Skeletal Muscle Mitochondrial Function/Dysfunction and Type 2 Diabetes

“Let food be your medicine and medicine be your food” stated Hippocrates, the father of Western medicine, in 400 B.C. This statement was based on the belief that food was able to influence disease, a concept that was revived several times in later years by painters, writers, scientists, and philosophers. One such philosopher, Ludwig Feuerbach, famously wrote in his 1863-4 essay “man is what he eats” introducing the idea that if we want to improve the spiritual conditions of people we must first improve their material conditions (Feuerbach, 2003). However, for years his warnings remained unheeded, at least in Western countries, in contrast to the teachings of Indian and Chinese medicine which for millennia have argued that a living organism has to assume a healthy diet. Like diet, physical activity has been also considered an important starting point for people's health. Hippocrates wrote in his book Regimen "if we could give every individual the right amount of nourishment and exercise, not too little and not too much, we would have found the safest way to health" (Hippocrates, 1955). Our knowledge about the links between diet, exercise, and disease has vastly increased since Hippocrates time. A healthy lifestyle based on diet and physical activity is now considered the keystone of disease prevention and the basis for a healthy aging. However, modern society has created conditions with virtually unrestricted access to food resources and reduced physical activity, resulting in a positive overall energy balance. This is far from the environment of our ”hunter-gathered ancestros” whose genes were modulated over thousands of years adapting our metabolism to survive when food was scarce and maximizing energy storage when food became available. In terms of evolution, this radical and sudden lifestyle change in modern society has led to a dramatic increase in the incidence of metabolic diseases including obesity and type 2 diabetes mellitus (T2DM). It seems clear that the development of T2DM has a genetic component that becomes obvious when individuals are exposed to western lifestyle. However, environment plays a critical role in the incidence of the disease being obesity the main etiological cause of T2DM. Thus, modest weight loss is enough for obese glucose intolerant subjects to prevent the development of T2DM (National Task Force on the Prevention and Treatment of Obesity, 2000)..

Protein tyrosine phosphatase-1B modulates pancreatic β-cell mass

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin signalling pathway. It has been demonstrated that PTP1B deletion protects against the development of obesity and Type 2 Diabetes, mainly through its action on peripheral tissues. However, little attention has been paid to the role of PTP1B in β-cells. Therefore, our aim was to study the role of PTP1B in pancreatic β-cells. Silencing of PTP1B expression in a pancreatic β-cell line (MIN6 cells) reveals the significance of this endoplasmic reticulum bound phosphatase in the regulation of cell proliferation and apoptosis. Furthermore, the ablation of PTP1B is able to regulate key proteins involved in the proliferation and/or apoptosis pathways, such as STAT3, AKT, ERK1/2 and p53 in isolated islets from PTP1B knockout (PTP1B (-)/(-)) mice. Morphometric analysis of pancreatic islets from PTP1B (-)/(-) mice showed a higher β-cell area, concomitantly with higher β-cell proliferation and a lower β-cell apoptosis when compared to islets from their respective wild type (WT) littermates. At a functional level, isolated islets from 8 weeks old PTP1B (-)/(-) mice exhibit enhanced glucose-stimulated insulin secretion. Moreover, PTP1B (-)/(-) mice were able to partially reverse streptozotocin-induced β-cell loss. Together, our data highlight for the first time the involvement of PTP1B in β-cell physiology, reinforcing the potential of this phosphatase as a therapeutical target for the treatment of β-cell failure, a central aspect in the pathogenesis of Type 2 Diabete

Calcineurin regulates skeletal muscle metabolism via coordinated changes in gene expression

