Regulation Of Pyruvate Dehydrogenase Research Articles

Regulation of muscle pyruvate dehydrogenase activity and fuel use during exercise in high-altitude deer mice.

Adult, lab-reared, highland deer mice acclimate to hypoxia by increasing reliance on carbohydrates to fuel exercise. Yet neither the underlying mechanisms for this shift in fuel use nor the impact of lifetime hypoxia exposure experienced in high alpine conditions, are fully understood. Thus, we assessed the use of fuel during exercise in wild highland deer mice running in their native environment. We examined a key step in muscle carbohydrate oxidation - the regulation of pyruvate dehydrogenase (PDH) - during exercise at altitude in wild highlanders and in first generation (G1) lab-born and -raised highlanders acclimated to normoxia or hypoxia. PDH activity was also determined in the gastrocnemius of G1 highlanders using an in situ muscle preparation. We found that wild highlanders had a high reliance on carbohydrates while running in their native environment, consistent with data from hypoxia-acclimated G1 highlanders. PDH activity in the gastrocnemius was similar post exercise between G1 and wild highlanders. However, when the gastrocnemius was stimulated at a light work rate in situ, PDH activity was higher in hypoxia-acclimated G1 highlanders and was associated with lower intramuscular lactate levels. These findings were supported by lower PDH kinase 2 protein production in hypoxia-acclimated G1 mice. Our findings indicate that adult phenotypic plasticity in response to low oxygen is sufficient to increase carbohydrate reliance during exercise in highland deer mice. Additionally, variation in PDH regulation with hypoxia acclimation contributes to shifts in whole-animal patterns of fuel use and is likely to improve exercise performance via elevated energy yield per mole of O2. .

Differential effects of Western diet and traumatic muscle injury on skeletal muscle metabolic regulation in male and female mice.

This study was designed to develop an understanding of the pathophysiology of traumatic muscle injury in the context of Western diet (WD; high fat and high sugar) and obesity. The objective was to interrogate the combination of WD and injury on skeletal muscle mass and contractile and metabolic function. Male and female C57BL/6J mice were randomized into four groups based on a two-factor study design: (1) injury (uninjured vs. volumetric muscle loss [VML]) and (2) diet (WD vs. normal chow [NC]). Electrophysiology was used to test muscle strength and metabolic function in cohorts of uninjured+NC, uninjured+WD, VML+NC and VML+WD at 8weeks of intervention. VML-injured male and female mice both exhibited decrements in muscle mass (-17%, P<0.001) and muscle strength (-28%, P<0.001); however, VML+WD females had a 28% greater muscle mass compared to VML+NC females (P=0.034), a compensatory response not detected in males. VML-injured male and female mice both had lower carbohydrate- and fat-supported muscle mitochondrial respiration (JO2 ) and less electron conductance through the electron transport system (ETS); however, male VML-WD had 48% lower carbohydrate-supported JO2 (P=0.014) and 47% less carbohydrate-supported electron conductance (P=0.026) compared to male VML+NC, and this diet-injury phenotype was not present in females. ETS electron conductance starts with complex I and complex II dehydrogenase enzymes at the inner mitochondrial membrane, and male VML+WD had 31% less complex I activity (P=0.004) and 43% less complex II activity (P=0.005) compared to male VML+NC. This was a diet-injury phenotype not present in females. Pyruvate dehydrogenase (PDH), β-hydroxyacyl-CoA dehydrogenase, citrate synthase, α-ketoglutarate dehydrogenase and malate dehydrogenase metabolic enzyme activities were evaluated as potential drivers of impaired JO2 in the context of diet and injury. There were notable male and female differential effects in the enzyme activity and post-translational regulation of PDH. PDH enzyme activity was 24% less in VML-injured males, independent of diet (P<0.001), but PDH enzyme activity was not influenced by injury in females. PDH enzyme activity is inhibited by phosphorylation at serine-293 by PDH kinase 4 (PDK4). In males, there was greater total PDH, phospho-PDHser293 and phospho-PDH-to-total PDH ratio in WD mice compared to NC, independent of injury (P≤0.041). In females, PDK4 was 51% greater in WD compared to NC, independent of injury (P=0.025), and was complemented by greater phospho-PDHser293 (P=0.001). Males are more susceptible to muscle metabolic dysfunction in the context of combined WD and traumatic injury compared to females, and this may be due to impaired metabolic enzyme functions.

