caused by the chronic
elevation of NEFAs.
In considering the implications of these various
conclusions it is clear that, in vivo, the combined effects
of elevated glucose and fatty acids may be more relevant
to the long-term effects on β-cell viability than the effects
of fatty acids alone. Moreover, it is also evident that any
reduction in viability associated with these stimuli occurs
over a long timescale. Therefore it is must be recognized
that any effects of fatty acids on β-cell viability seen
during exposure in vitro cannot be fully representative
of the situation in vivo, but this does not mean that
the mechanisms involved are irrelevant or that the effects
are purely artefactual. Rather, the in vitro system should
be viewed as a model in which the pathological processes
are increased in speed, but which can still yield important
information.
What, then, of the suggestion that lipotoxicity is
essentially an artefact of culturing pancreatic β-cells in
the presence of exogenous fatty acids in vitro? This
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suggestion has been made most forcibly by Moffitt et al.
[76] based on their studies of TAG formation during
incubation of a β-cell line INS-1 with exogenous fatty
acids. These authors observed that exposing β-cells to
exogenous fatty acids leads to increased TAG formation
and, as such, they have confirmed earlier results obtained
by several other groups [73,82–85]. However, Moffitt et
al. [76] have extended their analysis to show that the
fatty acid composition of the TAG generated in the cells
reflects the chemical structure of the predominant fatty
acid species present in the incubation medium, and they
noted further that, in the case of long-chain saturated
fatty acids (e.g. palmitate), this leads to the formation
of TAG species that are essentially solid at physiological
temperatures. As a result, these lipid species accumulate
in the cell and effectively cause a physical disruption of
cellular integrity by virtue of their inflexible physical
state. Hence, Moffitt et al. [76] argue that lipotoxicity may
be an artefact of the in vitro incubation of β-cells with
single species of long-chain saturated fatty acids which
are not representative of the fatty acid composition of the
extracellular fluids encountered by β-cells in vivo.
The latter proposition may be correct, but the idea
that fatty acid toxicity elicited in vitro does not represent
a relevant mechanism from which important mechanistic
information can be deduced is more contentious. In fact,
the implications derived by Moffitt et al. [76] sit at the
heart of a, still raging, debate about the functional role of
TAG in fatty acid toxicity in β-cells. On one side of
the argument are those who consider that fatty-acidinduced TAG formation is beneficial to β-cell survival (by
sequestration of potentially toxic fatty acid derivatives)
[78,84] whereas, on the other, there are groups who
suggest that it is causative to β-cell death (by promoting
the physical disruption of the cells) [85–89]. All agree
that β-cell TAG levels are increased following exposure
to fatty acids, but they diverge dramatically on the
interpretation of this finding.
One possibility that might explain the functional
impact of TAG accumulation in β-cells is enhanced
TAG/fatty acid cycling. This can be expected to consume
considerable amounts of glucose (as a supplier of glycerol
3-phosphate for esterification) and ATP, and the diversion
of ATP to TAG/fatty acid cycling and release of
potentially high intracellular concentrations of NEFAs
may negatively impact on β-cell function and viability.
POTENTIAL MEDIATORS OF β-CELL FATTY
ACID TOXICITY
NEFAs are rapidly transported into cells and the bulk of
these are then esterified to form acyl-CoA for subsequent
metabolism by β-oxidation or storage as neutral lipids.
There is general agreement that long-chain saturated
(rather than shorter chain saturated or long-chain
Role of non-esterified fatty acids in β-cell function and integrity
unsaturated) fatty acids are the most toxic to β-cells and
that the initial esterification step appears to be critical,
as agents such as Triacsin-C, which block this step,
effectively prevent β-cell death [75,90,91]. Thus it is not
the NEFA itself which causes cell death, but a derivative
generated as a result of acyl-CoA synthesis. In support of this, the methyl derivative of palmitate in which
the carboxy group is unavailable for esterification to
CoA is not toxic (H. J. Welters and N. G. Morgan,
unpublished work). Moreover, bromopalmitate, which
can be esterified to CoA, but cannot then be oxidized
further, is, in our hands, also non-toxic to β-cells,
although this has not been universally observed [84].
