Minireview: implication of mitochondria in insulin secretion and action

(1)

Article

Reference

Minireview: implication of mitochondria in insulin secretion and action

WIEDERKEHR, Andreas, WOLLHEIM, Claes

Abstract

Mitochondria are essential for intermediary metabolism as well as energy production in the cell. Their aerobic metabolism permits oxidation of glucose and fatty acids for the generation of ATP and other intermediates that are exchanged with the cytoplasm for various biosynthetic and secretory processes. In the pancreatic beta-cell, glucose carbons are quantitatively funneled to the mitochondria, where signals for the initiation and potentiation of insulin secretion are generated. After mitochondrial activation, the plasma membrane is depolarized with ensuing cytosolic calcium transients and exocytosis of insulin. Calcium also acts in a feed-forward manner on mitochondrial metabolism, which contributes to sustained second phase insulin secretion. Patients with mitochondrial diabetes and a corresponding mouse model display defective glucose-stimulated insulin secretion and reduced beta-cell mass, leading to overt diabetes. Normal mitochondrial activity appears to be equally important in the action of insulin on its target tissues. The development of insulin resistance may involve impairment of glucose oxidation after short [...]

WIEDERKEHR, Andreas, WOLLHEIM, Claes. Minireview: implication of mitochondria in insulin secretion and action. Endocrinology , 2006, vol. 147, no. 6, p. 2643-9

DOI : 10.1210/en.2006-0057 PMID : 16556766

Available at:

http://archive-ouverte.unige.ch/unige:47613

Disclaimer: layout of this document may differ from the published version.

1 / 1

(2)

Minireview: Implication of Mitochondria in Insulin Secretion and Action

Andreas Wiederkehr and Claes B. Wollheim University of Geneva Medical Center, Geneva, Switzerland

Mitochondria are essential for intermediary metabolism as well as energy production in the cell. Their aerobic metabo- lism permits oxidation of glucose and fatty acids for the gen- eration of ATP and other intermediates that are exchanged with the cytoplasm for various biosynthetic and secretory processes. In the pancreatic␤-cell, glucose carbons are quan- titatively funneled to the mitochondria, where signals for the initiation and potentiation of insulin secretion are generated.

After mitochondrial activation, the plasma membrane is de- polarized with ensuing cytosolic calcium transients and exo- cytosis of insulin. Calcium also acts in a feed-forward manner on mitochondrial metabolism, which contributes to sustained second phase insulin secretion. Patients with mitochondrial

diabetes and a corresponding mouse model display defective glucose-stimulated insulin secretion and reduced␤-cell mass, leading to overt diabetes. Normal mitochondrial activity ap- pears to be equally important in the action of insulin on its target tissues. The development of insulin resistance may in- volve impairment of glucose oxidation after short exposure to increased levels of circulating free fatty acids. Insulin resis- tance in the elderly and in relatives of type 2 diabetic patients has also been associated with mitochondrial dysfunction.

Both prevention and treatment of type 2 diabetes should focus on mitochondrial targets for the improvement of nutrient- stimulated insulin secretion and their utilization in periph- eral tissues. (Endocrinology147: 2643–2649, 2006)

M

ITOCHONDRIA NOT ONLY play a crucial role in cellular energy production, but also participate in intermediary metabolism, Ca^2⫹handling, as well as apoptosis (1, 2). It is well established that␤-cell mitochondria are key determinants in glucose-stimulated insulin secretion and participate in the actions of insulin on its target tissues.

Recently, particular interest has focused on mitochondria in

␤-cells and target tissues in the development of type 2 diabetes (3), prompted by novel genetic findings in tissues from diabetic subjects. This article will highlight the implication of mitochondria in normal metabolism secretion coupling in the␤-cell and how the process may be impaired in the diabetic state. It will also briefly address certain aspects of mitochondrial function in insulin action in type 2 diabetes.

Glucose Metabolism in the␤-Cell

␤-Cells are designed to sense blood glucose and other secretagogues to adjust insulin secretion according to the needs of the organism. Rather than activating specific receptor molecules, glucose is metabolized to generate downstream signals that stimulate insulin secretion (4 – 6). Glucose enters ␤-cells by facilitated diffusion through the glucose transporter (GLUT2 in rodents; mainly GLUT1 in humans)

and is retained inside the cell through its phosphorylation by glucokinase (Fig. 1). This glucose sensor mechanism adjusts glycolytic flux to the plasma glucose concentration in hepa- tocytes and islet cells (7).

Pyruvate is the main end product of glycolysis in␤-cells, because the cell contains extremely low levels of lactate dehydrogenase (8). Pyruvate is efficiently channeled into mitochondria, where more than 90% of the glucose-derived carbons are converted to CO2; this high proportion of aerobic metabolism is at least 3-fold greater than that in most other cell types (9).

In the mitochondrial matrix, pyruvate has two main fates:

oxidation by pyruvate dehydrogenase to form acetyl-coenzyme A (acetyl-CoA; oxidative pathway) or carboxylated by pyruvate carboxylase to form oxaloacetate (anaplerotic pathway) (9, 10). Acetyl-CoA enters the tricarboxylic acid (TCA) cycle to undergo additional oxidation steps generating CO₂ and the reducing equivalents, flavin adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH).

These reducing equivalents enter the respiratory chain to form an electrochemical gradient across the inner mitochondrial membrane. The final electron acceptor of these reactions is molecular oxygen. The proton motive force then drives ATP synthesis at complex V (ATP synthase), thereby linking respiration to the synthesis of ATP from ADP and inorganic phosphate (2) (Fig. 1).

