Anaplerosis and insulin release

An increased blood glucose concentration stimulates pancreatic β-cells to secrete insulin but the molecular aspects concerning GIIS are not completely understood. Recent findings have proposed an oscillatory pattern of downstream events, leading to an increase of cytoplasmic Ca²⁺ levels, insulin granule docking and fusion with the plasma membrane, enhanced cyclic AMP production, amplified Ca signalling and activation of secretory mechanisms by Ca²⁺ ions⁽Reference Dyachok, Idevall-Hagren and Sågetorp^2, Reference Jones, Fyles and Howell^36–Reference MacDonald and Rorsman³⁸⁾. Several mechanisms have been proposed involving the K_ATP- and Ca²⁺-dependent pathway, the K⁺_ATP-independent and Ca²⁺-dependent pathway, and possibly a K⁺_ATP- and Ca²⁺-independent pathway⁽Reference Prentki^39–Reference Gembal, Gilon and Henquin⁴³⁾. However, little is known about the metabolic regulation associated with these pathways.

Improved TACI anaplerosis–cataplerosis flux is needed to enhance intracellular ATP content⁽Reference MacDonald, Fahien and Brown^4, Reference Fransson, Rosengren and Schuit^5, Reference Ronnebaum, Ilkayeva and Burgess⁴⁴⁾. Although the mechanism involved remains unknown, a strong correlation has been documented between PC activity and GIIS⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference Curi, Carpinelli and Malaisse⁴⁵⁾. This enzyme, located in the mitochondrial matrix, catalyses the ATP-dependent carboxylation of pyruvate to form oxaloacetate⁽Reference Khan, Ling and Landau⁴⁶⁾. PC is highly expressed in the liver and kidney, and participates with phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase of gluconeogenesis⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference Curi^47, Reference Schuit, De Vos and Farfari⁴⁸⁾. The lack of phosphoenolpyruvate carboxykinase activity and relatively low lipogenic capacity suggest an important role of PC in β-cells during oxidative energy metabolism. A proportion of about 40–50 % pyruvate enters β-cells during mitochondrial metabolism through PC reactions at stimulating glucose concentrations, a very high flux for a non-gluconeogenic tissue. The anaplerosis of glucose carbons, therefore, highly correlates with GIIS by β-cells⁽Reference MacDonald, Fahien and Brown^4, Reference Schuit, De Vos and Farfari⁴⁸⁾. In INS cells (from the insulinoma cell line), anaplerosis was reported to be increased followed by a high basal insulin secretion after exposure to glucose. However, at a low glucose concentration, PC expression was demonstrated to be markedly decreased⁽Reference Lu, Mulder and Zhao^8, Reference Jensen, Joseph and Ilkayeva⁴⁹⁾. Farfari et al. ⁽Reference Farfari, Schulz and Corkey⁵⁰⁾ demonstrated that phenylacetic acid, a PC inhibitor, reduced GIIS in INS cells and pancreatic islets, an effect that was associated with reduced citrate accumulation. Similarly, Fransson et al. ⁽Reference Fransson, Rosengren and Schuit⁵⁾, examining the phenylacetic acid-induced effect on PC during insulin release in rat islets, demonstrated that anaplerosis via PC is required for an appropriated rise in the ATP:ADP ratio and insulin secretion. However, caution must be taken from the above studies once phenylacetic acid specificity is limited toward PC⁽Reference Ronnebaum, Ilkayeva and Burgess⁴⁴⁾. Very recently, Hasan et al. ⁽Reference Hasan, Longacre and Stoker⁵¹⁾, testing the hypothesis that anaplerosis via PC is important for GIIS in cell lines, verified that reduced expression of this enzyme using transfection of short hairpin RNA was associated with reduced PC activity and insulin release in response to glucose and other secretagogues. Although the mechanism remains to be elucidated, the influx of carbon intermediates into the TAC is critical for appropriate ATP generation in β-cells⁽Reference Curi, Carpinelli and Malaisse⁴⁵⁾. The proposed mechanisms have been extensively studied and supported by different metabolic pathways in which pyruvate is metabolised and/or recycled⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference Jensen, Joseph and Ilkayeva^49, Reference Farfari, Schulz and Corkey^50, Reference MacDonald^52, Reference Guay, Madiraju and Aumais⁵³⁾. As in most mammalian tissues, pyruvate may follow two different routes, feeding the TAC with acetyl-CoA via pyruvate dehydrogenase and oxaloacetate via PC in pancreatic β-cells⁽Reference Curi, Carpinelli and Malaisse⁴⁵⁾.

Pyruvate dehydrogenase contributes with the TAC by the production of acetyl-CoA. In this reaction, one carbon from pyruvate is lost as CO₂ and two are converted in the acetyl-CoA molecule, which will condensate with oxaloacetate to form citrate by citrate synthase (CS)⁽Reference MacDonald⁵²⁾. The oxaloacetate molecule is regenerated with the TACI, remaining constant at the expense of acetyl-CoA. Although much attention has been given to PC, the flux of pyruvate decarboxylation through pyruvate dehydrogenase in β-cells is estimated to be similar to that observed during pyruvate carboxylation by PC⁽Reference Guay, Madiraju and Aumais⁵³⁾. Sustained glucose supply raises the intracellular citrate content in β-cells, suggesting an association between PC and pyruvate dehydrogenase during the anaplerosis process⁽Reference Schuit, De Vos and Farfari⁴⁸⁾. However, the high K _m of the CS for acetyl-CoA compared with oxaloacetate suggests that the flux of oxaloacetate from pyruvate via PC is greatly favoured, increasing the content of intermediates in the second span of TAC⁽Reference Srere⁵⁴⁾. Moreover, acetyl-CoA is a well known allosteric activator of PC, favouring the anaplerosis process and consequently the rate of intracellular ATP production⁽Reference Newsholme and Leech⁵⁵⁾. However, the elevated TACI content exerts an inhibitory effect on most of the regulatory sites from the TAC⁽Reference MacDonald, Fahien and Brown^4, Reference Newsholme and Leech^55, Reference MacDonald, Fahien and Buss⁵⁶⁾, which will further allow the TACI export from the mitochondria to the cytosol. Once accumulated, TACI not only provide ATP through the mitochondrial electron transport chain but also are exported (cataplerosis) to the cytosol⁽Reference MacDonald, Fahien and Brown⁴⁾. In this latter process, carbon derived from pyruvate carboxylation exits from the mitochondria to the cytosol, primarily as malate⁽Reference MacDonald, Fahien and Brown^4, Reference Guay, Madiraju and Aumais⁵³⁾. In the cytosol, malate is converted to pyruvate in a NADPH generation reaction catalysed by cytosolic malic enzyme (MEc), which can be transported back to the mitochondria⁽Reference MacDonald, Fahien and Brown⁴⁾. This cycle, also known as the pyruvate–malate shuttle, occurs inside the mitochondrial matrix and exerts a critical role in β-cells during both the anaplerosis and cataplerosis processes by recycling pyruvate and NADPH production, a potent metabolic coupling factor⁽Reference MacDonald^52, Reference Guay, Madiraju and Aumais⁵³⁾ (Fig. 1). Evidence of pyruvate–malate shuttle activity was recently demonstrated during GIIS in INS cells. The inhibition of the malate dicarboxylate transporter by pharmacological inhibition and/or by RNA interference (RNAi) markedly reduced the GIIS⁽Reference Guay, Madiraju and Aumais⁵³⁾.