Moreover, we find that bromopalmitate attenuates the
toxicity associated with exposure to palmitate. The simplest explanation for this is that bromopalmitate sequesters the pool of free CoA thereby reducing the rate
of esterification of palmitate and minimizing its toxicity,
although it cannot be excluded that bromopalmitate may
also influence other aspects of palmitate utilization (e.g.
protein acylation).
The specificity of fatty acid toxicity in β-cells has not
been studied very extensively and most investigators have
employed palmitate and/or oleate as the fatty acids of
choice. This is probably because these two fatty acids
represent the major species present in human serum
and, therefore, they are the principal molecules to which
β-cells might be exposed in vivo [92,93]. However, a
consideration of the specificity of the effects of fatty acids
on β-cell viability is informative, as it has been shown
that the C14 saturated molecule myristate is tolerated to a
much greater extent than palmitate [94,95], implying that
some important feature of the way these two molecules
are handled determines their relative toxicity in β-cells.
Myristate oxidation has not been extensively studied in
β-cells and it remains to be confirmed that this proceeds
at an equivalent rate to that of palmitate. Arachidonate is
also relatively well-tolerated by β-cells and has even been
reported to exert beneficial effects on insulin secretion
and β-cell mass [73,96]. This may reflect the fact that
arachidonate occurs with relatively low abundance in
β-cells (approx. 2 % of total) and that provision of
exogenous arachidonate leads to exchange of more ‘toxic’
species (e.g. palmitate) for arachidonate, thereby exerting
beneficial effects.
Whatever the nature of the metabolite that is responsible for initiating the loss of viability caused by palmitate, it seems probable that a succession of additional
steps are required to cause the final demise of the cell. A
variety of signals have been implicated in this respect; the
most studied of which include NO, ceramide formation,
inhibition of PKB (protein kinase B)/Akt activity [97,98],
activation of calpain-10 [99] or activation of PKCδ
[53,97,100].
NO is widely accepted to play a role in the death of βcells exposed to pro-inflammatory cytokines implicated
in the development of Type 1 diabetes, and some
investigators have considered NO as a common signal
that might also be elevated by fatty acids [101]. Indeed,
NO formation has been reported in fatty-acid-treated
β-cells by several different groups and this has been
correlated with the induction of iNOS (inducible form
of NO synthase) in some cases [102–106]. By contrast, a
number of other studies have failed to find any direct evidence that fatty acids promote a rise in NO formation
in β-cells or in other cell types [84,94,107–109]. The
hypothesis of a cell death mechanism involving activation
of NF-κB (nuclear factor κB; which regulates the
transcription of iNOS) in β-cells exposed to fatty acids
has been proposed [101,110,111], but it was shown
recently that NF-κB-dependent genes are not induced
in response to fatty acids [110]. Thus there is no firm
consensus as to whether NO formation represents a
common factor that might mediate fatty acid toxicity
and it remains enigmatic why some studies report iNOS
induction and NO formation in fatty-acid-treated β-cells
while others do not.
The sphingolipid ceramide is a second candidate that
has been proposed as a mediator of palmitate-induced βcell toxicity, and this idea sits well with some experimental
observations; notably the finding that the rate-limiting
step in its biosynthesis shows a marked preference
for saturated fatty acids having a chain length of C16
(compared with C14 ) [112]. Thus ceramide levels are
expected to increase in cells exposed to palmitate, whereas
they are less likely to be changed when myristate is
provided. This difference could provide a convenient
explanation for the differential specificity of these two
fatty acids as mediators of β-cell death if ceramide
formation is involved. The precise mechanisms by which
changes in ceramide might cause β-cell death are not
entirely clear, although it seems likely that alterations
in the disposition of plasma membrane survival and/or
death receptor signalling complexes could be involved.
Palmitate has been shown to promote ceramide
formation in β-cells [113], and treatment of islet cells
with ceramide analogues can lead to loss of viability
[102,114], although the magnitude of this effect is often
rather modest [115]. In addition, an inhibitor of ceramide
synthesis, fumonisin-1B, has been reported to attenuate
palmitate-induced cytotoxicity in both rodent [102,116]
and human [117,118] islet cells, suggesting that ceramide
formation might be involved in fatty-acid-induced death
under some circumstances. However, this agent was
not effective in pure populations of β-cells [94,107],
which implies that ceramide formation may not account
completely for the cytotoxic response to palmitate.