Synthesis of oxaloacetate from pyruvate serves a separate, possibly as important, function in mitochondrial metabolism, especially in the␤-cell that has high pyruvate carboxylase activity (9, 11). As suggested by the term anaplerotic pathway, oxaloacetate fills up or adds new intermediate metabolites to the TCA cycle. Unlike oxaloacetate itself, some of the intermediates derived from oxaloacetate, such as malate, citrate, isocitrate, and 2-oxoglutarate, can leave the First Published Online March 23, 2006

Abbreviations: acetyl-CoA, Acetyl-coenzyme A; AMPK, AMP-dependent kinase; GLUT, glucose transporter; GSIS, glucose-stimulated insulin secretion; mtDNA, mitochondrial DNA; NADH, nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine nucleotide phosphate; OXPHOS, oxidative phosphorylation; PGC1␣, peroxisome proliferator-activated receptor-␥ coactivator-1␣; ROS, reactive oxygen species; TCA, tricarboxylic acid; TFAM, mitochondrial transcription factor A; UCP2, uncoupling protein 2.

Endocrinologyis published monthly by The Endocrine Society (http://

www.endo-society.org), the foremost professional society serving the endocrine community.

doi: 10.1210/en.2006-0057

2643

(3)

TCA cycle and cross the inner mitochondrial membrane (12).

This process depends on the mitochondrial membrane potential. Thus, these metabolites can serve as precursors for many essential molecules in the mitochondrial matrix as well as in the cytoplasm. Importantly, glutamate aspartate transport is rate limiting to mitochondrial metabolism, because overexpression of aralar1 enhances glucose-stimulated insulin secretion (GSIS) (13). Anaplerotic metabolism in the

␤-cell may be used to generate coupling factors that contrib- ute to GSIS (see below). Due to the very low lactate dehydrogenase expression in␤-cells, NADH formed during glycolysis would accumulate and reduce glycolytic flux unless NADH is reoxidized. Therefore, the hydrogen shuttles (malate/aspartate and glycerophosphate shuttles) that transport reducing equivalents from the cytoplasm to the mitochondria are particularly active in the␤-cell (8, 14).

Mitochondrially Derived ATP in Metabolism Secretion Coupling

Mitochondrial ATP production is essential for GSIS, which is abolished by inhibitors of mitochondrial ATP synthesis.

Such inhibitors also attenuate ␤-cell glycolysis as cytosolic NADH accumulates (15). This is also shown directly in mitochondrial DNA (mtDNA)-deficient Rho⁰cells (14), which lack mitochondrially encoded subunits of the respiratory chain complexes I, III, IV, and V. Based on these defects, mitochondrial ATP production is absent in Rho⁰cells. GSIS is abolished in such cells, which, however, still generate enough nonmitochondrial ATP for insulin synthesis, storage, and the triggering of secretion by Ca^2⫹-elevating agents (14, 16). Furthermore, stimulation of ␤-cells with substrates that activate mitochondrial metabolism directly, such as membrane- permeant methyl-succinate, is sufficient to effectively induce insulin secretion (17). Pyruvate itself evokes insulin secretion in islets transduced with the plasma membrane monocarboxylate transporter, which is normally not expressed in ␤-cells (17).

Optimal stimulatory concentrations of the different carbon sources stimulate insulin secretion to a similar extent, suggest- ing that mitochondrial metabolism becomes rate limiting and sets an upper limit on insulin secretion in the presence of excess substrate (18).

Thus, compelling evidence demonstrates that ATP is a key factor coupling mitochondrial metabolism to insulin secretion. ATP production is accelerated after glucose stimulation to provide the energy for insulin exocytosis, replenishing of insulin granules, and establishment of a new steady state of ion gradients. Despite increased ATP consumption, cytosolic ATP levels in␤-cells are increased by about 40% after a shift from basal to high glucose (17). ATP synthesized in the mitochondrial matrix is transported to the cytosol in exchange for cytosolic ADP via adenine nucleotide transloca- tors. The resulting increase in the cytosolic ATP:ADP ratio reduces the opening probability of the plasma membrane K_ATPchannel. Among the molecular components regulating insulin secretion, the K_ATPchannel has proven to be a very useful target, because its opening is blocked by sulfonylurea compounds (5). These are widely used in the treatment of type 2 diabetes. Functional K_ATP channels are essential for GSIS, and a common polymorphism in the K_ATPchannel has been linked to type 2 diabetes (19). Closure of K_ATPchannels depolarizes the plasma membrane, triggering the opening of voltage-dependent Ca^2⫹channels (Fig. 1). As in other exocytotic fusion events, Ca^2⫹is an essential trigger of insulin granule exocytosis (20). The Ca^2⫹signal resulting from mitochondrial activation completes the chain of events between glucose uptake into the␤-cells and insulin exocytosis.

Role of Ca^2ⴙin Mitochondrial Activation Cytoplasmic Ca^2⫹increases after the opening of voltage- gated Ca^2⫹ channels. Ca^2⫹ exerts multiple functions in

␤-cells in addition to its role in exocytosis. The cytoplasmic Ca^2⫹signal is relayed to the mitochondria (Fig. 1). This is FIG. 1. Role of mitochondria in metab-

olism-secretion coupling of the ␤-cell.

Respiratory chain complexes I–IV (or- ange), complex V/ATP synthase (green), calcium transport systems (yellow), transporters/shuttles of the inner mitochondrial membrane (pale blue), and open and closed forms of the K_ATPchan- nel (dark blue) are indicated. ACC, Acetyl-CoA carboxylase; GDH, glutamate dehydrogenase; GK, glucokinase;

ME, malic enzyme; TCA, TCA cycle.

2644 Endocrinology, June 2006, 147(6):2643–2649 Wiederkehr and Wollheim • Minireview

(4)

amplified by hyperpolarization of the inner mitochondrial membrane, increasing the driving force for Ca^2⫹uptake (17).