The metabolic property of skeletal muscle adapts in response to an increased physiological demand by altering substrate utilization and gene expression. The calcium-regulated serine/threonine protein phosphatase calcineurin has been implicated in the transduction of motor neuron signals to alter gene expression programs in skeletal muscle. We utilized transgenic mice that overexpress an activated form of calcineurin in skeletal muscle (MCK-CnA*) to investigate the impact of calcineurin activation on metabolic properties of skeletal muscle. Activation of calcineurin increased glucose incorporation into glycogen and lipid oxidation in skeletal muscle. Activated calcineurin suppressed skeletal muscle glucose oxidation and increased lactate release. The enhancement in lipid oxidation was supported by increased expression of genes for lipid metabolism and mitochondrial oxidative phosphorylation. In a reciprocal fashion, several genes of glycolysis were down-regulated, whereas pyruvate dehydrogenase kinase 4 was markedly induced. This expression pattern was associated with decreased glucose utilization and enhanced glycogen storage. The peroxisome proliferator-activated receptors (PPARs) and PPARgamma coactivator 1alpha (PGC1alpha) are transcription regulators for the expression of metabolic and mitochondrial genes. Consistent with changes in the gene-regulatory program, calcineurin promoted the expression of PPARalpha, PPARdelta, and PPARgamma coactivator 1alpha in skeletal muscle. These results provide evidence that calcineurin-mediated skeletal muscle reprogramming induces the expression of several transcription regulators that coordinate changes in the expression of genes for lipid and glucose metabolism, which in turn alters energy substrate utilization in skeletal muscle

Gain-of-function R225Q mutation in AMP-activated protein kinase gamma3 subunit increases mitochondrial biogenesis in glycolytic skeletal muscle

AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma), that works as a cellular energy sensor. The existence of multiple heterotrimeric complexes provides a molecular basis for the multiple roles of this highly conserved signaling system. The AMPK gamma3 subunit is predominantly expressed in skeletal muscle, mostly in type II glycolytic fiber types. We determined whether the AMPK gamma3 subunit has a role in signaling pathways that mediate mitochondrial biogenesis in skeletal muscle. We provide evidence that overexpression or ablation of the AMPK gamma3 subunit does not appear to play a critical role in defining mitochondrial content in resting skeletal muscle. However, overexpression of a mutant form (R225Q) of the AMPK gamma3 subunit (Tg-AMPKgamma3(225Q)) increases mitochondrial biogenesis in glycolytic skeletal muscle. These adaptations are associated with an increase in expression of the co-activator PGC-1alpha and several transcription factors that regulate mitochondrial biogenesis, including NRF-1, NRF-2, and TFAM. Succinate dehydrogenase staining, a marker of the oxidative profile of individual fibers, was also increased in transversal skeletal muscle sections of white gastrocnemius muscle from Tg-AMPKgamma3(225Q) mice, independent of changes in fiber type composition. In conclusion, a single nucleotide mutation (R225Q) in the AMPK gamma3 subunit is associated with mitochondrial biogenesis in glycolytic skeletal muscle, concomitant with increased expression of the co-activator PGC-1alpha and several transcription factors that regulate mitochondrial proteins, without altering fiber type composition

The disruption of mitochondrial axonal transport is an early event in neuroinflammation

Background: in brain inflammatory diseases, axonal damage is one of the most critical steps in the cascade that leads to permanent disability. Thus, identifying the initial events triggered by inflammation or oxidative stress that provoke axonal damage is critical for the development of neuroprotective therapies. Energy depletion due to mitochondrial dysfunction has been postulated as an important step in the damage of axons. This prompted us to study the effects of acute inflammation and oxidative stress on the morphology, transport, and function of mitochondria in axons. Methods: mouse cerebellar slice cultures were challenged with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) ex vivo for 24 h. Axonal mitochondrial morphology was evaluated by transmission electron microscopy (TEM) and mitochondrial transportation by time-lapse imaging. In addition, mitochondrial function in the cerebellar slice cultures was analyzed through high-resolution respirometry assays and quantification of adenosine triphosphate (ATP) production. Results: both conditions promoted an increase in the size and complexity of axonal itochondria evident in electron microscopy images, suggesting a compensatory response. Such compensation was reflected at the tissue level as increased respiratory activity of complexes I and IV and as a transient increase in ATP production in response to acute inflammation. Notably, time-lapse microscopy indicated that mitochondrial transport (mean velocity) was severely impaired in axons, increasing the proportion of stationary mitochondria in axons after LPS challenge. Indeed, the two challenges used produced different effects: inflammation mostly reducing retrograde transport and oxidative stress slightly enhancing retrograde transportation. Conclusions: neuroinflammation acutely impairs axonal mitochondrial transportation, which would promote an inappropriate delivery of energy throughout axons and, by this way, contribute to axonal damage. Thus, preserving axonal mitochondrial transport might represent a promising avenue to exploit as a therapeutic target for neuroprotection in brain inflammatory diseases like multiple sclerosis