The NADase CD38 may not dictate NAD levels in brain mitochondria of aged mice but regulates hydrogen peroxide generation

Aging is a time-related functional decline that affects many species. One of the hallmarks of aging is mitochondrial dysfunction, which leads to metabolic decline. The NAD decline during aging, in several tissues, correlates with increase in NADase activity of CD38. Knock out or pharmacological inhibition of CD38 activity can rescue mitochondrial function in several tissues, however, the role of CD38 in controlling NAD levels and metabolic function in the aging brain is unknown. In this work, we investigated CD38 NADase activity controlling NAD levels and mitochondrial function in mice brain with aging. We demonstrate that NADase activity of CD38 does not dictate NAD total levels in brain of aging mice and does not control mitochondrial oxygen consumption nor other oxygen parameters markers of mitochondrial dysfunction. However, for the first time we show that CD38 regulates hydrogen peroxide (H2O2) generation, one of the reactive oxygen species (ROS) in aging brain, through regulation of pyruvate dehydrogenase and alfa-ketoglutarate dehydrogenase, as mitochondria H2O2 leakage sites. The effect may be related to mitochondrial calcium handling differences in CD38 absence. Our study highlights a novel role of CD38 in brain energy metabolism and aging.

AMP-activated protein kinase - a journey from 1 to 100 downstream targets.

A casual decision made one evening in 1976, in a bar near the Biochemistry Department at the University of Dundee, led me to start my personal research journey by following up a paper that suggested that acetyl-CoA carboxylase (ACC) (believed to be a key regulatory enzyme of fatty acid synthesis) was inactivated by phosphorylation by what appeared to be a novel, cyclic AMP-independent protein kinase. This led me to define and name the AMP-activated protein kinase (AMPK) signalling pathway, on which I am still working 46 years later. ACC was the first known downstream target for AMPK, but at least 100 others have now been identified. This article contains some personal reminiscences of that research journey, focussing on: (i) the early days when we were defining the kinase and developing the key tools required to study it; (ii) the late 1990s and early 2000s, an exciting time when we and others were identifying the upstream kinases; (iii) recent times when we have been studying the complex role of AMPK in cancer. The article is published in conjunction with the Sir Philip Randle Lecture of the Biochemical Society, which I gave in September 2022 at the European Workshop on AMPK and AMPK-related kinases in Clydebank, Scotland. During the early years of my research career, Sir Philip acted as a role model, due to his pioneering work on insulin signalling and the regulation of pyruvate dehydrogenase.

Modeling the role of HIF in the regulation of metabolic key genes LDH and PDH: Emergence of Warburg phenotype

AbstractOxygenation of tumors and the effect of hypoxia on cancer cell metabolism is a widely studied subject. Hypoxia‐inducible factor (HIF), the main actor in the cell response to hypoxia, represents a potential target in cancer therapy. HIF is involved in many biological processes such as cell proliferation, survival, apoptosis, angiogenesis, iron metabolism, and glucose metabolism. This protein regulates the expressions of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), both essential for the conversion of pyruvate to be used in aerobic and anaerobic pathways. HIF upregulates LDH, increasing the conversion of pyruvate into lactate which leads to higher secretion of lactic acid by the cell and reduced pH in the microenvironment. HIF indirectly downregulates PDH, decreasing the conversion of pyruvate into acetyl coenzyme A, which leads to reduced usage of the tricarboxylic acid (TCA) cycle in aerobic pathways. Upregulation of HIF may promote the use of anaerobic pathways for energy production even in normal extracellular oxygen conditions. Higher use of glycolysis even in normal oxygen conditions is called the Warburg effect. In this paper, we focus on HIF variations during tumor growth and study, through a mathematical model, its impact on the two metabolic key genes PDH and LDH, to investigate its role in the emergence of the Warburg effect. Mathematical equations describing the enzyme regulation pathways were solved for each cell of the tumor represented in an agent‐based model to best capture the spatio‐temporal oxygen variations during tumor development caused by cell consumption and reduced diffusion inside the tumor. Simulation results show that reduced HIF degradation in normoxia can induce higher lactic acid production. The emergence of the Warburg effect appears after the first period of hypoxia before oxygen conditions return to a normal level. The results also show that targeting the upregulation of LDH and the downregulation of PDH could be relevant in therapy.