Inside the organelle, Ca^2⫹activates several matrix dehydro- genases (pyruvate dehydrogenase, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase). This is reflected by Ca^2⫹-driven oscillations of reduced nicotinamide adenine nucleotide phosphate, NAD(P)H, in glucose-stimulated

␤-cells (21). Ca^2⫹has also been suggested to stimulate ATP synthase directly (22). These coordinated actions should re- inforce GSIS, possibly during the second phase of insulin secretion. Both first and second phases of GSIS are attenuated in type 2 diabetic patients and first degree relatives (for review, see Ref. 23). Ca^2⫹can also be sensed at the outer surface of the inner mitochondrial membrane, because the above-mentioned glutamate/aspartate transporter arlar1 is regulated by cytosolic Ca^2⫹(24). Together, these mechanisms converge to ensure sustained ATP generation during cell activation.

Ca^2⫹accumulated by mitochondria must be removed. In- creases in matrix Ca^2⫹are counteracted principally by the Na^⫹/Ca^2⫹exchanger. Its activity is an important determinant of the extent and duration of mitochondrial Ca^2⫹transients. Inhibition of the Na^⫹/Ca^2⫹exchanger has been ex- ploited as a potential target in diabetes research. An inhibitor of the exchanger (CGP37157) was shown to prolong mitochondrial Ca^2⫹signals and increase insulin secretion (25).

The same compound has been shown to prolong mitochondrial Ca^2⫹signals and ATP production in fibroblasts from patients with complex I deficiency (26).

Coupling Factors in the Amplifying Pathway of Insulin Exocytosis

Glucose-induced insulin secretion depends on additional, as yet unidentified mitochondrial signals. When the K_ATP channel opener diazoxide is added, Ca^2⫹increases, and GSIS are completely blocked. Because Ca^2⫹signals are essential for granule exocytosis, cytoplasmic Ca^2⫹ concentrations were elevated artificially to create a semipermissive condi- tion for granule fusion. Using this experimental setup, it could be demonstrated that glucose metabolism can stimulate insulin secretion independent of K_ATPchannel closure (27). In␣-toxin-permeabilized cells, permitting clamping of cytoplasmic Ca^2⫹and ATP, succinate stimulates insulin secretion through mitochondrial activation (28, 29). Interest- ingly, glycerophosphate was unable to stimulate insulin secretion under the same conditions. Via the glycerol phosphate shuttle, glycerophosphate can provide reducing equivalents, but not carbons, to the mitochondria, demon- strating the need for anaplerotic input to the TCA cycle for the potentiation of insulin secretion. Thus, the synthesis of coupling factors supporting the action of ATP could be linked to the anaplerotic pathway of pyruvate metabolism.

In support of this possibility, inhibition of pyruvate carboxylase impairs GSIS (11, 30). Several mitochondria-derived molecules have been proposed to serve such a role.

Citrate exported from mitochondria can act as an allosteric activator of acetyl-CoA synthase, which leads to a rapid increase in the malonyl-CoA concentration after glucose stimulation of the␤-cell (31). This suggests that malonyl-CoA

could have a direct impact on insulin secretion. Suppressing the increases in malonyl-CoA by the overexpression of malonyl-CoA carboxylase, however, did not impair the glucose responsiveness of␤-cells. Although these results argue against a direct involvement of malonyl-CoA in GSIS, it is likely to facilitate fatty acid-mediated potentiation by di- minishing fatty acid oxidation, favoring their actions on exocytosis (31, 32). Although short-term exposure of islets to palmitate and other free fatty acids acutely enhances GSIS, longer exposure of islets is inhibitory. It is of interest in this context that infusion of free fatty acids for 4 d into healthy members of families with a history of type 2 diabetes causes marked inhibition of GSIS, contrasting with slight stimulation in control subjects (33).

Another possible metabolic event linked to insulin secretion is pyruvate cycling. This process is initiated in response to glucose as pyruvate is carboxylated to form oxaloacetate.

In pyruvate cycling, malate and/or citrate derived from oxaloacetate are transported out of the mitochondria, where they can be converted to pyruvate. The last step of these reactions is mediated by malic enzyme, which oxidatively decarboxylates malate, also generating NADPH (Fig. 1).

Pyruvate completes the cycle to be used again as substrate for mitochondrial matrix enzymes. As measured by¹³C nuclear magnetic resonance, pyruvate cycling is stimulated 2- to 3-fold after a shift of INS-1 cells to elevated glucose. Using different INS-1 clones that vary in their secretory response, it was also shown that pyruvate cycling, but not pyruvate oxidation, was proportional to GSIS (11). However the cor- relation between pyruvate cycling and insulin secretion is lost in the presence of free fatty acids (34). Both malonyl-CoA and pyruvate cycling in insulin secretion merit additional investigation.

Pyruvate cycling, as described above, generates reducing equivalents in the cytosol in the form of NADPH. A recent study suggests that NADPH can stimulate Ca^2⫹ induced insulin exocytosis, as monitored by capacitance changes (35).

This is an attractive mechanism, because glucose increases the ratios of both NADH:NAD^⫹and NADPH:NADP^⫹. In- terestingly, the effect was specific for NADPH, because in- jection of NADH did not influence capacitance. However, glutathione as well as glutaredoxin could also stimulate exocytosis (35). In summary, the results suggest that the redox state of the cell could have a direct impact on the granule fusion machinery.