Fig. 1 Anaplerosis and cataplerosis pathways during insulin secretion in β-cells. The pyruvate cycling is the main cytosolic site of NADPH production. NADPH is one of the most import antioxidants in β-cells. This anaplerotic process includes the pyruvate–malate shuttle, pyruvate–citrate cycle and pyruvate–isocitrate–α-ketoglutarate cycle. NADPHox, NADPH–oxidase enzymic complex; SOD, superoxide dismutase; O₂^− ∙, superoxide anion; GSSG, oxidised glutathione; GPX, glutathione peroxidase; GSH, glutathione; GR, glutathione reductase; cME, cytosolic malic enzyme; glucose 6-P, glucose 6-phosphate; MDH, malate dehydrogenase; CPT, carnitine palmitoyltransferase; mME, mitochondrial malic enzyme; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; ETC, electron transport chain; Pi, inorganic phosphate.

Although the export of citrate is not increased as much as malate, during β-cell anaplerotic activity, citrate content has also been shown to suffer a larger range of oscillation compared with other TACI, an effect that was correlated with ATP and NAD(P)H oscillations⁽Reference MacDonald, Fahien and Buss⁵⁶⁾. Elevated malate and citrate concentrations suggest that the K _m of malate dehydrogenase and CS for oxaloacetate must be of similar magnitude, favouring rapid TAC expansion⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference MacDonald^52, Reference Guay, Madiraju and Aumais⁵³⁾. Similarly to malate, citrate after exiting the mitochondria is converted by ATP–citrate lyase to oxaloacetate and acetyl-CoA⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference Guay, Madiraju and Aumais⁵³⁾. Oxaloacetate in a sequence of two reactions is reduced to malate by malate dehydrogenase, which will further be converted to pyruvate by MEc. In this reaction, NADP⁺ is used as cofactor favouring the rise of cytosolic NADPH:NADP⁺ and pyruvate transport back to the mitochondria. This cycle is defined as the pyruvate–citrate cycle⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference Farfari, Schulz and Corkey^50, Reference Guay, Madiraju and Aumais⁵³⁾ (Fig. 1). Recent studies have demonstrated that oscillatory ATP production is strongly associated with cytosolic citrate content⁽Reference MacDonald, Fahien and Buss⁵⁶⁾. This relationship suggests that citrate might be by itself coordinating the activities of TAC and anaerobic glycolysis. Inside the mitochondrial matrix, citrate has been described as an important inhibitor of CS⁽Reference Newsholme and Leech⁵⁵⁾. This effect will favour its mitochondrial accumulation and consequently its exit toward the cytosol. Once accumulated into the cytosol, citrate might inhibit glycolysis via allosteric modulation of phosphofructokinase⁽Reference Newsholme and Leech^55, Reference Tornheim, Andrés and Schultz⁵⁷⁾. This mechanism, associated with elevated cytosolic NADPH:NADP⁺, could explain, at least in part, the oscillatory pattern of insulin secretion in β-cells.

An increased NADPH:NADP⁺ inhibits the pentose phosphate pathway. Although this route is described as exhibiting low activity in β-cells, inhibition of the pentose phosphate pathway might exert a metabolic-saving effect, driving the glucose-6-phosphate to glycolysis⁽Reference Farfari, Schulz and Corkey⁵⁰⁾. On the other hand, acetyl-CoA is converted to malonyl-CoA by acetyl-CoA carboxylase, a well-known inhibitor of FA metabolism⁽Reference Coyle, Hargreaves and Thompson^58, Reference Silveira, Fiamoncini and Hirabara⁵⁹⁾. In a tissue with elevated anaplerosis capacity, the cytosolic NADH:NAD⁺ ratio is reduced, allowing sustained glyceraldehyde 3-phosphate dehydrogenase activity and glycolytic flux, leading to an increase in the ATP:ADP ratio⁽Reference MacDonald, Fahien and Brown^4, Reference Newsholme and Leech⁵⁵⁾. In β-cells, glucose oxidation provides a higher mitochondrial electrochemical gradient (Δψ) and ATP:ADP ratio than FA⁽Reference Willis and Jackman⁶⁰⁾, whereas FA oxidation seems to induce mitochondrial uncoupling and reduced ATP synthesis⁽Reference Spacek, Santorová and Zacharovová⁶¹⁾. There is strong evidence linking PC activity and the pyruvate–citrate cycle with insulin secretion⁽Reference Fransson, Rosengren and Schuit^5, Reference Lu, Mulder and Zhao^8, Reference Guay, Madiraju and Aumais^53, Reference Cline, Lepine and Papas⁶²⁾. PC inhibition resulted in decreased GIIS, which was positively correlated with citrate concentration⁽Reference Farfari, Schulz and Corkey^50, Reference Hasan, Longacre and Stoker⁵¹⁾. Furthermore, impairment of mitochondria citrate metabolism by either inhibition of mitochondrial di- and tricarboxylate carriers or deletion of citrate lyase and MEc genes leads to reduced GIIS, with no change in glucose oxidation, probably by a reduction in malonyl-CoA and NADPH synthesis⁽Reference Guay, Madiraju and Aumais⁵³⁾.