One hypothesis that has been considered in some detail
is that activation of a specific isoform of PKC (PKCδ)
may underlie the ability of palmitate to promote βcell death. This is supported by evidence that PKCδ is
translocated from the cytosol to the nucleus as an early
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event in palmitate-treated cells [53,97,100] and that PKCδ
is required for apoptosis in various cell types [119–121].
This may be mediated, at least in some cells, by PKCδinduced phosphorylation and activation of caspase 3
[122]. Caspase 3 activity is increased in cells treated
with palmitate which would be consistent with such a
mechanism [75,97,123]; however, much of the evidence
implicating PKCδ in palmitate-induced β-cell apoptosis
has come from experiments conducted with rottlerin, a
purportedly selective PKCδ inhibitor [124], which has
been shown to suppress palmitate toxicity very effectively
[97,125]. Taken at face value, this would be consistent
with a central role for PKCδ in mediating the response
to palmitate, but this conclusion may be premature.
For example, studies in BRIN-BD11 β-cells suggest that
PKCδ can be successfully down-regulated by treatment
with PMA, but that this manoeuvre fails to alter their
susceptibility to palmitate toxicity or their sensitivity to
rottlerin [125]. Although it is known that rottlerin can
affect other intracellular signalling components [126],
so far none of these has been directly correlated with
palmitate toxicity.
In addition to rottlerin, we have also shown [127] that
inhibitors of gene transcription and mRNA translation
can markedly attenuate the ability of saturated fatty acids
to cause β-cell death, suggesting that early changes in
gene expression may be instrumental in the process.
Indeed, exposure of β-cells to palmitate is associated
with a range of alterations in gene expression and some
of the candidates have been identified [118,128–131].
Among these is the nuclear receptor Nur77, which has
been suggested as a possible regulator of lipotoxicity in
response to fatty acids in β-cells, although its precise
role has not been defined [132]. In some cells, Nur77
serves to regulate lipid metabolism [133] and it is possible,
therefore, that its up-regulation in β-cells represents
principally an adaptive response to fatty acid availability.
One potential means to identify the pathway by which
saturated fatty acids cause a loss of β-cell viability is
to grow cells in medium containing fatty acids and to
use this as a means to exert selection pressure such that
long-term survivors can be propagated. Such cells can be
expected to exhibit a variety of underlying mechanistic
alterations, but some may display specific changes in
the pro-apoptotic pathway by which fatty acids induce
apoptosis. This approach has recently been taken by
Busch et al. [134], who succeeded in identifying MIN-6
β-cell clones that are resistant to palmitate toxicity. The
resistant cells display elevated rates of palmitate oxidation
which implies that oxidation itself is not the means of
toxicity, but they also have enhanced expression of a
desaturase enzyme involved in cholesterol ester synthesis.
As a consequence, the cells also have elevated ratios
of oleate to palmitate and this may be related to their
resistance to lipotoxicity, since certain unsaturated fatty
acids are often protective (see below). However, it is
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also possible that the diversion of palmitate metabolism
towards cholesterol ester formation forms part of the
protective mechanism, which supports the concept that
palmitate utilization (other than to generate cholesterol
esters) is normally important in causing its lipotoxic
effects.
A further pathway that should be considered as a
potential mediator of lipotoxicity is the ER (endoplasmic
reticulum) stress response that is known to be associated
with increased apoptosis in a number of situations. ER
stress leads to the altered expression of a variety of
characteristic genes involved in the regulation of protein synthesis and may also promote the generation of
ceramide. It is primarily a protective response designed to
minimize cell damage by inhibition of protein synthesis
in the face of unfavourable conditions, but prolonged
ER stress is ultimately detrimental (reviewed in [135]). In
various cell types, including β-cells, exposure to palmitate
induces a pattern of protein alterations that is consistent
with long-term ER stress [136–138], and imposition of
ER stress may, therefore, be a contributory factor to fattyacid-induced apoptosis in β-cells.