Glutamate is generated from the TCA cycle intermediate 2-oxoglutarate by glutamate dehydrogenase, from other transamination reactions, or from glutamine in the cytoplasm. In response to glucose, glutamate levels rise in islets (29, 36), as also shown by nuclear magnetic resonance in clonal␤-cells (11, 37). The same technique was used to demonstrate anaplerotic input to the TCA cycle from glucose and cataplerotic generation of glutamate via glutamate dehydrogenase under conditions favoring GSIS (10). As argued above, glutamate, therefore, has the potential to act as an intermediate that stimulates insulin secretion. Because glutamate levels rise only 5–10 min after glucose addition, it is likely to impact mainly on second phase insulin secretion (29). Consistent with a role in insulin secretion, the cell membrane-permeable glutamate analog dimethylglutamate in-

(5)

creases glucose sensitivity, resulting in insulin secretion. Ad- ditional evidence for the implication of glutamate in GSIS is suggested by down-regulation of glutamate dehydrogenase, resulting in a 50% inhibition of insulin secretion in response to glucose (6). Glucose-derived glutamate is generated by mitochondria and acts downstream as a possible link between mitochondrial metabolism and the exocytotic machinery (Fig. 1). Glutamate directly enhances Ca^2⫹-stimulated exocytosis, as demonstrated in both permeabilized cells and capacitance recordings, under conditions of clamped ATP (29, 38). The underlying mechanism remains to be established. It is of interest that familial hyperinsulinism has been linked to gain of function mutations in glutamate dehydrogenase, which render the␤-cell sensitive to glutamine (normally not insulinotropic) as a secretagogue (39).

In summary, a good case has been made for several mitochondrially derived factors that could potentiate GSIS.

Changes in mitochondrial metabolism after glucose stimulation of the␤-cell is associated with changes in the concentration of many metabolites. Several of these metabolites could synergize to optimally stimulate insulin secretion; for example, NADPH and glutamate may affect different processes required for insulin exocytosis. Although malonyl- CoA would potentiate the effect of fatty acids, glutamate could enhance the second phase of GSIS.

Unfortunately, mechanistically we know rather little about the downstream mediators of the proposed coupling factors.

The identification of components involved in insulin secretion that are directly regulated by coupling factors would certainly open up new possibilities for the treatment of type 2 diabetes.

Fatigue of␤-Cell Mitochondria in Type 2 Diabetes A critical role for mitochondria in normal␤-cell function has also become evident from the study of mitochondrial diabetes (40, 41). This hereditary disorder accounts for only about 1% of all diabetes cases, but observations from the analysis of mitochondrial diabetes may well aid in under- standing some of the ␤-cell changes in the more common forms of the disease. Patients with mitochondrial diabetes have mutations or deletions in mtDNA, which is maternally inherited. Each cell contains many mitochondria and many copies of mtDNA (100 –1000 copies/cell). Patient mitochondria display heteroplasmy with regard to mtDNA, the degree of which determines the impact of the mutation. The mitochondrial genome only encodes 37 genes, including 13 subunits of the respiratory chain complexes, ribosomal RNAs, and tRNAs. Most mitochondrial proteins are encoded by nuclear DNA and are imported into the mitochondria post- translationally. Defects in mitochondrial DNA consequently result in impaired oxidative phosphorylation (OXPHOS).

The impact of mitochondrial DNA mutations is most pronounced in tissues with a low mitotic rate, such as brain, muscle, and islet cells. Cells depending on high ATP production, such as ␤-cells, are particularly susceptible to a diminished fraction of functional mitochondria, which tend to decompensate with time. This leads to apoptosis and decreased␤-cell mass, which, together with defective secretion coupling, cause impaired GSIS. These patients may even

require insulin therapy (41, 42). Mitochondrial diabetes il- lustrates how the gradual loss of mitochondrial activity is deleterious for␤-cell secretory function and survival.

In analogy, decreased mitochondrial activity in ␤-cells would predispose individuals to develop␤-cell dysfunction in type 2 diabetes. In individuals with a family history of type 2 diabetes, mitochondrial respiratory function may be sufficient during a normal workload, but may deteriorate under conditions that put stress on the␤-cells, such as insulin resistance or already slightly increased glycemia (43). These states require increased insulin output to maintain glucose homeostasis.

This confirms that both genetic predisposition and environmental factors could precipitate␤-cell dysfunction.

The prevalence of type 2 diabetes increases with age. Un- fortunately, measurements of mitochondrial activity during aging are only available for insulin target tissues. One of the take-home messages is that mitochondrial function decreases with increasing age (see below).

A key determinant of aging is the production of reactive oxygen species (ROS) (44). Irreversible damage caused by ROS interferes with mitochondrial function. Interestingly, it is the mitochondria themselves that in large part are respon- sible for ROS production resulting in damage to their lipids, proteins, and mtDNA. ROS are produced in the mitochondria as a side reaction of respiration. Rather than reducing molecular oxygen to water at complex IV, some electrons escape to reduce oxygen directly to form superoxide. The superoxide radical can then react further to give rise to H₂O₂ and hydroxyl radicals. The mtDNA is vulnerable to the formed ROS. First, ROS are generated nearby in the matrix space, and second, mtDNA is not protected by histones. A role for mtDNA damage in aging has been convincingly demonstrated in a mouse model expressing a mitochondrial DNA polymerase lacking proof-reading activity (45). These mice accumulate more mtDNA mutations and show all the phenotypes of premature aging. mtDNA mutations have also been shown to accumulate in humans as a function of age (46).␤-Cell-targeted disruption of the frataxin gene, en- coding a mitochondrial iron-binding protein, causes diabetes in the mouse. This was linked to increased islet ROS production and decreased␤-cell mass. Patients with mutations in the frataxin gene develop a neurodegenerative disease, associated with type 2 diabetes in 20% of cases (47). Whether these patients or patients with common forms of type 2 diabetes have reduced␤-cell mass as a consequence of mitochondrial dysfunction remains to be firmly established.

In the␤-cell, glucose has been reported to promote ROS production through its hyperpolarizing effect on mitochondrial membrane potential (48, 49). When a similar methodology was combined with measurements of reduced pyrimidine and flavin nucleotide fluorescence in␤-cells, sur- prisingly, physiological as well as high glucose concentrations lowered superoxide production. This was related to the initial metabolic state of the cells,i.e.suppression was most pronounced in cells with the highest initial NADPH levels (50). In view of these conflicting results, ROS generation as well as their putative detrimental actions in the␤-cell need additional study.