The importance of the anaplerotic PC–pyruvate–citrate cycle pathway was reinforced when β-cells were incubated in the presence of weak insulin secretagogues including acetoacetate, β-hydroxybutyrate, monometyl succinate and lactate. When incubated together, these metabolites increased acetyl-CoA, oxaloacetate and consequently citrate concentrations, that were able to enhance insulin release by 10- to 20-fold, almost the same effect observed for GIIS⁽Reference MacDonald, Longacre and Stoker⁷⁾. However, a strong correlation between GIIS and a non-PC-derived anaplerosis pathway in insulinoma cell lines was observed, suggesting that other pathways, probably aspartate aminotransferase, act in combination with PC in TAC anaplerosis-induced GIIS, enhancing oxaloacetate levels through aspartate and α-ketoglutarate consumption, respectively⁽Reference Simpson, Khokhlova and Oca-Cossio⁶³⁾. MacDonald et al. ⁽Reference MacDonald, Stoker and Hasan⁶⁾ demonstrated that incorporation of carbon from pyruvate into lipids was not lowered in the citrate lyase-deficient INS-1 cell line, suggesting that citrate is not the only cataplerotic carbon carrier from the mitochondria to cytosol. Further studies are, therefore, needed to clarify other anaplerotic and cataplerotic pathways and substrates that regulate GIIS.

Alternatively, pyruvate cycling during TACI expansion can also occur via pyruvate, isocitrate and α-ketoglutarate⁽Reference Ronnebaum, Ilkayeva and Burgess⁴⁴⁾. Unlike the pyruvate–malate shuttle, oxaloacetate formed by PC is converted to citrate and isocitrate, which then leave the mitochondria forming oxaloacetate and acetyl-CoA through citrate lyase or α-ketoglutarate through cytosolic NADP⁺-dependent isocitrate dehydrogenase (ICDc). The pyruvate cycling can take place via conversion of oxaloacetate to malate and further to pyruvate by malate dehydrogenase and MEc, respectively⁽Reference Ronnebaum, Ilkayeva and Burgess^44, Reference Jensen, Joseph and Ilkayeva^49, Reference Guay, Madiraju and Aumais⁵³⁾. In addition, α-ketoglutarate can re-enter the mitochondria forming malate by following the TAC reactions, which will be converted to pyvurate via MEc or mitochondrial malic enzyme with elevated NADPH production (Fig. 1). Ronnebaum et al. ⁽Reference Ronnebaum, Ilkayeva and Burgess⁴⁴⁾, examining the role of ICDc in control of GIIS in β-cells using ICDc iRNA, observed that suppression of ICDc attenuated the glucose-induced increment in pyruvate cycling and intracellular NADPH content. These findings, therefore, suggest that the pyruvate cycling pathway involving ICDc plays an important role in the control of GIIS.

In addition to the pyruvate cycling shuttle enzymes, the role of glutamate dehydrogenase (GDH) during anaplerosis-induced insulin release has recently been reviewed⁽Reference Maechler, Carobbio and Rubi⁶⁴⁾. Mice with overexpression of GDH had enhanced insulin release⁽Reference Carobbio, Ishihara and Fernandez-Pascual⁶⁵⁾. Carobbio et al. ⁽Reference Carobbio, Frigerio and Rubi⁶⁶⁾, using GDH knockout rats, showed a near 37 % reduction in GIIS. Studies using leucine, a GDH-positive allosteric modulator, as an insulin secretagogue, showed enhanced insulin release⁽Reference Filiputti, Ferreira and Souza^32, Reference Zawalich, Yamazaki and Zawalich⁶⁷⁾. To date, it is not known if GDH acts as an anaplerotic enzyme producing α-ketoglutarate or has a cataplerotic function generating glutamate at the expense of α-ketoglutarate during insulin release. Accumulated evidence has shown the link between GDH and GIIS⁽Reference Maechler, Carobbio and Rubi⁶⁴⁾. In addition to the possible anaplerotic and cataplerotic role during insulin release, GDH produces NADPH, another important metabolic coupling factor⁽Reference MacDonald, Fahien and Brown⁴⁾.

Anaplerosis, intracellular redox status and insulin release in β-cells

Interestingly, most alterations in anaplerotic- and cataplerotic-related mechanisms are involved with NADPH production (Fig. 2). As first reported by MacDonald⁽Reference MacDonald⁵²⁾, the pyruvate cycling may be the main cytosolic site of NADPH production with much higher capacity than the pentose phosphate pathway in β-cells. In this sense, the cataplerosis of malate and citrate would increase NADPH production. The β-cells’ needs for this reducing agent are still not known. However, NADPH is involved in FA synthesis and cellular redox modulation. As β-cells exhibit a relatively low rate of FA synthesis⁽Reference Brun, Roche and Assimacopoulos-Jeannet⁶⁸⁾, the main NADPH function could be attributed to the regulation of intracellular redox status. Also, one would speculate that the low pentose phosphate pathway activity must be of relevance for β-cells, as this pathway is highly associated with lipid synthesis in different tissues. However, a high intracellular lipid content might be potentially toxic for β-cells, in which an elevated anaplerosis and cataplerosis process is required during GIIS.

Fig. 2 The β-cell antioxidant system. The NADPH production during anaplerosis provides a substrate for glutathione and thioredoxin systems, which in turn will favour the intracellular redox status. GSH, glutathione; GSSG, oxidised glutathione; SOD, superoxide dismutase; O₂^− ∙, superoxide anion; ^∙OH, hydroxyl radical; GPX, glutathione peroxidase; GR, glutathione reductase; CAT, catalase; S-S, thiol and disulfide (-S-S-).

Redox alteration of β-cells to a more reduced status is needed for proper insulin secretion⁽Reference Ramirez, Rasschaert and Sener⁶⁹⁾. The NADP⁺:NADPH ratio seems to play an important role in the redox modulation of insulin release, since NADPH is the substrate for several pro- and antioxidant enzymes⁽Reference MacDonald, Fahien and Brown⁴⁾. The expansion of TACI during GIIS, therefore, seems to be important not only for energy production but also to regulate the intracellular redox balance.

