The role of mitochondria in the aetiology of insulin resistance and type 2 diabetes

Background

The prevalence of type 2 diabetes is rapidly increasing world-wide and insulin resistance is central to the aetiology of this disease. The biology underpinning the development of insulin resistance is not completely understood and the role of impaired mitochondrial function in the development of insulin resistance is controversial.

Scope of review

This review will provide an overview of the major processes regulated by mitochondria, before examining the evidence that has investigated the relationship between mitochondrial function and insulin action. Further considerations aimed at clarifying some controversies surrounding this issue will also be proposed.

Major conclusions

Controversy on this issue is fuelled by our lack of understanding of some of the basic biological interactions between mitochondria and insulin regulated processes in the context of insults thought to induce insulin resistance. Aspects that have not yet been considered are tissue/cell type specific responses, mitochondrial responses to site-specific impairments in mitochondrial function and as yet uncharacterised retrograde signalling from mitochondria.

General significance

Further investigation of the relationship between mitochondria and insulin action could reveal novel mechanisms contributing to insulin resistance in specific patient subsets. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Akt signalling in health and disease

Akt (also known as protein kinase B or PKB) comprises three closely related isoforms Akt1, Akt2 and Akt3 (or PKBα/β/γ respectively). We have a very good understanding of the mechanisms by which Akt isoforms are activated by growth factors and other extracellular stimuli as well as by oncogenic mutations in key upstream regulatory proteins including Ras, PI3-kinase subunits and PTEN. There are also an ever increasing number of Akt substrates being identified that play a role in the regulation of the diverse array of biological effects of activated Akt; this includes the regulation of cell proliferation, survival and metabolism. Dysregulation of Akt leads to diseases of major unmet medical need such as cancer, diabetes, cardiovascular and neurological diseases. As a result there has been substantial investment in the development of small molecular Akt inhibitors that act competitively with ATP or phospholipid binding, or allosterically. In this review we will briefly discuss our current understanding of how Akt isoforms are regulated, the substrate proteins they phosphorylate and how this integrates with the role of Akt in disease. We will furthermore discuss the types of Akt inhibitors that have been developed and are in clinical trials for human cancer, as well as speculate on potential on-target toxicities, such as disturbances of heart and vascular function, metabolism, memory and mood, which should be monitored very carefully during clinical trial.     

Skeletal muscle: Energy metabolism, fiber types, fatigue and adaptability

Skeletal muscles cope with a large range of activities, from being able to support the body weight during long periods of upright standing to perform explosive movements in response to an unexpected threat. This requires systems for energy metabolism that can provide energy during long periods of moderately increased energy consumption as well as being able to rapidly increasing the rate of energy production more than 100-fold in response to explosive contractions. In this short review we discuss how muscles can deal with these divergent demands. We first outline the major energy metabolism pathways in skeletal muscle. Next we describe metabolic differences between different muscle fiber types. Contractile performance declines during intense activation, i.e. fatigue develops, and we discuss likely underlying mechanisms. Finally, we discuss the ability of muscle fibers to adapt to altered demands, and mechanisms behind these adaptations. The accumulated experimental evidence forces us to conclude that most aspects of energy metabolism involve multiple and overlapping signaling pathways, which indicates that the control of energy metabolism is too important to depend on one single molecule or mechanism.     

Genistein stimulates fatty acid oxidation in a leptin receptor-independent manner through the JAK2-mediated phosphorylation and activation of AMPK in skeletal muscle

Obesity is a public health problem that contributes to the development of insulin resistance, which is associated with an excessive accumulation of lipids in skeletal muscle tissue. There is evidence that soy protein can decrease the ectopic accumulation of lipids and improves insulin sensitivity; however, it is unknown whether soy isoflavones, particularly genistein, can stimulate fatty acid oxidation in the skeletal muscle. Thus, we studied the mechanism by which genistein stimulates fatty acid oxidation in the skeletal muscle. We showed that genistein induced the expression of genes of fatty acid oxidation in the skeletal muscle of Zucker fa/fa rats and in leptin receptor (ObR)-silenced C2C12 myotubes through AMPK phosphorylation. Furthermore, the genistein-mediated AMPK phosphorylation occurred via JAK2, which was possibly activated through a mechanism that involved cAMP. Additionally, the genistein-mediated induction of fatty acid oxidation genes involved PGC1α and PPARδ. As a result, we observed that genistein increased fatty acid oxidation in both the control and silenced C2C12 myotubes, as well as a decrease in the RER in mice, suggesting that genistein can be used in strategies to decrease lipid accumulation in the skeletal muscle.     