Despite the reported low expression of superoxide dis- mutase and catalase in␤-cells under normal conditions, an-

(6)

tioxidant systems are sufficient to prevent oxidative damage (51). Exogenous antioxidants have been investigated for the treatment of diabetes (52). For detailed discussion of␤-cell apoptosis in general and implication of mitochondria in particular, the reader is referred to the recent literature (53, 54).

How can we boost the fatigued␤-cell? Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane protein re- ceiving considerable attention as target for improved␤-cell function (3). UCP2 is homologous to UCP1, which is expressed in brown adipose tissue. UCP1 has been shown to uncouple mitochondria to generate heat by transporting pro- tons down their electrochemical gradient. By doing so, UCP1 dissipates the energy, disconnecting respiration from ATP synthesis. Whether UCP2 in ␤-cells and UCP3 in skeletal muscle carry out an identical molecular function is not yet known. It has been clearly demonstrated that increased expression of UCP2 has a negative impact on GSIS bothin vitro and in vivo (3). A common human polymorphism in the promoter of UCP2 that leads to its overexpression attenuates GSIS bothin vivoand in incubated islets (55). Most interestingly, in UCP2^⫺/⫺mice, GSIS is stimulated (56). Introduction of the UCP2^⫺/⫺ mutation in the ob/obmouse background improved glycemic control as well as GSIS in vitro. It was therefore suggested that inhibition of UCP2 function increases the coupling efficiency of respiration and ATP production. The more pronounced increase in mitochondrial membrane potential also augmented ROS production (49).

Superoxide can activate UCP2. This may be a trick of nature to prevent excessive mitochondrial damage in cells that tend to hyperpolarize their inner mitochondrial membrane. Indeed, overexpression of UCP2 reduces cytokine induced ROS production and apoptosis (57). Treatment of type 2 diabetes by inhibition of UCP2 may therefore be fraught with danger because of the long-term deleterious effects on␤-cells.

Among the possible treatments for type 2 diabetes is increased biogenesis of active mitochondria to replace those harboring mtDNA defects. The nuclear-encoded mitochondrial transcription factor A (TFAM) is essential for control of mtDNA copy number and transcription (58).␤-Cell-specific disruption of TFAM in the mouse results in a phenotype resembling human mitochondrial diabetes with early impairment of GSIS and late loss of␤-cell mass (59). Dominant negative suppression of the maturity-onset diabetes of the young 4 gene, Pdx1, in islets reduced the expression of TFAM and consequently mitochondrially encoded genes. Such islets are refractory to glucose-stimulated ATP generation and insulin secretion (60). Moreover, Pdx1^⫹/⫺ mice show age- dependent ␤-cell apoptosis (61). Conversely, increasing TFAM expression should, in theory, increase mitochondrial function. Unfortunately, TFAM-transgenic mice with up- regulated mtDNA copy number do not display increased number and function of mitochondria (58). It should be tested whether other nuclear factors are more efficient in promoting mitochondrial biogenesis.

Fatigue of Target Tissue Mitochondria in Type 2 Diabetes

Blood glucose is lowered by the action of insulin on its receptor on target cells. The responsiveness of insulin-sensitive

tissues determines the quantity of secreted insulin required to properly regulate blood glucose concentrations. Diabetes is generally associated with insulin resistance in the main target tissues, including liver, muscle, fat, and hypothalamus (for review, see Ref. 62). Environmental factors strongly influence the development of insulin resistance. The two most important are overnutrition and lack of exercise. The resulting overweight is always associated with insulin resistance. In this article, we restrict ourselves to a brief discussion of the role of mitochondria in the pathophysiology of insulin action. Recent evidence suggests that fat tissue-derived factors play important roles in the modulation of insulin responsiveness. More classically, intracellular fatty acids, including acyl-CoA, diacylglycerol, and other metabolites, have been found to act as messengers of the well-fed state that lead to insulin resistance.

Fatty acid oxidation takes place inside mitochondria. In a well-balanced system, the mitochondria, for example of skeletal muscle, will oxidize fatty acids efficiently to prevent the intramyocellular accumulation of triglycerides. Flooding the system with free fatty acids may overwhelm the mitochondria, leading to the accumulation of intracellular lipids and the development of insulin resistance (63). Elevation of free fatty acids during a hyperinsulinemic euglycemic clamp can induce insulin resistance over a time frame of only a few hours (64). More modest elevation of fatty acids over longer periods of time in combination with a diminished oxidative capacity of mitochondria may also lead to the accumulation of triglycerides inside liver muscle or␤-cells.

With increasing age, mitochondrial activity decreases, with a consequent reduction in fatty acid oxidation. This has been demonstrated, for instance, by comparing lean subjects in different age groups. The elderly (61– 84 yr) in this study had developed insulin resistance, showed increased levels of intramyocellular and intrahepatic lipid accumulation, and, most importantly, had a 46% decrease in the mitochondrial ATP synthesis rate (65). Similarly, in young lean offspring of patients with type 2 diabetes, intramyocellular triglycerides were elevated, and muscle ATP synthesis was reduced (66).

The reduced ATP synthesis in these experiments may either be the cause or the consequence of impaired insulin action.

Reduced insulin signaling or the accumulated triglycerides may affect mitochondrial biogenesis or function (67). Alter- natively, mitochondrial dysfunction could cause insulin resistance. Most likely, these two mechanisms act together in a vicious cycle of insulin resistance. Glucose tolerance remains normal, provided the␤-cells can compensate by main- taining elevated circulating insulin. The development of type 2 diabetes is mainly determined by genetic factors controlling

␤-cell function and occurs in approximately 20% of overweight, insulin-resistant subjects. Compensation occurs by both hypersecretion of insulin and increased␤-cell mass (66) (53). In summary, partial loss of mitochondrial activity goes hand in hand with the development of insulin resistance.