Phosphorylation‐based regulation of mitochondrial metabolism

Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7‐/‐ mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7‐/‐ tissues have decreased mitochondrial function concomitant with a post‐transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7‐/‐tissues suggest novel functional connections between Pptc7‐mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear‐encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix‐localized phosphatase in the post‐translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.

A Warburg-like metabolic program coordinates Wnt, AMPK, and mTOR signaling pathways in epileptogenesis.

Epilepsy is a complex neurological condition characterized by repeated spontaneous seizures and can be induced by initiating seizures known as status epilepticus (SE). Elaborating the critical molecular mechanisms following SE are central to understanding the establishment of chronic seizures. Here, we identify a transient program of molecular and metabolic signaling in the early epileptogenic period, centered on day five following SE in the pre-clinical kainate or pilocarpine models of temporal lobe epilepsy. Our work now elaborates a new molecular mechanism centered around Wnt signaling and a growing network comprised of metabolic reprogramming and mTOR activation. Biochemical, metabolomic, confocal microscopy and mouse genetics experiments all demonstrate coordinated activation of Wnt signaling, predominantly in neurons, and the ensuing induction of an overall aerobic glycolysis (Warburg-like phenomenon) and an altered TCA cycle in early epileptogenesis. A centerpiece of the mechanism is the regulation of pyruvate dehydrogenase (PDH) through its kinase and Wnt target genes PDK4. Intriguingly, PDH is a central gene in certain genetic epilepsies, underscoring the relevance of our elaborated mechanisms. While sharing some features with cancers, the Warburg-like metabolism in early epileptogenesis is uniquely split between neurons and astrocytes to achieve an overall novel metabolic reprogramming. This split Warburg metabolic reprogramming triggers an inhibition of AMPK and subsequent activation of mTOR, which is a signature event of epileptogenesis. Interrogation of the mechanism with the metabolic inhibitor 2-deoxyglucose surprisingly demonstrated that Wnt signaling and the resulting metabolic reprogramming lies upstream of mTOR activation in epileptogenesis. To augment the pre-clinical pilocarpine and kainate models, aspects of the proposed mechanisms were also investigated and correlated in a genetic model of constitutive Wnt signaling (deletion of the transcriptional repressor and Wnt pathway inhibitor HBP1). The results from the HBP1-/- mice provide a genetic evidence that Wnt signaling may set the threshold of acquired seizure susceptibility with a similar molecular framework. Using biochemistry and genetics, this paper outlines a new molecular framework of early epileptogenesis and advances a potential molecular platform for refining therapeutic strategies in attenuating recurrent seizures.

The extensive and functionally uncharacterized mitochondrial phosphoproteome

More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis.

A pyruvate dehydrogenase kinase inhibitor prevents retinal cell death and improves energy metabolism in rat retinas after ischemia/reperfusion injury

We aimed to assess the neuroprotective effect of a pyruvate dehydrogenase kinase (PDK) inhibitor, Nov3r after ischemia/reperfusion (IR) injury in rats. IR injury was induced by applying 150 mmHg of intraocular pressure for 50 min. Nov3r was orally administered (100 mg/kg) 3 h before and 24 h after IR injury. TUNEL-positive cells increased and immunoreactive RBPMS-positive cells decreased in the rat retinas after IR injury. Administration of Nov3r significantly ameliorated the increase in TUNEL-positive cells and prevented the RBPMS-positive cell decrease. Similarly, the number of IR-induced Iba1-positive microglial cells was significantly reduced with Nov3r treatment. Among metabolic parameters, IR damage induced the elevation of lactate and pyruvate, and the reduction of ATP. Oral administration of Nov3r ameliorated these changes. Our data suggest that the Nov3r had a retinal neuroprotective effect in IR injury in rats. This finding suggests that the regulation of pyruvate dehydrogenase (PDH) activity has potential therapeutic value by enabling metabolic reprograming in diseases associated with ischemic retinal damage, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ischemic optic neuropathy and glaucoma.