During basal secretion, β-cells present a relatively low antioxidant capacity⁽Reference Newsholme, Haber and Hirabara⁷⁰⁾, but acute glucose loading leads to a fast induction of superoxide dismutase and glutathione peroxidase (GPX) activities, indicating an elevated production of reactive oxygen species (ROS)⁽Reference Oliveira, Curi and Carpinelli^71, Reference Poitout and Robertson⁷²⁾. Glutathione reductase and thioredoxin, two NADPH-consuming enzymes, together with glutaredoxin are highly expressed in pancreatic islets⁽Reference Nagaoka, Iuchi and Ikeda^73, Reference Ivarsson, Quintens and Dejonghe⁷⁴⁾, indicating the importance of reduced glutathione turnover in these cells. These findings, therefore, suggest that NADPH production during the export of TACI to the cytosol modulates Ca²⁺-dependent insulin secretion by regulating the intracellular redox balance⁽Reference Ivarsson, Quintens and Dejonghe^74, Reference Reinbothe, Ivarsson and Li⁷⁵⁾. Oliveira et al. ⁽Reference Oliveira, Verlengia and Carvalho⁷⁶⁾ also showed that β-cells express phagocyte-like NAD(P)H oxidase, a NADPH-consuming enzyme, which is known to produce ROS. Although the physiological role of β-cell NAD(P)H oxidase remains unclear, its activity may play a role, as demonstrated in other cell types, during Ca²⁺ release and consequently insulin secretion⁽Reference Hu, Zheng and Zweier⁷⁷⁾. Indeed, Morgan et al. ⁽Reference Morgan, Rebelato and Abdulkader⁷⁸⁾ provided primary evidence for β-cell NAD(P)H oxidase-induced ROS production regulation of glucose flux and oxidation as well as Ca²⁺ intracellular response.

Robertson & Harmon⁽Reference Robertson and Harmon⁷⁹⁾ described the importance of GPX for the control of β-cell redox status. This enzyme acts in combination with glutathione reductase. Under low NADPH content, GPX has its ROS detoxification capacity markedly compromised. NO has also been proposed to have a protective effect upon the endoplasmic reticulum under ROS-induced stress⁽Reference Kitiphongspattana, Khan and Ishii-Schrade⁸⁰⁾. In addition, NO has been reported to stimulate insulin gene transcription⁽Reference Campbell, Richardson and Ferris⁸¹⁾. NO is synthesised by NO synthase, which also uses NADPH as a substrate. The redox imbalance leading to β-cell oxidative stress has been implicated in several dysfunctions including low insulin release, cell proliferation and death, which are directly related to type 2 diabetes development⁽Reference Newsholme, Haber and Hirabara⁷⁰⁾.

Protein malnutrition, β-cell molecular alterations and insulin release

Protein-deficient diets lead to impaired insulin secretion in response to oral or intravenous glucose infusion and to other secretagogues⁽Reference Mann, Becker and Pimstone^24–Reference Parra, Klish and Cuellar^26, Reference Latorraca, Reis and Carneiro^29–Reference Filiputti, Ferreira and Souza^32, Reference Weinkove, Weinkove and Pimstone^82–Reference Leon-Quinto, Magnan and Portha⁸⁵⁾. Under such conditions, the expression of pancreatic and duodenal homeobox-1, a transcription factor that plays a role in the maintenance of β-cell homeostasis, is markedly reduced in association with reduced pancreatic islet area and insulin release⁽Reference Arantes, Teixeira and Reis⁸⁶⁾. Expression of signalling proteins, such as protein kinase A and protein kinase C, is also reduced during protein malnutrition⁽Reference Ferreira, Filiputti and Arantes^87, Reference Ferreira, Barbosa and Stoppiglia⁸⁸⁾. Several genes involved in insulin production and secretion mechanisms also have their expression altered⁽Reference Delghingaro-Augusto, Ferreira and Bordin³³⁾. Recently, it was demonstrated that Ca²⁺ uptake and insulin mRNA content were also reduced in undernourished rats, leading to reduced insulin release in response to glucose⁽Reference Latorraca, Carneiro and Mello^89, Reference de Barros Reis, Arantes and Cunha⁹⁰⁾. These outcomes might in part be attributed to the reduced expression of both constitutive and inducible NO synthase isozymes under protein malnutrition⁽Reference Wu, Flynn and Flynn⁹¹⁾. NO has been demonstrated to protect against endoplasmic reticulum stress and has a stimulating effect upon insulin gene transcription under regular fed state⁽Reference Kitiphongspattana, Khan and Ishii-Schrade^80, Reference Campbell, Richardson and Ferris⁸¹⁾. In addition, β-cells from rats fed with a protein-deficient diet have decreased expression of protein kinase B (PKB or Akt), mammalian target of rapamycin (mTOR) and p70s6k⁽Reference Filiputti, Ferreira and Souza³²⁾.

Although substrate availability might be reduced, capacity for oxidative ATP synthesis seems to be enhanced in protein deficiency. This statement is based on the findings of an almost threefold increase in ATP-synthase F1 complex expression, whereas GLUT-2 expression in β-cells was decreased⁽Reference Delghingaro-Augusto, Ferreira and Bordin³³⁾. The rise in ATP synthase gene expression may reflect an adaptation to low substrate availability, reduced mitochondrial metabolism and anaplerotic capacity. In contrast, the content of β-cell GLUT-1 and GLUT-2, intracellular glucose availability and glycolytic flux in fetuses from undernourished rats and adult undernourished rats were not different from control. However, mitochondrial glucose oxidation was found to be directly related to pancreatic and duodenal homeobox-1 expression and insulin secretion in undernourished rats, providing a possible link between metabolic and molecular mechanisms of insulin production and secretion in protein malnutrition⁽Reference Martín, Fernández and Pascual-Leone⁹²⁾.

Protein malnutrition, anaplerosis and insulin release

Concerning metabolic aspects of protein malnutrition-induced reduction in insulin release, Sener et al. ⁽Reference Sener, Reusens and Remacle⁹³⁾ have shown that low-protein-fed rats have reduced glucose oxidation as demonstrated by a decreased metabolite flux through the glycerol phosphate shuttle in β-cells from malnourished rats, probably by a low mitochondrial FAD-linked glycerophosphate dehydrogenase activity⁽Reference Rasschaert, Reusens and Dahri⁹⁴⁾. In addition, at high glucose concentration, low-protein-fed rats showed an elevated glycolytic flux. However, leucine transamination to α-ketoisocaproic acid and further production of α-ketoglutarate was significantly reduced, indicating a poor anaplerotic capacity⁽Reference Sener, Reusens and Remacle⁹³⁾.