AMP-dependent kinase (AMPK) has been proposed as a target for prevention of the metabolic syndrome, character- ized by dislipidemia, obesity, hypertension, and insulin resistance, eventually leading to type 2 diabetes. Malonyl-CoA is the gatekeeper of fatty acid oxidation by inhibition of mitochondrial carnithine-palmitoyl transferase I. The activation of AMPK by exercise, leptin, or the antidiabetic drug

(7)

metformin lowers the concentration of malonyl-CoA in the muscle through several mechanisms, including inhibition of acetyl-CoA carboxylase by phosphorylation (reviewed in Ref. 68). In contrast, metformin-induced activation of AMPK is associated with inhibition of GSIS in human islets (69). This is one of several examples of differential responses in␤-cells and insulin target cells.

AMPK also signals to increase the expression of OXPHOS genes. This is mediated by the peroxisome proliferator- activated receptor-␥coactivator 1␣(PGC1␣). This transcription factor coordinately regulates the expression of a large set of OXPHOS genes, enhancing mitochondrial activity (70).

Interestingly, an impressive 89% (94 of 106) genes classified as OXPHOS genes showed lower expression in muscle of type 2 diabetic patients. Two thirds of the identified OXPHOS genes are coregulated by PGC1␣. The results clearly demonstrate that transcriptional regulation contributes to mitochondrial defects in the target tissues of diabetic patients (67, 70). This role of PGC1␣is particularly interesting, because it was also found to be a positive regulator of mitochondrial biogenesis. However, the effects of PGC1␣are tissue specific. For instance, overexpression of PGC1␣in ␤-cells decreased the levels of several glycolytic enzymes, causing glucose intolerance after islet trans- plantationin vivoand impaired GSISin vitro(71).

Nonetheless, improving mitochondrial respiration and fatty acid oxidation in insulin target tissues are attractive drug targets in type 2 diabetes. In contrast, increased levels of malonyl-CoA in the hypothalamus result in reduced food intake and weight loss in obese animals (72). This and the above-mentioned examples illustrate the difficulty of finding novel drugs for the prevention and improved treatment of type 2 diabetes. One of the difficulties lies in the multiorgan regulation of normal blood glucose homeostasis.

Acknowledgments

Received January 18, 2006. Accepted February 21, 2006.

Address all correspondence and requests for reprints to: Dr. Claes B.

Wollheim, Department of Cell Physiology and Metabolism, University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail:

claes.wollheim@medecine.uinge.ch.

This work was supported by the Swiss National Science Foundation (to C.B.W.).

References

1. Maechler P2002 Mitochondria as the conductor of metabolic signals for insulin exocytosis in pancreatic␤-cells. Cell Mol Life Sci 59:1803–1818

2. Duchen MR2004 Mitochondria in health and disease: perspectives on a new mitochondrial biology. Mol Aspects Med 25:365– 451

3. Lowell BB, Shulman GI2005 Mitochondrial dysfunction and type 2 diabetes.

Science 307:384 –387

4. Wollheim CB, Maechler P2002␤-Cell mitochondria and insulin secretion:

messenger role of nucleotides and metabolites. Diabetes 51(Suppl 1):S37–S42 5. Henquin JC2004 Pathways in␤-cell stimulus-secretion coupling as targets for

therapeutic insulin secretagogues. Diabetes 53(Suppl 3):S48 –S58

6. Maechler P, Carobbio S, Rubi B2006 In␤-cells, mitochondria integrate and generate metabolic signals controlling insulin secretion. Int J Biochem Cell Biol 38(5– 6):696 –709

7. Matschinsky FM2002 Regulation of pancreatic␤-cell glucokinase: from basics to therapeutics. Diabetes 51(Suppl 3):S394 –S404

8. Sekine N, Cirulli V, Regazzi R, Brown LJ, Gine E, Tamarit-Rodriguez J, Girotti M, Marie S, MacDonald MJ, Wollheim CB1994 Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic␤-cells. Potential role in nutrient sensing. J Biol Chem 269:4895– 4902 9. Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, Prentki M1997

Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in␤cells. J Biol Chem 272:18572–18579

10. Cline GW, Lepine RL, Papas KK, Kibbey RG, Shulman GI2004¹³C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells. J Biol Chem 279:

44370 – 44375

11. Lu D, Mulder H, Zhao P, Burgess SC, Jensen MV, Kamzolova S, Newgard CB, Sherry AD2002 ¹³C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS).

Proc Natl Acad Sci USA 99:2708 –2713

12. Arco AD, Satrustegui J2005 New mitochondrial carriers: an overview. Cell Mol Life Sci 62:2204 –2227

13. Rubi B, del Arco A, Bartley C, Satrustegui J, Maechler P2004 The malate- aspartate NADH shuttle member aralar1 determines glucose metabolic fate, mitochondrial activity, and insulin secretion in␤cells. J Biol Chem 279:55659 – 55666

14. Noda M, Yamashita S, Takahashi N, Eto K, Shen LM, Izumi K, Daniel S, Tsubamoto Y, Nemoto T, Iino M, Kasai H, Sharp GW, Kadowaki T2002 Switch to anaerobic glucose metabolism with NADH accumulation in the

␤-cell model of mitochondrial diabetes. Characteristics of␤HC9 cells deficient in mitochondrial DNA transcription. J Biol Chem 277:41817– 41826 15. 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, Kadowaki T1999 Role of NADH shuttle system in glucose- induced activation of mitochondrial metabolism and insulin secretion. Science 283:981–985

16. Kennedy ED, Maechler P, Wollheim CB1998 Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells. Diabetes 47:374 –380

17. Ishihara H, Maechler P, Gjinovci A, Herrera PL, Wollheim CB2003 Islet␤-cell secretion determines glucagon release from neighbouring␣-cells. Nat Cell Biol 5:330 –335