Metabolic reorganization in winter: Regulation of pyruvate dehydrogenase (PDH) during long-term freezing and anoxia

Wood frogs, Rana sylvatica, can undergo prolonged periods of whole body freezing during winter, locking as much as 65–70% of total body water into extracellular ice and imposing both anoxia and dehydration on their cells. Metabolic rate depression (MRD) is an adaptation used by R. sylvatica to survive these environmental stresses, where a finite amount of ATP generated through anaerobic metabolism is directed towards maintaining pro-survival functions, while most ATP-expensive cellular processes are temporarily reduced in function. Pyruvate dehydrogenase (PDH) is a vital metabolic enzyme that links anaerobic glycolysis to the aerobic TCA cycle and is an important regulatory site in MRD. PDH enzymatic activity is regulated via reversible protein phosphorylation in response to energetic demands of cells. This study explored the posttranslational regulation of PDH at three serine sites (S232, S293, S300) on the catalytic E1α subunit along with protein expression of four pyruvate dehydrogenase kinases (PDHK1-4) in response to 24 h Freezing, 8 h Thaw, 24 h Anoxia, and 4 h Recovery in the liver and skeletal muscle of R. sylvatica using Luminex multiplex technology and western immunoblotting. Overall, inhibitory regulation of PDH was evident during 24 h Freezing and 24 h Anoxia, which could indicate a notable reduction in glycoytic flux and carbon entry into the tricarboxylic acid cycle as part of MRD. Furthermore, the expression of PDHK1-4 and phosphorylation of PDH at S232, S293, and S300 were highly tissue and stress-specific, indicative of how different tissues respond differently to stress within the same organism.

Molecular Mechanisms in Skeletal Muscle Underlying Insulin Resistance in Women Who Are Lean With Polycystic Ovary Syndrome.

Skeletal muscle molecular mechanisms underlying insulin resistance in women with polycystic ovary syndrome (PCOS) are poorly understood. To provide insight into mechanisms regulating skeletal muscle insulin resistance in women who are lean with PCOS. A hyperinsulinemic-euglycemic clamp with skeletal muscle biopsies was performed. Thirteen women who are lean who have hyperandrogenism and PCOS and seven age- and body mass index-matched healthy control subjects were enrolled. Skeletal muscle protein expression and phosphorylation were analyzed by Western blotting and intramuscular lipid content was measured by thin-layer chromatography. Women with PCOS had 25% lower whole-body insulin sensitivity and 40% lower plasma adiponectin concentration than in control subjects. Intramuscular triacylglycerol, sn-1.3 diacylglycerol, and ceramide contents in skeletal muscle were higher (40%, 50%, and 300%, respectively) in women with PCOS than in control subjects. Activation of insulin signaling did not differ between groups. In women with PCOS, the insulin-stimulated glucose oxidation was reduced and insulin-stimulated dephosphorylation of pyruvate dehydrogenase (PDH) Ser293 was absent. AMP-activated protein kinase (AMPK) α2 protein expression and basal Thr172 phosphorylation were 45% and 50% lower in women with PCOS than in control subjects, respectively. Whole-body insulin resistance in women who are lean who have hyperandrogenism and PCOS was not related to changes in the proximal part of the insulin signaling cascade in skeletal muscle despite lipid accumulation. Rather, reduced insulin sensitivity was potentially related to plasma adiponectin levels playing a modulating role in human skeletal muscle via AMPK. Furthermore, abnormal PDH regulation may contribute to reduced whole-body metabolic flexibility and thereby insulin resistance.

Microenvironmental control of glucose metabolism in tumors by regulation of pyruvate dehydrogenase.

During malignant progression cancer cells undergo a series of changes, which promote their survival, invasiveness and metastatic process. One of them is a change in glucose metabolism. Unlike normal cells, which mostly rely on the tricarboxylic acid cycle (TCA), many cancer types rely on glycolysis. Pyruvate dehydrogenase complex (PDC) is the gatekeeper enzyme between these two pathways and is responsible for converting pyruvate to acetyl-CoA, which can then be processed further in the TCA cycle. Its activity is regulated by PDP (pyruvate dehydrogenase phosphatases) and PDHK (pyruvate dehydrogenase kinases). Pyruvate dehydrogenase kinase exists in 4 tissue specific isoforms (PDHK1-4), the activities of which are regulated by different factors, including hormones, hypoxia and nutrients. PDHK1 and PDHK3 are active in the hypoxic tumor microenvironment and inhibit PDC, resulting in a decrease of mitochondrial function and activation of the glycolytic pathway. High PDHK1/3 expression is associated with worse prognosis in patients, which makes them a promising target for cancer therapy. However, a better understanding of PDC's enzymatic regulation in vivo and of the mechanisms of PDHK-mediated malignant progression is necessary for the design of better PDHK inhibitors and the selection of patients most likely to benefit from such inhibitors.

Training state and fasting-induced PDH regulation in human skeletal muscle.