The impaired insulin release in malnourished rats might be, therefore, related to the lower mitochondrial oxidative and anaplerotic capacity (Fig. 3). The reduced β-cell anaplerosis in malnutrition is supported by findings that insulin synthesis is also regulated by succinate and/or succinyl-CoA cataplerosis⁽Reference Alarcon, Wicksteed and Prentki^95, Reference Attali, Parnes and Ariav⁹⁶⁾. This finding is in accordance with reduced β-cell insulin mRNA levels in undernourished rats⁽Reference de Barros Reis, Arantes and Cunha^90, Reference Martín, Fernández and Pascual-Leone⁹²⁾. Moreover, malonyl-CoA content, another insulin release signalling molecule, has been shown to be reduced under protein malnutrition⁽Reference de Barros Reis, Arantes and Cunha⁹⁰⁾. Malonyl-CoA is produced through mitochondrial citrate cataplerosis, and one of its actions is to limit FA oxidation, stimulating GIIS⁽Reference MacDonald, Fahien and Brown⁴⁾. There seems to be a pyruvate–citrate cycle impairment under protein malnutrition, since intracellular malonyl-CoA content is very low. Thus, PC expression might be altered under this situation and the reasons are still unknown. Aspartate aminotransferase is identified as another main anaplerotic pathway under physiological conditions⁽Reference Simpson, Khokhlova and Oca-Cossio⁶³⁾. However, it has not been investigated in protein restriction conditions. Under physiological conditions, glutamate is described as a non-essential anaplerotic substrate⁽Reference Simpson, Khokhlova and Oca-Cossio⁶³⁾ but, under protein malnutrition, this amino acid may exert a role during anaplerosis. The hypothesis for a main glutamate anaplerotic route is based on the higher activity of glutamate–pyruvate transaminase shown in undernourished rats⁽Reference Rasschaert, Reusens and Dahri⁹⁴⁾. In this condition, an increased insulin release is observed in response to leucine stimulation, a positive GDH allosteric modulator⁽Reference Filiputti, Ferreira and Souza³²⁾. One possible reason could be an alternative pathway for pyruvate, α-ketoglutarate and NADPH synthesis. In agreement, we have recently demonstrated that GDH protein expression is reduced in rats submitted to protein undernourishment. However, leucine supplementation restored GDH expression as well as GIIS to control levels⁽Reference Silva, Filiputti and Zoppi⁹⁷⁾, probably by enhanced α-ketoglutarate and NADPH production.

Fig. 3 Effect of protein malnutrition on anaplerosis and cataplerosis pathways during insulin secretion in β-cells. The pyruvate cycling including the pyruvate–malate shuttle, pyruvate–citrate cycle and pyruvate–isocitrate–α-ketoglutarate cycle is severely affected. Glucose anaplerosis might be affected by the decrease observed in FAD-linked glycerophosphate dehydrogenase activity. This observed decrease might impair mitochondrial reoxidation of cytosolic NADH, reducing glycolytic flux and pyruvate availability. The amino acid anaplerotic route seems to be also decreased by the reduced glutamate dehydrogenase expression which decreases α-ketoglutarate enhancement. Reduced anaplerotic capacity will result in lowered ATP:ADP and cataplerosis as well. Decreased cataplerosis flux would result in reduced metabolic coupling factors, such as malonyl-CoA and NADPH production. These metabolic alterations might impair insulin release.

Reusens et al. ⁽Reference Reusens, Sparre and Kalbe⁹⁸⁾, examining the effect of a low-protein diet in pregnant rats, demonstrated that the gene expression of most TAC proteins was substantially up-regulated, an effect that was accompanied by a reduced expression of superoxide dismutase and heat shock protein-1, -1a and -1b. However, cytochrome c oxidase activity and ATP production were markedly reduced. Preliminary data from our laboratory provided evidence for a reduction of catalase activity, but GPX activity remained unchanged (APG Cappelli, CC Zoppi, A Trevisan, TM Batista, PMR da Silva and EM Carneiro, unpublished results). It seems reasonable, therefore, that during protein deficiency anaplerosis might be compromised, leading to reduced NADH:NAD⁺ and NADPH:NADP⁺ ratios and insulin synthesis and release. Further studies are needed to investigate the expression and content of anaplerotic enzymes as well as their substrate oscillations during insulin release events in undernourished β-cells.

Anaplerosis and peripheral insulin resistance

Skeletal muscle operates in a coordinated way with pancreatic islets in the control of glucose levels⁽Reference Phielix and Mensink¹³⁾. The binding of insulin to its receptor induces insulin receptor tyrosine kinase activity. Tyrosine phosphorylation of insulin receptor substrate (IRS)-1 results in activation of the p85 regulatory subunit of phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase and activates the p110 catalytic subunit, which increases phosphoinositides such as PI3. This leads to activation of phosphoinositide-dependent protein kinase and downstream PKB (Akt) and/or atypical protein kinase C⁽Reference Ishiki and Klip⁹⁹⁾. Phosphorylation of Akt substrate 160 (AS160), which has a GTPase-activating domain (Rab4), facilitates translocation of GLUT-4 to the sarcolemma, favouring glucose uptake⁽Reference Wei, Chen and Whaley-Connell¹⁰⁰⁾.

Intramuscular TAG (IMTG) accumulation leads to an increased concentration of FA metabolites including diacylglycerol, fatty acyl-CoA and ceramides that in turn activate serine kinase leading to IRS-1 phosphorylation of serine residues, and consequently inhibition of insulin downstream events⁽Reference Gual, Le Marchand-Brustel and Tanti¹⁰¹⁾. In addition, higher levels of IMTG may increase ROS production and induce inflammation⁽Reference Wei, Chen and Whaley-Connell^100, Reference Schenk, Saberi and Olefsky¹⁰²⁾.

Abnormal mitochondrial metabolism has been described in the insulin-resistant state⁽Reference Turner and Heilbronn^12, Reference De Filippis, Alvarez and Berria^103–Reference Rabøl, Boushel and Dela¹⁰⁵⁾. However, IMTG accumulation with no mitochondrial dysfunction has also been associated with the development of peripheral insulin resistance⁽Reference De Feyter, Van den Broek and Praet^106–Reference Kraegen, Cooney and Turner¹⁰⁹⁾. Therefore, whether mitochondrial dysfunction is a cause or consequence of peripheral insulin resistance development is still unknown⁽Reference Phielix and Mensink¹³⁾.