New functions of mitochondria associated membranes in cellular signaling

In all eukaryotic cells, the endoplasmic reticulum (ER) and the mitochondria establish a tight interplay, which is structurally and functionally modulated through a proteinaceous tether formed at specific subdomains of the ER membrane, designated mitochondria-associated membranes or MAMs. The tethering function of the MAMs allows the regulation of lipid synthesis and rapid transmission of calcium (Ca^2 +) signals between the ER and mitochondria, which is crucial to shape intracellular Ca^2 + signaling and regulate mitochondrial bioenergetics. Research on the molecular characterization and function of MAMs has boomed in the last few years and the list of signaling and structural proteins dynamically associated with the ER–mitochondria contact sites in physiological and pathological conditions, is rapidly increasing along with the realization of an unprecedented complexity underlying the functional role of MAMs. Besides their established role as a signaling hub for Ca^2 + and lipid transfer between ER and mitochondria, MAMs have been recently shown to regulate mitochondrial shape and motility, energy metabolism and redox status and to be central to the modulation of various key processes like ER stress, autophagy and inflammasome signaling. In this review we will discuss some emerging cell-autonomous and cell non-autonomous roles of the MAMs in mammalian cells and their relevance for important human diseases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

The complete mitochondrial genome of the leafminer Liriomyza sativae (Diptera: Agromyzidae): great difference in the A+T-rich region compared to Liriomyza trifolii

The complete mitochondrial genome sequence of Liriomyza sativae Blanchard (15,551 bp) was determined and analyzed in this study. The circular genome contained 37 genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and an A + T-rich region. The initiation codons of COI and ND1 were ‘ATCA’ and ‘GTG’, respectively. ND2 gene used the truncated termination codon ‘T’. All the tRNA genes had the typical cloverleaf secondary structures except for tRNA^Ser(AGN) gene, which was found with the absence of a DHU arm. In addition, a tRNA-like secondary structure (tRNA^Met) was found in the A + T-rich region. The great difference was that the length of L. sativae A + T-rich region was 597 bp shorter than that of Liriomyza trifolii (Burgess). Meanwhile, some minor differences such as ‘TATA’ block were also observed in L. sativae in contrast to ‘TACA’ block in L. trifolii. There were also some essential structure elements such as ‘TATA’ block, ‘G(A)_nT’ block, poly-T stretch and stem-and-loop structure in the A + T-rich region of L. sativae mitochondrial genome.     

Role of AMP-activated protein kinase in the metabolic syndrome and in heart disease

Obesity, type 2 diabetes and the metabolic syndrome are disorders of energy balance, which the AMP-activated protein kinase (AMPK) regulates both at the cellular and whole body levels. AMPK switches cells from an anabolic state where nutrients are taken up and stored, to a catabolic state where they are oxidized. Drugs that activate AMPK indirectly (metformin and thiazolidinediones) are now the mainstay of treatment for type 2 diabetes, but more direct AMPK activators may have fewer side effects. However, activating mutations in AMPK can cause heart disease, and it will be important to look for adverse effects in the heart.     

PPAR delta as a therapeutic target in metabolic disease

PPAR delta is the only member in the PPAR subfamily of nuclear receptors that is not a target of current drugs. Animal studies demonstrate PPAR delta activation exerts many favorable effects, including reducing weight gain, increasing skeletal muscle metabolic rate and endurance, improving insulin sensitivity and cardiovascular function and suppressing atherogenic inflammation. These activities stem largely from the ability of PPAR delta to control energy balance, reduce fat burden and protect against lipotoxicity caused by ectopic lipid deposition. Therefore, PPAR delta represents a novel therapeutic target and the development of PPAR delta gonists/modulators may be useful for treating the whole spectrum of metabolic syndrome.     

Protein kinase C-beta: An emerging connection between nutrient excess and obesity

There is currently a global epidemic of obesity as a result of recent changes in lifestyle. Excess body fat deposition is caused by an imbalance between energy intake and energy expenditure due to interactions between genetic and environmental factors. The signals and biological mechanisms that trigger fat accumulation by disrupting energy homeostasis are not well understood. There is considerable evidence now supporting a possible role of protein kinase C beta (PKCβ) in energy homeostasis. This review highlights recent findings on the role of PKCβ activation in the genesis and progression of obesity, and of PKCβ repression in mediating the beneficial effects of physical exercise. Available data support a model in which adipose PKCβ activation is among the initiating events that disrupt mitochondrial function through interaction with p66^shc and amplify fat accumulation and adipose dysfunction, with systemic consequences. Manipulation of PKCβ levels, activity, or signaling could provide a therapeutic approach to combat obesity and associated metabolic disorders.     

The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin

Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely available analysis software, to measure f-actin content per myofibre and per nucleus over two weeks of ex vivo maintenance. We characterize the 35% per week atrophy of the isolated myofibre in terms of early changes in gene expression and investigate the effects on loss of muscle mass of modulatory agents, including Myostatin and Follistatin. By tracing the incorporation of a nucleotide analogue we show that the observed atrophy is not associated with loss or replacement of myonuclei. Such a completely controlled investigation can be conducted with the myofibres of a single muscle. With this novel method we can distinguish those features and mechanisms of atrophy and hypertrophy that are intrinsic to the muscle fibre from those that include activities of other tissues and systemic agents.     

Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria

Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H₂O₂ scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation,platelet aggregation,and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.