The aim of the present study was to examine the influence of training state on fasting-inducedskeletal muscle pyruvate dehydrogenase (PDH) regulation, including PDH phosphorylation. Trained and untrained subjects, matched for skeletal muscle CS activity and OXPHOS protein, fasted for 36h after receiving a standardized meal. Respiratory exchange ratio (RER) was measured and blood as well as vastus lateralis muscle biopsies were obtained 2, 12, 24, and 36h after the meal. RER decreased with fasting only in untrained individuals, while PDHa activity decreased from 12h after the meal in untrained, but only tended to decrease at 36h in trained. PDH-E1α, PDP1 protein, PDH phosphorylation, and PDH acetylation in skeletal muscle was higher in trained than untrained subjects, but did not change with fasting, while PDK4 protein was higher at 36h than at 2h after the meal in both groups. In conclusion, the present results suggest that endurance exercise training modifies the fasting-induced regulation of PDHa activity in skeletal muscle and the substrate switch towards fat oxidation. PDH phosphorylation could not explain the fasting-induced regulation of PDHa activity suggesting other post translational modifications.

Effects of training status on PDH regulation in human skeletal muscle during exercise.

Pyruvate dehydrogenase (PDH) is the gateway enzyme for carbohydrate-derived pyruvate feeding into the TCA cycle. PDH may play a central role in regulating substrate shifts during exercise, but the influence of training state on PDH regulation during exercise is not fully elucidated. The purpose of this study was to investigate the impact of training state on post-translational regulation of PDHa activity during submaximal and exhaustive exercise. Eight untrained and nine endurance exercise-trained healthy male subjects performed incremental exercise on a cycle ergometer: 40min at 50% incremental peak power output (IPPO), 10min at 65% (IPPO), followed by 80% (IPPO) until exhaustion. Trained subjects had higher (P<0.05) PDH-E1α, PDK1, PDK2, PDK4, and PDP1 protein content as well as PDH phosphorylation and PDH acetylation. Exercising at the same relative intensity led to similar muscle PDH activation in untrained and trained subjects, whereas PDHa activity at exhaustion was higher (P<0.05) in trained than untrained. Furthermore, exercise induced similar PDH dephosphorylation in untrained and trained subjects, while PDH acetylation was increased (P<0.05) only in trained subjects. In conclusion, PDHa activity and PDH dephosphorylation were well adjusted to the relative exercise intensity during submaximal exercise. In addition, higher PDHa activity in trained than untrained at exhaustion seemed related to differences in glycogen utilization rather than differences in PDH phosphorylation and acetylation state, although site-specific contributions cannot be ruled out.

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus)

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.

PGC-1α and fasting-induced PDH regulation in mouse skeletal muscle.

The purpose of the present study was to examine whether lack of skeletal muscle peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α) affects the switch in substrate utilization from a fed to fasted state and the fasting‐induced pyruvate dehydrogenase (PDH) regulation in skeletal muscle. Skeletal muscle‐specific PGC‐1α knockout (MKO) mice and floxed littermate controls were fed or fasted for 24 h. Fasting reduced PDHa activity, increased phosphorylation of all four known sites on PDH‐E1α and increased pyruvate dehydrogenase kinase (PDK4) and sirtuin 3 (SIRT3) protein levels, but did not alter total acetylation of PDH‐E1α. Lack of muscle PGC‐1α did not affect the switch from glucose to fat oxidation in the transition from the fed to fasted state, but was associated with lower and higher respiratory exchange ratio (RER) in the fed and fasted state, respectively. PGC‐1α MKO mice had lower skeletal muscle PDH‐E1α, PDK1, 2, 4, and pyruvate dehydrogenase phosphatase (PDP1) protein content than controls, but this did not prevent the fasting‐induced increase in PDH‐E1α phosphorylation in PGC‐1α MKO mice. However, lack of skeletal muscle PGC‐1α reduced SIRT3 protein content, increased total lysine PDH‐E1α acetylation in the fed state, and prevented a fasting‐induced increase in SIRT3 protein. In conclusion, skeletal muscle PGC‐1α is required for fasting‐induced upregulation of skeletal muscle SIRT3 and maintaining high fat oxidation in the fasted state, but is dispensable for preserving the capability to switch substrate during the transition from the fed to the fasted state and for fasting‐induced PDH regulation in skeletal muscle.

