5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [¹³C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.     

Enhanced Fasting Glucose Turnover in Mice with Disrupted Action of TUG Protein in Skeletal Muscle

Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO₂), carbon dioxide production (VCO₂), and energy expenditure were increased by 12–13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.     

How Metabolism Generates Signals during Innate Immunity and Inflammation

The interplay between immunity, inflammation, and metabolic changes is a growing field of research. Toll-like receptors and NOD-like receptors are families of innate immune receptors, and their role in the human immune response is well documented. Exciting new evidence is emerging with regard to their role in the regulation of metabolism and the activation of inflammatory pathways during the progression of metabolic disorders such as type 2 diabetes and atherosclerosis. The proinflammatory cytokine IL-1β appears to play a central role in these disorders. There is also evidence that metabolites such as NAD⁺ (acting via deacetylases such as SIRT1 and SIRT2) and succinate (which regulates hypoxia-inducible factor 1α) are signals that regulate innate immunity. In addition, the extracellular overproduction of metabolites such as uric acid and cholesterol crystals acts as a signal sensed by NLRP3, leading to the production of IL-1β. These observations cast new light on the role of metabolism during host defense and inflammation.     

Cholecystokinin impact on rainbow trout glucose homeostasis: possible involvement of central glucosensors

Although the role of cholecystokinin (CCK) on fish appetite regulation has been widely studied, its involvement in the regulation of glucose metabolism had been little explored to date. In the present study we have carried out different experimental approaches to study CCK effects in rainbow trout (a so-called 'glucose intolerant' fish species) glucose homeostasis. We have found that for the first time in a vertebrate species, systemic or central CCK administration causes hyperglycemia, which is at least in part related to the presence of an ancestral gut-brain axis in which CCK is involved. By using capsaicin we have found that part of the action of CCK on glucose homeostasis is mediated by vagal and splanchnic afferents. Changes in hepatic metabolism after systemic CCK administration suggest that the effects are not directly taking place on the liver, but probably in other tissues, while after the central CCK administration, the glycogenolytic response observed in liver could be mediated by the activation of the sympathetic system. In hypothalamus and hindbrain changes elicited by CCK-8 treatment are likely related to the glucosensor response to the increased glycemia and/or vagal/splanchnic afferences whereas in hindbrain a possible action through specific CCK-1 receptors cannot be excluded. All these processes result in changes in metabolic parameters related with glucose homeostasis control. Further studies are needed to fully understand the role of this peptide on glucose homeostasis control in fish.     

Metabolic reprogramming of the heart through stearoyl-CoA desaturase

Stearoyl-CoA desaturase (SCD), a central enzyme in lipid metabolism that synthesizes monounsaturated fatty acids, has been linked to tissue metabolism and body adiposity regulation. Recent studies showed that SCD has the ability to reprogram cardiac metabolism, thereby regulating heart function. In the heart, the lack of SCD1 enhances glucose transport and metabolism at the expense of fatty acid (FA) uptake and oxidation. The metabolic changes associated with SCD1 deficiency protect cardiac myocytes against both necrotic and apoptotic cell death and improve heart function. Furthermore, SCD4, a heart-specific isoform of SCD, is specifically repressed by leptin and the lack of SCD1 function in leptin-deficient ob/ob mice results in a decrease in the accumulation of neutral lipids and ceramide and improves the systolic and diastolic function of a failing heart. Large-population human studies showed that the plasma SCD desaturation index is positively associated with heart rate, and cardiometabolic risk factors are modulated by genetic variations in SCD1. The current findings indicate that SCD may be used to reprogram myocardial metabolism to improve cardiac function. Here, we review recent advances in understanding the role of SCD in the control of heart metabolism and its involvement in the pathogenesis of lipotoxic cardiomyopathies.     

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no ¹³C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated ¹³C-MFA using the optimal tracers [1,2-¹³C]glucose, [1,6-¹³C]glucose, [1,2-¹³C]xylose and [5-¹³C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-¹³C]glucose and [U-¹³C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO₂ originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH₂, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.     