One of the changes in gene expression that may be
indicative of a state of ER stress is increased expression
of the transcription factor SREBP-1c, which acts as a
lipogenic molecule in β-cells [139]. Overexpression of
SREBP-1c in the β-cells of transgenic mice leads to
increased TAG accumulation and a reduction in β-cell
mass [140], and this may recapitulate some of the features
of lipotoxicity as SREBP-1c is also elevated in isolated
β-cells exposed to fatty acids. Additionally, Yamashita
et al. [141] overexpressed SREBP-1c in INS-1 cells,
which resulted in enhanced TAG accumulation, blunted
GSIS and enhanced expression of UCP-2. siRNA (small
interfering RNA) to UCP-2 increased the ATP/ADP
ratio and partially rescued GSIS but it did not affect
the TAG content. This would argue in favour of increased TAG/fatty acid cycling under conditions of
SREBP1c overexpression which, due to elevated cytosolic
NEFA concentrations, would activate UCP-2 activity,
thus decreasing mitochondrial efficiency. An alternative
target for SREBP-1c is the promoter region of IRS-2
(insulin receptor substrate-2) [139], a tyrosine kinase
substrate that regulates survival pathways controlled by
PI3K (phosphoinositide 3-kinase) and PKB/Akt. Indeed,
increased expression of SREBP-1c leads to reduced
transcription of IRS-2 in β-cells (probably be displacing
certain forkhead transcription factors that are required
to mediate transcription), suggesting that loss of IRS-2,
and thereby attenuated signalling through the PI3K
survival pathway, might be one consequence of exposure
of β-cells to fatty acids. This hypothesis warrants further
investigation since it has also been proposed that control
of IRS-2 levels might represent a common step at
which multiple pro-apoptotic pathways converge in the
β-cell [142] and blockade of the PI3K pathway may
Role of non-esterified fatty acids in β-cell function and integrity
enhance fatty-acid-induced death in β-cells under some
circumstances [107]. Sustained expression of SREBP1c (perhaps initiated by the ER stress response) may
therefore prove to be a critical factor that causes the
ultimate loss of viability in β-cells exposed to saturated
fatty acids.
Increasing evidence suggests that another key organelle
that can regulate the entry of cells into apoptosis is the
mitochondrion [143]. It is well established that the inner
mitochondrial membrane contains a group of proteins
that are regulated to form a pore [the MPTP (mitochondrial permeability transition pore] through which
certain, critical, pro-apoptotic proteins can exit under
specific conditions [144]. Among these is cytochrome c
which is normally resident within the inner mitochondrial
membrane and does not access the cytosol. However,
under pro-apoptotic conditions cytochrome c can be
released, via the MPTP, to form a cytosolic complex
with Apaf-1 (apoptotic protease-activating factor-1) and
caspase 9, leading to the activation of the latter enzyme
and thereby promoting the effector arm of apoptosis.
It has been reported that exposure of β-cells to saturated fatty acids leads to loss of mitochondrial membrane
potential and the release of cytochrome c, suggesting that
the mitochondrial pathway may be important for fattyacid-induced cytotoxicity [105,116,118]. If this is the case,
then additional alterations at the level of the mitochondria
are likely to precede the release of cytochrome c, as this
molecule is normally anchored at the inner face of the
lipid bilayer of the mitochondrial inner membrane by
cardiolipin. Reduction in the levels of cardiolipin have
been implicated in palmitate-induced apoptosis in other
cell types [90,145], but has received very little attention
as a possible mediator of fatty-acid-induced apoptosis in
β-cells. Cardiolipin could, though, play a significant role
since its composition will be altered by the prevailing
fatty acid milieu. This is because cardiolipin contains
four fatty acid molecules in its structure and, normally,
these are primarily unsaturated molecules as cardiolipin
synthase displays a marked preference for unsaturated
fatty acids. If these are replaced with saturated species,
then the binding properties of cardiolipin are altered
and its affinity for cytochrome c is markedly reduced.
This will tend to promote dissociation of cytochrome
c from cardiolipin which, in turn, will allow it to exit
from the mitochondrion via the MPTP. Thus alterations
in the binding of cytochrome c to cardiolipin, arising from
changes in the fatty acid composition of its side chains,
may account for the increased propensity of saturated
fatty acids to induce mitochondrial cytochrome c release
(reviewed in [146]). This mechanism could also explain,
in part, why unsaturated fatty acids are better tolerated
by β-cells than their saturated counterparts.