Muscle interleukin-6 and fasting-induced PDH regulation in mouse skeletal muscle.

Fasting prompts a metabolic shift in substrate utilization from carbohydrate to predominant fat oxidation in skeletal muscle, and pyruvate dehydrogenase (PDH) is seen as a controlling link between the competitive oxidation of carbohydrate and fat during metabolic challenges like fasting. Interleukin (IL)-6 has been proposed to be released from muscle with concomitant effects on both glucose and fat utilization. The aim was to test the hypothesis that muscle IL-6 has a regulatory impact on fasting-induced suppression of skeletal muscle PDH. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) were either fed or fasted for 6 or 18 h. Lack of muscle IL-6 elevated the respiratory exchange ratio in the fed and early fasting state, but not with prolonged fasting. Activity of PDH in the active form (PDHa) was higher in fed and fasted IL-6 MKO than in control mice at 18 h, but not at 6 h, whereas lack of muscle IL-6 did not prevent downregulation of PDHa activity in skeletal muscle or changes in plasma and muscle substrate levels in response to 18 h of fasting. Phosphorylation of three of four sites on PDH-E1α increased with 18 h of fasting, but was lower in IL-6 MKO mice than in control. In addition, both PDK4 mRNA and protein increased with 6 and 18 h of fasting in both genotypes, but PDK4 protein was lower in IL-6 MKO than in control. In conclusion, skeletal muscle IL-6 seems to regulate whole body substrate utilization in the fed, but not fasted, state and influence skeletal muscle PDHa activity in a circadian manner. However, skeletal muscle IL-6 is not required for maintaining metabolic flexibility in response to fasting.

Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise.

Pyruvate dehydrogenase (PDH) plays a key role in the regulation of skeletal muscle substrate utilization. IL-6 is produced in skeletal muscle during exercise in a duration dependent manner and has been reported to increase whole body fatty acid oxidation, muscle glucose uptake and decrease PDHa activity in skeletal muscle of fed mice. The aim of the present study was to examine whether muscle IL-6 contributes to exercise-induced PDH regulation in skeletal muscle. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) completed a single bout of treadmill exercise for 10, 60 or 120 min, with rested mice of each genotype serving as basal controls. The respiratory exchange ratio (RER) was overall higher (P<0.05) in IL-6 MKO than control mice during the 120 min of treadmill exercise, while RER decreased during exercise independent of genotype. AMPK and ACC phosphorylation also increased with exercise independent of genotype. PDHa activity was in control mice higher (P<0.05) at 10 and 60 min of exercise than at rest but remained unchanged in IL-6 MKO mice. In addition, PDHa activity was higher (P<0.05) in IL-6 MKO than control mice at rest and 60 min of exercise. Neither PDH phosphorylation nor acetylation could explain the genotype differences in PDHa activity. Together, this provides evidence that skeletal muscle IL-6 contributes to the regulation of PDH at rest and during prolonged exercise and suggests that muscle IL-6 normally dampens carbohydrate utilization during prolonged exercise via effects on PDH.

Abstract A61: Tumor metabolic remodeling can modulate anticancer drug response: CPI-613 attack on tumor cell mitochondrial metabolism is mediated by metabolite availability