18. Antinozzi PA, Ishihara H, Newgard CB, Wollheim CB2002 Mitochondrial metabolism sets the maximal limit of fuel-stimulated insulin secretion in a model pancreatic␤cell: a survey of four fuel secretagogues. J Biol Chem 277:11746 –11755

19. Schwanstecher C, Meyer U, Schwanstecher M2002 K(IR)6.2 polymorphism predisposes to type 2 diabetes by inducing overactivity of pancreatic␤-cell ATP-sensitive K^⫹channels. Diabetes 51:875– 879

20. Rorsman P, Renstrom E2003 Insulin granule dynamics in pancreatic␤cells.

Diabetologia 46:1029 –1045

21. Pralong WF, Spat A, Wollheim CB1994 Dynamic pacing of cell metabolism by intracellular Ca^2⫹transients. J Biol Chem 269:27310 –27314

22. Territo PR, Mootha VK, French SA, Balaban RS2000 Ca^2⫹activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase. Am J Physiol 278:C423–C435

23. Wollheim CB2000␤-Cell mitochondria in the regulation of insulin secretion:

a new culprit in type II diabetes. Diabetologia 43:265–277

24. Pardo B, Contreras L, Serrano A, Ramos M, Kobayashi K, Iijima M, Saheki T, Satrustegui J2006 Essential role of aralar in the transduction of small Ca^⫹ signals to neuronal mitochondria. J Biol Chem 281:1039 –1047

25. Lee B, Miles PD, Vargas L, Luan P, Glasco S, Kushnareva Y, Kornbrust ES, Grako KA, Wollheim CB, Maechler P, Olefsky JM, Anderson CM2003 Inhibition of mitochondrial Na^⫹-Ca^2⫹exchanger increases mitochondrial metabolism and potentiates glucose-stimulated insulin secretion in rat pancreatic islets. Diabetes 52:965–973

26. Visch HJ, Rutter GA, Koopman WJ, Koenderink JB, Verkaart S, de Groot T, Varadi A, Mitchell KJ, van den Heuvel LP, Smeitink JA, Willems PH2004 Inhibition of mitochondrial Na^⫹-Ca^2⫹exchange restores agonist-induced ATP production and Ca^2⫹handling in human complex I deficiency. J Biol Chem 279:40328 – 40336

27. Gembal M, Gilon P, Henquin JC1992 Evidence that glucose can control insulin release independently from its action on ATP-sensitive K⫹channels in mouse B cells. J Clin Invest 89:1288 –1295

28. Maechler P, Kennedy ED, Pozzan T, Wollheim CB1997 Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic

␤-cells. EMBO J 16:3833–3841

29. Maechler P, Wollheim CB1999 Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis. Nature 402:685– 689

30. Farfari S, Schulz V, Corkey B, Prentki M2000 Glucose-regulated anaplerosis and cataplerosis in pancreatic␤-cells: possible implication of a pyruvate/

citrate shuttle in insulin secretion. Diabetes 49:718 –726

31. Deeney JT, Prentki M, Corkey BE2000 Metabolic control of␤-cell function.

Semin Cell Dev Biol 11:267–275

32. Deeney JT, Gromada J, Hoy M, Olsen HL, Rhodes CJ, Prentki M, Berggren PO, Corkey BE2000 Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI␤-cells). J Biol Chem 275:9363–9368

33. Kashyap S, Belfort R, Gastaldelli A, Pratipanawatr T, Berria R, Pratipana- watr W, Bajaj M, Mandarino L, DeFronzo R, Cusi K2003 A sustained increase in plasma free fatty acids impairs insulin secretion in nondiabetic subjects genetically predisposed to develop type 2 diabetes. Diabetes 52:2461–2474 34. Boucher A, Lu D, Burgess SC, Telemaque-Potts S, Jensen MV, Mulder H,

(8)

Wang MY, Unger RH, Sherry AD, Newgard CB2004 Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and re- versal with a malate analogue. J Biol Chem 279:27263–27271

35. Ivarsson R, Quintens R, Dejonghe S, Tsukamoto K, in ’t Veld P, Renstrom E, Schuit FC2005 Redox control of exocytosis: regulatory role of NADPH, thioredoxin, and glutaredoxin. Diabetes 54:2132–2142

36. Bertrand G, Ishiyama N, Nenquin M, Ravier MA, Henquin JC2002 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

37. Broca C, Brennan L, Petit P, Newsholme P, Maechler P2003 Mitochondria- derived glutamate at the interplay between branched-chain amino acid and glucose-induced insulin secretion. FEBS Lett 545:167–172

38. Hoy M, Maechler P, Efanov AM, Wollheim CB, Berggren PO, Gromada J 2002 Increase in cellular glutamate levels stimulates exocytosis in pancreatic

␤-cells. FEBS Lett 531:199 –203

39. Kelly A, Li C, Gao Z, Stanley CA, Matschinsky FM2002 Glutaminolysis and insulin secretion: from bedside to bench and back. Diabetes 51(Suppl 3):S421–

S426

40. Maechler P, Wollheim CB2001 Mitochondrial function in normal and diabetic

␤-cells. Nature 414:807– 812

41. Maassen JA, Janssen GM, t Hart LM2005 Molecular mechanisms of mitochondrial diabetes (MIDD). Ann Med 37:213–221

42. Kobayashi T, Nakanishi K, Nakase H, Kajio H, Okubo M, Murase T, Kosaka K1997 In situ characterization of islets in diabetes with a mitochondrial DNA mutation at nucleotide position 3243. Diabetes 46:1567–1571

43. Weir GC, Bonner-Weir S2004 Five stages of evolving␤-cell dysfunction during progression to diabetes. Diabetes 53(Suppl 3):S16 –S21

44. Balaban RS, Nemoto S, Finkel T2005 Mitochondria, oxidants, and aging. Cell 120:483– 495

45. Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S, Oldfors A, Wibom R, Tornell J, Jacobs HT, Larsson NG2004 Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429:417– 423