In a recent study [147], mitochondrial DNA damage
was reported to occur in INS-1 β-cells exposed to fatty
acids, and this effect could be overcome by targeted
expression of the DNA repair enzyme OGG1 (8oxoguanine DNA glycosylase/apurinic lyase). Moreover,
increased expression of OGG1 minimized the induction
of apoptosis in the fatty-acid-treated cells, implying
that damage to mitochondrial DNA might be a critical
component of the cytotoxicity. The protective effects of
OGG1 expression were also accompanied by reduced
cytochrome c release from the mitochondria, implying
that mitochondrial damage could be causative factor in
mediating the lipotoxic response under the conditions
employed.
Further evidence for the involvement of mitochondria
in NEFA-induced apoptosis comes from studies of
the expression of Bcl-2 and related proteins. Bcl-2 is
a member of the large family of apoptosis-regulator
proteins that either facilitate cell survival (e.g. Bcl-2,
Bcl-XL , Bcl-w and others) or promote cell death (Bax,
Bak, Bad etc.). The relative amounts of these proteins
plays a key role in determining cell fate [148,149], and
apoptosis of islet cells in response to saturated fatty acids
is accompanied by a marked reduction in Bcl-2 expression
[75,83,118,131] and up-regulation of Bax [105].
PROTECTIVE EFFECTS OF FATTY ACIDS
AGAINST β-CELL APOPTOSIS: THE
LONG-CHAIN FATTY ACID PARADOX
One disconcerting feature that emerges from an examination of the literature covering effects of fatty acids in
β-cells is that there is often very little consensus about the
net outcome as reported by different sets of investigators.
This frustration is compounded by the fact that it
appears to bear no obvious relationship to the model
under investigation (whether a cell line or isolated islets;
one species compared with another) and the differential
effects reported are frequently diametrically opposed
(rather than being subtle alterations of response). This
failure to achieve consensus can be extremely unsettling
but it should not be readily dismissed. One factor
that almost certainly contributes (but does not provide
a full explanation) is that fatty acids are intrinsically
difficult molecules to work with; not least because they
are readily altered in culture (e.g. by peroxidation) and
because they can bind in different proportions to many
components that are typically present in cell cultures,
including proteins (especially albumin and other serum
proteins) and even to tissue culture plastics. Thus experiments that are apparently performed under identical conditions in different laboratories may not be equivalent.
Indeed, in our hands, even batches of the same albumin
formulation purchased from a given supplier can give rise
to quite different responses when fatty acids are employed
in vitro.
However, despite these differences, one common
and striking theme that emerges from the literature
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is that saturated and monounsaturated fatty acids of
equivalent chain length exert distinct effects on β-cells.
As discussed in preceding sections, there is little debate
that saturated long-chain molecules are toxic to βcells during chronic exposure but, equally, an area of
reasonably strong consensus is that monounsaturated
(and most polyunsaturated) molecules are well tolerated
[84,94,107,116,118,150,151]. Thus there appears to be
a critical difference in response that derives from the
introduction of a single double bond into a long-chain
fatty acid molecule. It might be easy to dismiss this
as the inevitable outcome of a change in a parameter
such as membrane fluidity; however, this would be
an oversimplification and there may be a much more
significant explanation that has important implications
for the understanding of β-cell biology.
This is illustrated most clearly from studies in
which both saturated and monounsaturated molecules
are provided to β-cells in combination. Thus, whereas
palmitate is toxic to β-cells when used in isolation, its
toxicity is dramatically attenuated (even abolished) by the
simultaneous presence of a monounsaturated molecule,
such as palmitoleate or oleate [105,116,118,150]. Hence
these latter species are not ‘inert’ as far as their
effects on β-cell viability is concerned; rather they are
actively protective. This is an important distinction that
has critical implications for an understanding of their
actions.
Perhaps the most obvious factor that might influence
the toxicity of a saturated fatty acid is the ability of
another agent (including an unsaturated analogue) to
alter some aspect of its metabolism. This could occur
at any one of several levels ranging from a change in
the rate of uptake into the cell (e.g. by competing for a
common transporter) to competition for esterification to
CoA and/or an alteration in the ultimate metabolic fate.