Abstract We will describe recent progress in understanding the role of cancer metabolic remodeling in determining the response of tumors and tumor cells to a novel, first-in-class clinical agent class selectively targeting cancer cell mitochondrial metabolism. The reconfiguration of mitochondrial metabolism in cancer in general, and the tricarboxylic acid (TCA) cycle in particular, presents a rich source of potential therapeutic targets. Pyruvate dehydrogenase (PDH) and alpha ketoglutarate dehydrogenase (KGDH) are two gatekeeper enzymes regulating carbon flux into the mitochondrial TCA cycle. A key component of the altered regulatory processes in cancer is lipoic acid (lipoate), which not only functions as a cofactor for both PDH and KGDH, but also as source of regulatory information. Moreover, the lipoate-sensitive regulatory components of PDH and KGDH are substantially reconfigured in tumor cells, providing cancer-specific targets that can be successfully attacked with properly designed lipoate analogs. We developed and characterized CPI-613, a lipoic acid analog, which selectively targets mitochondrial metabolism in tumor cells by mobilizing lipoate-sensitive, cancer-specific regulation of PDH and KGDH, shutting down the two enzymes and thereby choking carbon flow through the TCA cycle. This attack on mitochondrial metabolism redundantly activates multiple cell death pathways (Zachar, et al., 2011, J Mol Med 89, 1137; Stuart, et al., 2014, Cancer&amp;Metab 2, 4). In vitro, CPI-613 is uniformly effective in killing tumor cell lines (more than 50 tumor lines tested to date). Moreover, these drugs have produced compelling clinical responses (including complete remissions) in some, but not all patients with advanced, refractory, heavily pre-treated cancers in initial Phase I/II clinical trials (Pardee et al., 2014, Clin Cancer Res 20:5255). In an effort to explore the mechanisms of CPI-613 activity and characterize the reasons for differential patient responses, we have shown that the kinetics of the cancer cell death response to CPI-613 is profoundly affected by the carbon sources available to the cell (Zachar, et al., 2011, J Mol Med 89, 1137). For example, in media where pyruvate and glutamine (capable of producing net ATP synthesis only in the mitochondrial compartment) are the major carbon sources, death is rapid, completed within 4 hours (op cit). In contrast, when glucose is substituted for pyruvate, the cell death programs take up to 48 hours to complete. We have analyzed the mechanistic basis of this and related observations, including steady state and flux metabolomics, pharmacological inhibitors of non-TCA metabolic pathways, and manipulation of media composition to systematically characterize these dramatic effects of nutrient availability on cancer cell drug response. Our results demonstrate that CPI-613 induced inactivation of tumor cell KGDH is modulated by the potency of mitochondrial redox detoxification (Stuart, et al., 2014, Cancer&amp;Metab 2, 4). Given that various metabolic pathways (for example, glycolysis) can ultimately contribute to mitochondrial ROS detoxification potential, it appears that substrate availability powerfully influences the cellular response to CPI-613. These insights open a number of fundamentally new approaches to increasing the fraction of patients who respond strongly to CPI-613 in the clinic. We will also report pre-clinical studies targeted at allowing planned future clinical trials to exploit these new insights. Citation Format: Shawn D. Stuart, Moises Guardado, Michael Dahan, Paul M. Bingham, Zuzana Zachar. Tumor metabolic remodeling can modulate anticancer drug response: CPI-613 attack on tumor cell mitochondrial metabolism is mediated by metabolite availability. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A61.

METABOLIC ARREST DURING HIBERNATION! CARDIAC REGULATION OF PYRUVATE DEHYDROGENASE (PDH) IN A HIBERNATING GROUND SQUIRREL (ICTIDOMYS TRIDECEMLINEATUS)

Many mammals use hibernation to survive freezing temperatures and lack of food during winter months by cycling through periods of torpor-arousal. Hibernating 13-line ground squirrels (Ictidomys tridecemlineatus) native to the North American prairies utilize variety of physiological adaptations to survive the harsh winter conditions. Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that links glycolysis and tricarboxylic acid cycle. Inhibition of PDH activity via three serine phosphorylation sites (p-S293, p-S300, and p-S232) regulates the metabolic flux through the TCA cycle, decrease glucose utilization, facilitate lipid metabolism, and overall provide metabolic adaptation to nutrient availability. The purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase mediated by mitochondrial serine/threonine kinases, PDHKs (1-4) during topor-arousal cycle in cardiac tissues of 13-line ground squirrels. Liver and muscle tissues were also included in the study for multi-tissue comparison purpose. A combination of Luminex Multiplex Assay and western immunoblotting were used to measure the protein expression levels of total PDH, three phospho-PDH modifications, and four PDH kinases in euthermic control, entrance, late torpor, and interbout arousal cycles (n=4, p<0.05). Heart showed significant 2-3 fold upregulation in PDH-pS293 and PDH-pS300 levels during torpor compared to the euthermic-control, indicating a possible shut down of PDH activity during hibernation. Subsequently, cardiac PDHK4 (with high affinity for pS293 and pS300) showed a 2-fold increase during torpor. Liver showed 30-fold increase in PDH-pS300 during torpor with PDHK1 showing 2-fold increase during torpor and arousal. As expected skeletal muscle showed limited PDH regulation during torpor-arousal cycle. Overall, heart and liver showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization through glycolysis and an increase in β-oxidation of lipids during times of extended starvation in 13-line ground squirrels. This study provides us with greater insight into the biochemical mechanisms controlling the teeter-totter between glucose oxidation and β-oxidation of fatty acids, a central theme behind recent cardiac hypertension and type-II diabetes studies.