Interestingly, when sedentary obese insulin-resistant subjects were submitted to a moderate exercise training programme, insulin sensitivity was increased despite enhanced IMTG stores. However, this effect was associated with a reduction in diacylglycerol and ceramide content followed by an increased mitochondrial oxidative capacity⁽Reference Dubé, Amati and Stefanovic-Racic¹⁸⁾. Similar results have been obtained in obese Zucker rats submitted to acute exercise. Despite an unchanged or increased concentration of intramuscular diacylglycerol and long-chain acyl-CoA, acute exercise improved insulin sensitivity, probably by the enhanced phosphorylation of AS160⁽Reference Thyfault²³⁾. Skeletal muscle contraction is a well-known stimulus to increase GLUT-4 translocation by an insulin-independent mechanism⁽Reference Thorell, Hirshman and Nygren¹¹⁰⁾. Although the full mechanisms involved in contraction-induced GLUT-4 translocation are still unclear, sarcoplasmic Ca²⁺ efflux and the AS160 phosphorylation by the Akt–AMP-activated kinase pathway seem to play a pivotal role also in glucose uptake⁽Reference Cartee and Wojtaszewski¹¹¹⁾.

If mitochondrial dysfunction is not the main cause of insulin resistance, it can, at least in part, contribute to the development of this condition. A conciliatory hypothesis has been proposed that the link between mitochondrial metabolism and IMTG lipotoxicity-induced insulin resistance would be confined to the level of IMTG turnover inside muscle fibres. Thus, a mismatch between IMTG hydrolysis (lipolysis) and mitochondrial β-oxidation increases the intracellular lipid content with detrimental effects on insulin signalling and glucose metabolism. So, regular physical exercise plays a key role to protect against IMTG accumulation-induced insulin resistance⁽Reference Moro, Bajpeyi and Smith¹¹²⁾.

The endproduct of FA β-oxidation is acetyl-CoA, and the further oxidation of this compound occurs inside the TAC. However, the first TAC reaction catalysed by CS is the condensation of acetyl-CoA with oxaloacetate, giving rise to citrate. Thus for a complete oxidation of FA molecules, a regular production of oxaloacetate is needed⁽Reference Siu, Donley and Bryner¹¹³⁾. Muoio & Koves⁽Reference Muoio and Koves¹¹⁴⁾ reviewed the molecular mechanism-induced mitochondrial metabolism dysfunction and proposed several mechanisms that may be involved in peripheral insulin resistance, including a reduced TAC flux. Indeed, type 2 diabetic patients have reduced TAC flux and consequently impaired complete FA oxidation⁽Reference Schrauwen and Hesselink¹¹⁵⁾. In addition, reduced ADP phosphorylation in mitochondria from type 2 diabetic patients might result from reduced TAC and electron transport chain flux⁽Reference Phielix, Schrauwen-Hinderling and Mensink¹¹⁶⁾.

One possible mechanism to explain the reduced TAC flux is an impaired anaplerotic capacity (Fig. 4). However, there are scarce data regarding anaplerotic pathways in insulin-resistant tissues. Befroy et al. ⁽Reference Befroy, Petersen and Dufour¹⁷⁾ examined whether resting skeletal muscle metabolism is altered in endurance-trained compared with sedentary subjects. These authors reported that trained subjects exhibit elevated substrate consumption at rest, suggesting a high anaplerotic and cataplerotic capacity. In contrast, elevated uncoupling protein-3 content has been demonstrated in insulin-resistant subjects, indicating an elevated IMTG content and consequently poor anaplerosis and cataplerosis capacity⁽Reference Schrauwen, Saris and Hesselink^15, Reference Schrauwen^16, Reference Schrauwen and Hesselink¹¹⁵⁾.

Fig. 4 Insulin resistance reduces tricarboxylic acid cycle (TAC) flux in peripheral tissues. The shadowed areas indicate the major pathways associated with low anaplerotic and cataplerotic capacity including glycolysis, the TAC and β-oxidation. Glucose 6-P, glucose 6-phosphate; CPT, carnitine palmitoyltransferase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; mME, mitochondrial malic enzyme; ETC, electron transport chain; O₂^− ∙, superoxide anion.

Reduced TAC flux can also be impaired by oxidative stress as previously reported in peripheral insulin resistance, a metabolic state which stimulates ROS production⁽Reference Silveira, Fiamoncini and Hirabara^59, Reference Nicolson^117–Reference Bonnard, Durand and Peyrol¹¹⁹⁾. In fact, elevated concentrations of the superoxide anion might inhibit aconitase activity⁽Reference Andersson, Leighton and Young¹²⁰⁾. Likewise, high levels of H₂O₂ reduce the activity of α-ketoglutarate dehydrogenase, one far from near-equilibrium regulatory enzyme of the TAC⁽Reference Tretter and Adam-vizi¹²¹⁾. In contrast to what is observed for β-cells, reduction in FA oxidation by malonyl-CoA is not desirable for skeletal muscle fibres. Under conditions of increased ROS production, aconitase inhibition may induce citrate accumulation and cataplerosis, resulting in decreased glycolytic flux and enhanced malonyl-CoA synthesis, leading to reduced FA oxidation. In agreement with this statement, malonyl-CoA synthesis has been shown to be increased in the situations of diabetes and insulin resistance⁽Reference Bandyopadhyay, Yu and Ofrecio¹²²⁾. Acetyl-CoA carboxylase knockout mice show enhanced insulin sensitivity and were prevented from fat-rich diet-induced obesity⁽Reference Choi, Savage and Abu-Elheiga¹²³⁾.