46. Michikawa Y, Mazzucchelli F, Bresolin N, Scarlato G, Attardi G1999 Aging- dependent large accumulation of point mutations in the human mtDNA control region for replication. Science 286:774 –779

47. Ristow M, Mulder H, Pomplun D, Schulz TJ, Muller-Schmehl K, Krause A, Fex M, Puccio H, Muller J, Isken F, Spranger J, Muller-Wieland D, Magnuson MA, Mohlig M, Koenig M, Pfeiffer AF2003 Frataxin deficiency in pancreatic islets causes diabetes due to loss of␤cell mass. J Clin Invest 112:527–534 48. Bindokas VP, Kuznetsov A, Sreenan S, Polonsky KS, Roe MW, Philipson LH

2003 Visualizing superoxide production in normal and diabetic rat islets of Langerhans. J Biol Chem 278:9796 –9801

49. Krauss S, Zhang CY, Scorrano L, Dalgaard LT, St-Pierre J, Grey ST, Lowell BB2003 Superoxide-mediated activation of uncoupling protein 2 causes pancreatic␤cell dysfunction. J Clin Invest 112:1831–1842

50. Martens GA, Cai Y, Hinke S, Stange G, Van de Casteele M, Pipeleers D2005 Glucose suppresses superoxide generation in metabolically responsive pancreatic␤cells. J Biol Chem 280:20389 –20396

51. Tiedge M, Lortz S, Drinkgern J, Lenzen S1997 Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46:1733–1742

52. Hsu CC, Yen HF, Yin MC, Tsai CM, Hsieh CH2004 Five cysteine-containing compounds delay diabetic deterioration in BALB/cA mice. J Nutr 134:3245–

3249

53. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC2003␤-Cell deficit and increased␤-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102–110

54. Donath MY, Ehses JA, Maedler K, Schumann DM, Ellingsgaard H, Eppler E, Reinecke M2005 Mechanisms of␤-cell death in type 2 diabetes. Diabetes 54(Suppl 2):S108 –S113

55. Sesti G, Cardellini M, Marini MA, Frontoni S, D’Adamo M, Del Guerra S,

Lauro D, De Nicolais P, Sbraccia P, Del Prato S, Gambardella S, Federici M, Marchetti P, Lauro R2003 A common polymorphism in the promoter of UCP2 contributes to the variation in insulin secretion in glucose-tolerant subjects.

Diabetes 52:1280 –1283

56. Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB2001 Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity,␤cell dysfunction, and type 2 diabetes.

Cell 105:745–755

57. Zengaffinen N, Perreten H, Lameloise N, Keller P, Muzzin P, Assimacopoulos- Jeannet F2005 Uncoupling protein 2 overexpression prevents cytokine-induced reactive oxygen species production and apoptosis in pancreatic␤cells. Dia- betologia 48:A38

58. Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, Rustin P, Gustafsson CM, Larsson NG2004 Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet 13:

935–944

59. Silva JP, Kohler M, Graff C, Oldfors A, Magnuson MA, Berggren PO, Larsson NG2000 Impaired insulin secretion and␤-cell loss in tissue-specific knockout mice with mitochondrial diabetes. Nat Genet 26:336 –340 60. Gauthier BR, Brun T, Sarret EJ, Ishihara H, Schaad O, Descombes P, Woll-

heim CB2004 Oligonucleotide microarray analysis reveals PDX1 as an essential regulator of mitochondrial metabolism in rat islets. J Biol Chem 279:

31121–31130

61. Johnson JD, Ahmed NT, Luciani DS, Han Z, Tran H, Fujita J, Misler S, Edlund H, Polonsky KS2003 Increased islet apoptosis in Pdx1^⫹/⫺mice. J Clin Invest 111:1147–1160

62. Biddinger SB, Kahn CR2005 From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol 68:123–158

63. Schrauwen P, Hesselink MK2004 Oxidative capacity, lipotoxicity, and mitochondrial damage in type 2 diabetes. Diabetes 53:1412–1417

64. Storgaard H, Jensen CB, Bjornholm M, Song XM, Madsbad S, Zierath JR, Vaag AA2004 Dissociation between fat-inducedin vivoinsulin resistance and proximal insulin signaling in skeletal muscle in men at risk for type 2 diabetes.

J Clin Endocrinol Metab 89:1301–1311

65. Petersen KF, Befroy D, Dufour S, Dziura J, Ariyan C, Rothman DL, DiPietro L, Cline GW, Shulman GI2003 Mitochondrial dysfunction in the elderly:

possible role in insulin resistance. Science 300:1140 –1142

66. Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI2004 Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med 350:664 – 671

67. Stump CS, Short KR, Bigelow ML, Schimke JM, Nair KS2003 Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts. Proc Natl Acad Sci USA 100:7996 – 8001

68. Ruderman N, Prentki M2004 AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome. Nat Rev Drug Discov 3:340 –351 69. Leclerc I, Woltersdorf WW, da Silva Xavier G, Rowe RL, Cross SE, Korbutt

GS, Rajotte RV, Smith R, Rutter GA2004 Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. Am J Physiol 286:E1023–E1031

70. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D, Groop LC2003 PGC-1␣-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34:267–273

71. Yoon JC, Xu G, Deeney JT, Yang SN, Rhee J, Puigserver P, Levens AR, Yang R, Zhang CY, Lowell BB, Berggren PO, Newgard CB, Bonner-Weir S, Weir G, Spiegelman BM2003 Suppression of␤cell energy metabolism and insulin release by PGC-1␣. Dev Cell 5:73– 83

72. Hu Z, Dai Y, Prentki M, Chohnan S, Lane MD2005 A role for hypothalamic malonyl-CoA in the control of food intake. J Biol Chem 280:39681–39683 Endocrinologyis published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the

endocrine community.