These various possibilities have only been investigated to
a limited extent in β-cells, but evidence has been obtained
[94] which supports the view that the anti-apoptotic
protection exerted by monounsaturated fatty acids does
not require their activation or subsequent metabolism in
the β-cell. Among these data are studies with etomoxir
and poorly metabolized fatty acid derivatives [94]. For
example, etomoxir completely failed to alter the ability of
palmitoleate to protect β-cells against serum-withdrawalinduced apoptosis, suggesting that mitochondrial entry
and subsequent β-oxidation of the monounsaturated
molecule is not a pre-requisite for its protective
action [94]. Moreover, the non-metabolizable analogue
methyl-palmitoleate attenuated β-cell cytotoxicity very
effectively [152], thereby providing additional evidence
that formation of fatty acyl-CoA is not required for the
protective response, since the presence of the methyl
group will mitigate against acyl-CoA formation. Thus
the protective mechanism must involve a process that is
selectively activated by (mono)unsaturated fatty acids,
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but which does not directly require their metabolism
within the cell.
The validity of this conclusion is illustrated most
dramatically by experiments in which the time of
addition of saturated and monounsaturated fatty acid to
cultured β-cells was varied to eliminate early changes
in metabolic fate arising from the combined presence of
both molecules. Under such conditions, it was established
that the unsaturated molecule palmitoleate could be
introduced as late as 6 h after the addition of palmitate
without dramatically altering its ability to protect β-cells
against the cytotoxic effects of the saturated molecule
[94]. This result has several implications. First, it confirms
that the toxicity of palmitate is not mediated by an instant
‘lytic’ effect (perhaps due to an action on membrane
permeability), as this would not be preventable by an
intervention occurring as much as 6 h later. Secondly, it
implies that the protective action of palmitoleate is likely
to occur rapidly since, in the absence of this molecule,
the toxicity of palmitate is already becoming evident
within 6–8 h and cell death is increasing at this time
during incubation in vitro. When considered in the context of the data (outlined above) showing that the
non-metabolizable derivative of palmitoleate, methylpalmitoleate, is equally effective as palmitoleate at protecting β-cells against palmitate toxicity, this suggests
very strongly that altered fatty acid metabolism does not
underlie the protective response.
Taken together, these observations suggest that the
monounsaturated fatty acids may control β-cell viability
in a more fundamental way and data to support this
have now been provided. Arguably, the most convincing
evidence in this regard has come from the unexpected
demonstration that monounsaturated molecules retain
their ability to exert protective actions when β-cell viability is reduced by alternative means that do not involve
incubation with saturated fatty acids. For example, the
loss of viability associated with withdrawal of serum
from cultures of β-cells is prevented by the addition of
palmitoleate, and the well-known cytotoxic actions
of pro-inflammatory cytokines are similarly attenuated
by the monounsaturated fatty acid [94]. Neither of these
responses would be expected to involve an alteration in
the metabolism of saturated fatty acids and, therefore,
they point to the conclusion that palmitoleate controls
β-cell viability in an entirely different way.
One obvious possibility is that monounsaturated fatty
acids may exert their protective effects by interaction
with either a cell-surface or intracellular receptor, rather
than by acting at an intracellular site. Consistent with
this idea is the fact that the protective actions occur at
very low concentrations of NEFAs, which are likely to
be in the low (or even sub) nanomolar range and are
at least 10-fold less than the concentration of saturated
molecules required to initiate cell death. (Note that
the total concentration of fatty acid is always much
Role of non-esterified fatty acids in β-cell function and integrity
Figure 2 Potential cytoprotective effects of NEFAs
Fatty acids (FFA) may enter the β-cell and become activated by conversion into acyl-CoA. The fatty acids may then become incorporated into TAG, be oxidized in the
mitochondrial matrix, be incorporated into cardiolipin or bind to and activate specific transcription factors or receptors. NEFA may also bind to GPR40 receptors and
stimulate relevant signal transduction pathway(s). All or some of these mechanisms may help protect the β-cell from potentially damaging nutrient or inflammatory
factors. AA, arachidonate; MUFA, monounsaturated fatty acid.
higher than the free concentration since most is not
biologically active due to its binding by other components
present in the incubation medium.) It is well known that
fatty acids can interact with the class of intracellular
PPARs (peroxisome-proliferator-activated receptors),
and there are reports that other agonists of these
molecules (including some thiazolidinedione PPARγ
agonists) can protect β-cells against NEFA-induced
damage [80,83,153], although this has not been seen in
all studies [127].