Chronic muscle contraction (i.e. exercise) remains as the pivotal preventive therapy against peripheral insulin resistance development. Voluntary exercise stimulates blood glucose uptake, contributing to glucose homeostasis as well by a non-insulin-dependent mechanism⁽Reference Thorell, Hirshman and Nygren^110, Reference Khayat, Patel and Klip¹²⁴⁾. In addition, exercise raises the expression of several glycolytic and oxidative enzymes involved in glucose and FA oxidation⁽Reference Laursen, Marsh and Jenkins^125–Reference Perry, Heigenhauser and Bonen¹²⁷⁾, contributing to high exercise and resting FA oxidation by the enhancement of TAC flux⁽Reference Befroy, Petersen and Dufour^17, Reference Holloszy, Kohrt and Hansen^128, Reference Mittendorfer and Klein¹²⁹⁾. In addition to the potential effect upon oxidative enzyme activities, exercise may contribute to a high mitochondrial FA oxidation capacity by regulating TAC flux and intermediate concentrations. Exercise also improves skeletal muscle antioxidant capacity, reducing the installation of oxidative stress-induced mitochondrial impairment⁽Reference Ji¹³⁰⁾. During moderate- to high-intensity exercise, TAC anaplerosis is increased and has a pivotal role in maintaining muscle contraction efficiency⁽Reference Bowtell, Marwood and Bruce^21, Reference Sahlin, Katz and Broberg²²⁾. TAC anaplerosis exerts an important role in regulating IMTG oxidation. The failure of this regulation might be implicated in peripheral insulin resistance development. In glycogen-depleted muscle, TACI concentration is maintained at the same level as during high glycogen store conditions during prolonged exercise⁽Reference Baldwin, Snow and Gibala¹³¹⁾. Under muscle glycogen-depleted exercise, FA metabolism is substantially favoured, suggesting a close relationship between anaplerosis and FA oxidation. However, acute down-regulation of TACI does not reduce oxidative ATP synthesis during exercise⁽Reference Dawson, Baker and Greenhaff¹³²⁾. Therefore, the anaplerosis process may not be directly associated with oxidative ATP production in muscle skeletal cells. Further studies are required to investigate the meaning of a possible link between anaplerosis, FA oxidation control and insulin resistance in skeletal muscle.

Protein malnutrition and peripheral insulin resistance

Protein-deficient diets have been shown to markedly affect skeletal muscle function. Several reports indicate that undernourished rats present low muscle weight and impaired morphological, metabolic and functional developments⁽Reference Alves, Dâmaso and Dal Pai^133–Reference Oumi, Miyoshi and Yamamoto¹³⁵⁾. Moreover, offspring from protein-malnourished dams also shows altered muscle structure and function that can affect animal posture and locomotion⁽Reference Toscano, Manhães-de-Castro and Canon³⁵⁾. Lehnert et al. ⁽Reference Lehnert, Byrne and Reverter¹³⁶⁾, using complementary DNA microarrays, showed a 2- to 6-fold reduction in the expression of several muscle structural proteins and metabolic enzymes in cattle submitted to severe undernutrition. Suppressed muscle growth in undernourished rats occurs probably due to a reduction in muscle and blood insulin-like growth factor-I content⁽Reference Yamaguchi, Fujikawa and Tateoka¹³⁷⁾. Based on the aforementioned, one would expect that a protein deficiency-induced decrease of muscle size and function would result in reduced muscle glucose and FA metabolism and predisposing to obesity and type 2 diabetes⁽Reference Stannard and Johnson^138, Reference Zambrano, Martínez-Samayoa and Bautista¹³⁹⁾.

Protein malnutrition shows diverse effects during the lifespan concerning peripheral insulin resistance development. Short-term protein undernutrition effects have been previously demonstrated to enhance muscle insulin sensitivity⁽Reference Escriva, Kergoat and Bailbé¹⁴⁰⁾. This effect has been shown to occur by improvement in several steps of the insulin cascade, such as increased p38 mitogen-activated protein kinase-induced GLUT-4 translocation, high levels of insulin receptor and IRS-1 tyrosine phosphorylation as well as increased IRS-1–PI3 kinase p85 subunit association and reduced IRS-1 serine phosphorylation⁽Reference Reis, Carneiro and Mello^141–Reference Gavete, Martín and Alvarez¹⁴³⁾.

Conversely, long-term protein undernutrition has the opposite effect, mainly when higher amounts of the nutrient become available⁽Reference Hales and Barker¹⁴⁴⁾. Adult rats submitted to protein restriction in early life, or offspring from undernourished mothers, develop hyperinsulinaemia, peripheral insulin resistance and type 2 diabetes⁽Reference Huang, Qiu and Shen^27, Reference Kappeler, De Magalhaes Filho and Leneuve^145–Reference Fernandez-Twinn, Wayman and Ekizoglou¹⁴⁷⁾. These alterations have been demonstrated to affect the second generation⁽Reference Pinheiro, Salvucci and Aguila¹⁴⁸⁾. Indeed, evidence points to a positive correlation between reduced fetal growth, a well-known protein-restriction feature, and peripheral insulin resistance development⁽Reference Ozanne and Hales^34, Reference Stocker, Arch and Cawthorne^149, Reference Newsome, Shiell and Fall¹⁵⁰⁾. In contrast to short-term effects, insulin signalling seems to be compromised in the adult stage of early protein undernourishment. Reduced protein kinase C-ζ expression has been reported in rats and young men. In addition, skeletal muscle PI3 kinase–Akt insulin signalling steps have been demonstrated to be altered in low-birth-weight men and rats⁽Reference Fernandez-Twinn, Wayman and Ekizoglou^147, Reference Jensen, Martin-Gronert and Storgaard^151, Reference Ozanne, Olsen and Hansen¹⁵²⁾.

Early protein restriction has been reported to programme the appetite in late adult life⁽Reference Bellinger and Langley-Evans¹⁵³⁾, reducing serotonin inhibitory action on food intake, and stimulating the preference for fat-rich foods⁽Reference Lopes de Souza, Orozco-Solis and Grit^154, Reference Bellinger, Lilley and Langley-Evans¹⁵⁵⁾. Furthermore, early protein undernutrition has also been shown to determine fat distribution and reduce physical activity levels, contributing to body and skeletal muscle fat storage in adult life⁽Reference Bellinger, Sculley and Langley-Evans¹⁵⁶⁾. The observed differences upon skeletal muscle glucose metabolism after early protein restriction might be associated with time- and diet-dependent metabolic changes. However, data on the metabolic regulation of protein malnutrition-induced peripheral insulin resistance are scant. To date, most studies have focused on neuroendocrine aspects and some have attempted to investigate skeletal muscle insulin signalling pathway changes. The effects of IMTG stores, incomplete FA oxidation and accumulation of metabolites leading to serine kinase activation and mitochondrial function are still unclear.