Regulation of plant glucosinolate metabolism

Glucosinolates and their degradation products are known to play important roles in plant interaction with herbivores and micro-organisms. In addition, they are important for human life. For example, some degradation products are flavor compounds and some exhibit anticarcinogenic properties. Recent years have seen great progress made in the understanding of glucosinolate biosynthesis in Arabidopsis thaliana. The core glucosinolate biosynthetic pathway has been revealed using biochemical and reverse genetics approaches. Future research needs to focus on questions related to regulation and control of glucosinolate metabolism. Here we review current status of studies on the regulation of glucosinolate metabolism at different levels, and highlight future research towards elucidating the signaling and metabolic network that control glucosinolate metabolism.     

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.     

Neurocircuits integrating hormone and nutrient signaling in control of glucose metabolism

As obesity, diabetes, and associated comorbidities are on a constant rise, large efforts have been put into better understanding the cellular and molecular mechanisms by which nutrients and metabolic signals influence central and peripheral energy regulation. For decades, peripheral organs as a source and a target of such cues have been the focus of study. Their ability to integrate metabolic signals is essential for balanced energy and glucose metabolism. Only recently has the pivotal role of the central nervous system in the control of fuel partitioning been recognized. The rapidly expanding knowledge on the elucidation of molecular mechanisms and neuronal circuits involved is the focus of this review.     

Polyamine Metabolism Is Sensitive to Glycolysis Inhibition in Human Neuroblastoma Cells

Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors.     

Role of Central Metabolism in the Osmoadaptation of the Halophilic Bacterium Chromohalobacter salexigens

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-¹³C]-, [2-¹³C]-, [6-¹³C]-, and [U-¹³C₆]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.     

Epigenomic links from metabolism—methionine and chromatin architecture

Chromatin and associated epigenetic marks provide important platforms for gene regulation in response to metabolic changes associated with environmental exposures, including physiological stress, nutritional deprivation, and starvation. Numerous studies have shown that fluctuations of key metabolites can influence chromatin modifications, but their effects on chromatin structure (e.g. chromatin compaction, nucleosome arrangement, and chromatin loops) and how they appropriately deposit specific chemical modification on chromatin are largely unknown. Here, focusing on methionine metabolism, we discuss recent developments of metabolic effects on chromatin modifications and structure, as well as consequences on gene regulation.     

Central nervous system regulation of adipocyte metabolism

The white adipose tissue was initially largely known only as an energy storage tissue. It is now well recognized that white adipose tissue is a major endocrine and secretory organ, which releases a wide range of protein signals and factors termed adipokines. The regulation of adipocyte metabolism is an important factor for the understanding of obesity, and some mechanisms are still unknown. Many homeostatic processes, including appetite and food intake, are controlled by neuroendocrine circuits involving the central nervous system. There is substantial evidence demonstrating that the central nervous system also directly regulates adipocyte metabolism. In this review, we discuss the central actions of some peptides with an important role in energy balance regulation on adipocyte metabolism and the physiological relevance of these actions.     

Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.     

The use of stable isotopes in the study of human pathophysiology

The growing prevalence of metabolic diseases including fatty liver disease and Type 2 diabetes has increased the emphasis on understanding metabolism at the mechanistic level and how it is perturbed in disease. Metabolomics is a continually expanding field that seeks to measure metabolites in biological systems during a physiological stimulus or a genetic alteration. Typically, metabolomics studies provide total pool sizes of metabolites rather than dynamic flux measurements. More recently there has been a resurgence in approaches that use stable isotopes (e.g. ²H and ¹³C) for the unambiguous tracking of individual atoms through compartmentalised metabolic networks in humans to determine underlying mechanisms. This is known as metabolic flux analysis and enables the capture of a dynamic picture of the metabolome and its interactions with the genome and proteome. In this review, we describe current approaches using stable isotope labelling in the field of metabolomics and provide examples of studies that led to an improved understanding of glucose, fatty acid and amino acid metabolism in humans, particularly in relation to metabolic disease. Examples include the use of stable isotopes of glucose to study tumour bioenergetics as well as brain metabolism during traumatic brain injury. Lipid tracers have also been used to measure non-esterified fatty acid production whilst amino acid tracers have been used to study the rate of protein digestion on whole body postprandial protein metabolism. In addition, we illustrate the use of stable isotopes for measuring flux in human physiology by providing examples of breath tests to measure insulin resistance and gastric emptying rates.     

Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo ^r protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications. This revised version was published online in July 2006 with corrections to the Cover Date.