The idea that NEFAs may exert some of their effects
by interaction with cell-surface receptors has emerged
only relatively recently, but is becoming increasingly
accepted with the description of at least two previously designated ‘orphan’ G-protein-coupled receptors
(GPR40 and GPR120), whose endogenous ligands appear
to be long-chain fatty acids (others with specificity for
short-chain fatty acids also exist) [61,154,155]. One of
these, GPR40, is known to be expressed in β-cells and
has been implicated in mediating the acute regulatory
effects of long-chain fatty acids on insulin secretion [61–
63,154,156]. However, this receptor appears to bind both
saturated and unsaturated molecules with approximately
equal affinity and, for this reason, appears unlikely to be
responsible for mediating the highly selective effects of
unsaturated molecules on cell viability. The possible role
of GPR120 has not yet been studied in β-cells, although,
in the original report describing it as a NEFA receptor,
it was suggested to be absent from the β-cell line MIN-6
[157]. These findings suggest that GPR120 may not be
a prime candidate in mediating the protective effects of
unsaturated fatty acids in β-cells, but this conclusion still
requires verification in other β-cell lines and in normal
islets. This is especially true since GPR120 has recently
been implicated as the mediator of fatty-acid-induced
protection during serum-withdrawal-mediated apoptosis
in the intestinal cell line STC-1 [155].
Given the (still hypothetical) possibility that monounsaturated fatty acids may protect β-cells via ligation
of a cell-surface receptor, this raises a question about
the second messenger system that might underlie the
response. One well-described receptor-mediated antiapoptotic signalling mechanism in β-cells is exemplified
by GLP-1, which appears to act by raising cAMP levels
(reviewed in [158,159]). Thus it is possible that cAMP
could represent a common anti-apoptotic signalling
molecule in the β-cell whose level might be regulated
by monounsaturated fatty acids. We have examined
this possibility directly [160] and have shown that
cAMP is not elevated in BRIN-BD11 β-cells exposed
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to palmitoleate under conditions when its potent antiapoptotic effect is evident. On this basis, we do not
consider that cAMP represents a strong candidate to
fulfil the requirements of the second messenger mediating
the protective actions of monounsaturated fatty acids.
In support of this, we have noted further that, when
β-cells are treated with saturated fatty acids under
conditions favouring a rise in cAMP, their viability is
not improved significantly [160]. This lack of protection
occurs despite a reduction in caspase 3 activity in cells
having elevated cAMP and appears to reflect a switch
from a primarily apoptotic mode of death, to necrosis,
under these conditions. This situation stands in marked
contrast with that seen when monounsaturated fatty acids
are employed as the protective agents, where cell viability
is maintained at a high level and secondary necrosis does
not occur [160].
Alternative intracellular pathways that could be involved in the cytoprotection mediated by monounsaturated fatty acids abound, but none has yet emerged as
a strong candidate. We have summarized some of the
possible cellular protective pathways in Figure 2. Indeed,
few protective mechanisms have been investigated in
depth, although Beeharry et al. [107] have reported that
inhibitors of the PKB/Akt pathway (acting at the level of
PI3K) can minimize the protective effects of unsaturated
fatty acids in β-cells. This could imply that PI3K and thus
pro-survival signalling is regulated by monounsaturated
fatty acids, but this conclusion remains to be confirmed.
Even if it proves to be the case, then this still leaves open
the question as to how unsaturated fatty acids initiate the
activation of this (or any other) survival pathway(s) and
why saturated molecules do not. Clearly, there is much
work still to be done to unravel the molecular basis of
these effects.
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ACKNOWLEDGMENTS
We thank the Health Research Board of Ireland,
Enterprise Ireland, Diabetes UK, Wellcome Trust,
Boehringer Ingelheim Pharma, Institut Recherches
Internationales Servier, GB Sasakawa Foundation and the
Northcott Devon Medical Foundation for funding our
research.
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Received 11 May 2006/26 June 2006; accepted 3 July 2006
Published on the Internet 1 December 2006, doi:10.1042/CS20060115
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