Short- and long-term protein malnutrition, anaplerosis and peripheral insulin resistance

Concerning metabolic aspects of short-term protein malnutrition, Fagundes et al. ⁽Reference Fagundes, Moura and Passos¹⁵⁷⁾ reported that young adult rats from low-protein-fed mothers showed low visceral and total body fat content, probably caused by increased lipolysis or decreased lipogenesis which might be related to the observed high plasma catecholamine and low insulin levels compared with control. Pups from low-protein-fed mothers showed reduced serum NEFA and similar skeletal muscle acetyl-CoA carboxylase, FA synthase and carnitine palmitoyl transferase-1 expression as compared with control⁽Reference Maloney, Gosby and Phuyal¹⁵⁸⁾. These results are in line with those reported by Toyoshima et al. ⁽Reference Toyoshima, Ohne and Takahashi¹⁴²⁾. These latter authors reported decreased levels of IRS-1 serine phosphorylation. The lower level of total body and consequently skeletal muscle fat might reduce the activation of specific serine kinases that impair skeletal muscle insulin downstream events. On the other hand, Gosby et al. ⁽Reference Gosby, Stanton and Maloney¹⁵⁹⁾ provided clues for long-term protein restriction to increase body fat content triggering peripheral insulin resistance events. Zhu et al. ⁽Reference Zhu, Ford and Means¹⁶⁰⁾ examined the long-term effects of undernutrition on skeletal muscle of offspring pregnant ewes and reported severe alterations in skeletal muscle metabolism. Despite an enhanced proportion of type 2 muscle fibres, GLUT-4 concentration was decreased and IMTG content was increased. In addition, a reduction in FA oxidation due to an almost 25 % decrease in carnitine palmitoyl transferase-1 activity, as well as a reduced expression of ATP synthase and antioxidant enzymes, probably by decreased mitochondria density, was reported. In agreement, Park et al. ⁽Reference Park, Kim and Kim¹⁶¹⁾ showed reduced mitochondrial DNA content and cytochrome c oxidase subunits I and III expression due to long-term effect of protein malnutrition during gestation and lactation.

Selak et al. ⁽Reference Selak, Storey and Peterside¹⁶²⁾, using the bilateral uterine artery ligation model in pregnant rats to induce intra-uterine growth retardation, reported a decrease of 43 % in muscle glycogen content compared with control. This effect was associated with decreased mitochondrial ATP synthesis and reduced pyruvate, α-ketoglutarate, glutamate and succinate oxidation in isolated muscle mitochondria during respiratory state 3. Interestingly, alterations in most of the respiratory chain-linked electron transfer and energy coupling in muscle mitochondria parameters between control and growth-retarded animals were not observed. In this sense, a reduction in TAC flux and FA oxidation would be expected, but unfortunately TACI content was not measured. Evidence for a reduced TAC flux was provided by Lane et al. ⁽Reference Lane, Chandorkar and Flozak¹⁶³⁾. These authors showed a decreased NAD⁺:NADH in growth-retarded rats, despite unaltered activities of mitochondrial isocitrate dehydrogenase and malate dehydrogenase⁽Reference Lane, Kelley and Ritov¹⁶⁴⁾. As discussed earlier, under nutrient deprivation, TAC flux and consequently mitochondrial metabolism are altered. Mehta et al. ⁽Reference Mehta, Chopra and Mehta¹⁶⁵⁾ demonstrated reduced oxidative enzyme activities in growing young monkeys submitted to a low energy intake, being associated with peripheral insulin resistance and type 2 diabetes. However, as proposed for ordinary nutrition conditions, TAC flux might be reduced by impaired specific TAC enzyme activities or anaplerosis capacity, and so further studies are needed to answer the remaining questions.

Elevated oxidative stress has also been reported after protein malnutrition in skeletal muscle. Despite conflicting results, antioxidant enzyme activities vary according to diet protein content, and lipid peroxidation was directly related to their detoxifying capacity⁽Reference Park, Kim and Kim^161, Reference Huang and Fwu¹⁶⁶⁾. Moreover, a twofold reduction in glutathione-S-transferase expression and glutathione content was observed in skeletal muscle from malnourished rats⁽Reference Zhu, Ford and Means¹⁶⁰⁾. These effects were observed to be in concert with reduced GPX scavenger capacity⁽Reference Oumi, Miyoshi and Yamamoto¹⁶⁷⁾.

The long-term metabolic effects of protein undernutrition seem, therefore, to reproduce the effects of obesity in regular-fed subjects and it is reasonable that peripheral insulin resistance and type 2 diabetes in regular feeding and protein undernutrition are induced by the same metabolic alterations. However, earlier, chronic and acute exercise is a powerful tool against peripheral insulin resistance, by its action upon restoration of insulin sensitivity. Nevertheless, early protein undernutrition seems to compromise exercise tolerance in men and rats⁽Reference Toscano, Manhães-de-Castro and Canon^35, Reference Bellinger, Sculley and Langley-Evans^156, Reference Kopple, Storer and Casburi¹⁶⁸⁾.

During adult life, reduced exercise capacity after early protein undernourishment was demonstrated to be associated with reduced intracellular levels of phosphocreatine and inorganic phosphate⁽Reference Thompson, Damyanovich and Madapallimattam¹⁶⁹⁾. This effect was accompanied by a faster depletion of muscle glycogen stores during exercise⁽Reference de-Mello¹⁷⁰⁾.

Conclusion

β-Cell elevated TACI concentration plays a key role for oxidative energy production and insulin release. The mechanism is supported by pyruvate metabolism (anaplerosis), mainly involving the enzyme PC. The citrate, malate and isocitrate produced exit the mitochondria to the cytosol (cataplerosis), increasing the cytosolic pyruvate concentration followed by elevated NADPH generation. Protein-deficient diets lead to impaired insulin secretion which is related to the lower mitochondrial oxidative and anaplerotic capacity. It seems reasonable, therefore, that in the protein-deficient state anaplerosis might be compromised, leading to reduced NADH:NAD⁺ and NADPH:NADP⁺ ratios, and thus further investigation is needed. In peripheral tissues, the mismatch between IMTG hydrolysis and mitochondrial β-oxidation increases the intracellular lipid content with detrimental effects on insulin signalling and glucose metabolism in normally fed and protein malnutrition states. In contrast to β-cells, skeletal muscle fibre TAC flux enhancement does not seem to be related to ATP:ADP increase, but with FA oxidation regulation. Despite no clear evidence of impaired anaplerotic and cataplerotic reactions being available in protein undernourishment, it is possible that peripheral insulin resistance could be triggered by reduced anaplerotic replenishment of the TACI, leading to inadequate FA oxidation. In addition, impaired TAC could also be induced by oxidative stress, followed by citrate cataplerosis and consequent malonyl-CoA accumulation. Therefore, based on the aforementioned data, future studies are needed to better focus on the role played by anaplerosis and cataplerosis reactions upon the control of β-cell insulin release mechanisms and peripheral insulin resistance in protein malnutrition states.