Interrelation of adipose tissue macrophages and fibrosis in obesity

Obesity is characterized by adipose tissue expansion, extracellular matrix remodelling and unresolved inflammation that contribute to insulin resistance and fibrosis. Adipose tissue macrophages represent the most abundant class of immune cells in adipose tissue inflammation and could be key mediators of adipocyte dysfunction and fibrosis in obesity. Although macrophage activation states are classically defined by the M1/M2 polarization nomenclature, novel studies have revealed a more complex range of macrophage phenotypes in response to external condition or the surrounding microenvironment. Here, we discuss the plasticity of adipose tissue macrophages (ATMs) in response to their microenvironment in obesity, with special focus on macrophage infiltration and polarization, and their contribution to adipose tissue fibrosis. A better understanding of the role of ATMs as regulators of adipose tissue remodelling may provide novel therapeutic strategies against obesity and associated metabolic diseases

Calcineurin does not mediate exercise-induced increase in muscle GLUT4

Exercise induces a rapid increase in expression of the GLUT4 isoform of the glucose transporter in skeletal muscle. One of the signals responsible for this adaptation appears to be an increase in cytosolic Ca(2+). Myocyte enhancer factor 2A (MEF2A) is a transcription factor that is involved in the regulation of GLUT4 expression. It has been reported that the Ca(2+)-regulated phosphatase calcineurin mediates the activation of MEF2 by exercise. It has also been shown that the expression of activated calcineurin in mouse skeletal muscle results in an increase in GLUT4. These findings suggest that increases in cytosolic Ca(2+) induce increased GLUT4 expression by activating calcineurin. However, we have obtained evidence that this response is mediated by a Ca(2+)-calmodulin-dependent protein kinase. The purpose of this study was to test the hypothesis that calcineurin is involved in mediating exercise-induced increases in GLUT4. Rats were exercised on 5 successive days using a swimming protocol. One group of swimmers was given 20 mg/kg body weight of cyclosporin, a calcineurin inhibitor, 2 h before exercise. A second group was given vehicle. GLUT4 protein was increased approximately 80%, GLUT4 mRNA was increased approximately 2.5-fold, MEF2A protein was increased twofold, and hexokinase II protein was increased approximately 2.5-fold 18 h after the last exercise bout. The cyclosporin treatment completely inhibited calcineurin activity but did not affect the adaptive increases in GLUT4, MEF2A, or hexokinase expression. We conclude that calcineurin activation does not mediate the adaptive increase in GLUT4 expression induced in skeletal muscle by exercise

Impact of GLP-1 receptor agonist versus omega-3 fatty acids supplement on obesity-induced alterations of mitochondrial respiration

ObjectiveTo compare administration of the glucagon-like peptide-1 (GLP-1) analogue, exenatide, versus dietary supplementation with the omega-3 fatty acid-rich Calanus oil on obesity-induced alterations in mitochondrial respiration. MethodsSix-week-old female C57BL/6JOlaHSD mice were given high fat diet (HFD, 45% energy from fat) for 12 weeks to induce obesity. Thereafter, they were divided in three groups where one received exenatide (10 mu g/kg/day) via subcutaneously implanted mini-osmotic pumps, a second group received 2% Calanus oil as dietary supplement, while the third group received HFD without any treatment. Animals were sacrificed after 8 weeks of treatment and tissues (skeletal muscle, liver, and white adipose tissue) were collected for measurement of mitochondrial respiratory activity by high-resolution respirometry, using an Oroboros Oxygraph-2k (Oroboros instruments, Innsbruck, Austria). ResultsIt was found that high-fat feeding led to a marked reduction of mitochondrial respiration in adipose tissue during all three states investigated - LEAK, OXPHOS and ETS. This response was to some extent attenuated by exenatide treatment, but not with Calanus oil treatment. High-fat feeding had no major effect on hepatic mitochondrial respiration, but exenatide treatment resulted in a significant increase in the various respiratory states in liver. Mitochondrial respiration in skeletal muscle was not significantly influenced by high-fat diet or any of the treatments. The precise evaluation of mitochondrial respiration considering absolute oxygen flux and ratios to assess flux control efficiency avoided misinterpretation of the results. ConclusionsExenatide increased hepatic mitochondrial respiration in high-fat fed mice, but no clear beneficial effect was observed in skeletal muscle or fat tissue. Calanus oil did not negatively affect respiratory activity in these tissues, which maintains its potential as a dietary supplement, due to its previously reported benefits on cardiac functio

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2+

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology