Origin and Function of Stress-Induced IL-6 in Murine Models.

Acute psychological stress has long been known to decrease host fitness to inflammation in a wide variety of diseases, but how this occurs is incompletely understood. Using mouse models, we show that interleukin-6 (IL-6) is the dominant cytokine inducible upon acute stress alone. Stress-inducible IL-6 is produced from brown adipocytes in a beta-3-adrenergic-receptor-dependent fashion. During stress, endocrine IL-6 is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis, which is necessary for anticipating and fueling "fight or flight" responses. This adaptation comes at the cost of enhancing mortality to a subsequent inflammatory challenge. These findings provide a mechanistic understanding of the ontogeny and adaptive purpose of IL-6 as a bona fide stress hormone coordinating systemic immunometabolic reprogramming. This brain-brown fat-liver axis might provide new insights into brown adipose tissue as a stress-responsive endocrine organ and mechanistic insight into targeting this axis in the treatment of inflammatory and neuropsychiatric diseases.

Metabolic Adaptation Establishes Disease Tolerance to Sepsis.

Sepsis is an often lethal syndrome resulting from maladaptive immune and metabolic responses to infection, compromising host homeostasis. Disease tolerance is a defense strategy against infection that preserves host homeostasis without exerting a direct negative impact on pathogens. Here, we demonstrate that induction of the iron-sequestering ferritin H chain (FTH) in response to polymicrobial infections is critical to establish disease tolerance to sepsis. The protective effect of FTH is exerted via a mechanism that counters iron-driven oxidative inhibition of the liver glucose-6-phosphatase (G6Pase), and in doing so, sustains endogenous glucose production via liver gluconeogenesis. This is required to prevent the development of hypoglycemia that otherwise compromises disease tolerance to sepsis. FTH overexpression or ferritin administration establish disease tolerance therapeutically. In conclusion, disease tolerance to sepsis relies on a crosstalk between adaptive responses controlling iron and glucose metabolism, required to maintain blood glucose within a physiologic range compatible with host survival.

Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting.

The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.

Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.

Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme.

How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

Insulin- and Lipopolysaccharide-Mediated Signaling in Adipose Tissue Macrophages Regulates Postprandial Glycemia through Akt-mTOR Activation.

The physiological role of immune cells in the regulation of postprandial glucose metabolism has not been fully elucidated. We have found that adipose tissue macrophages produce interleukin-10 (IL-10) upon feeding, which suppresses hepatic glucose production in cooperation with insulin. Both elevated insulin and gut-microbiome-derived lipopolysaccharide in response to feeding are required for IL-10 production via the Akt/mammalian target of rapamycin (mTOR) pathway. Indeed, myeloid-specific knockout of the insulin receptor or bone marrow transplantation of mutant TLR4 marrow cells results in increased expression of gluconeogenic genes and impaired glucose tolerance. Furthermore, myeloid-specific Akt1 and Akt2 knockout results in similar phenotypes that are rescued by additional knockout of TSC2, an inhibitor of mTOR. In obesity, IL-10 production is impaired due to insulin resistance in macrophages, whereas adenovirus-mediated expression of IL-10 ameliorates postprandial hyperglycemia. Thus, the orchestrated response of the endogenous hormone and gut environment to feeding is a key regulator of postprandial glycemia.

Interconversion between Anticipatory and Active GID E3 Ubiquitin Ligase Conformations via Metabolically Driven Substrate Receptor Assembly.

Cells respond to environmental changes by toggling metabolic pathways, preparing for homeostasis, and anticipating future stresses. For example, in Saccharomyces cerevisiae, carbon stress-induced gluconeogenesis is terminated upon glucose availability, a process that involves the multiprotein E3 ligase GIDSR4 recruiting N termini and catalyzing ubiquitylation of gluconeogenic enzymes. Here, genetics, biochemistry, and cryoelectron microscopy define molecular underpinnings of glucose-induced degradation. Unexpectedly, carbon stress induces an inactive anticipatory complex (GIDAnt), which awaits a glucose-induced substrate receptor to form the active GIDSR4. Meanwhile, other environmental perturbations elicit production of an alternative substrate receptor assembling into a related E3 ligase complex. The intricate structure of GIDAnt enables anticipating and ultimately binding various N-degron-targeting (i.e., "N-end rule") substrate receptors, while the GIDSR4 E3 forms a clamp-like structure juxtaposing substrate lysines with the ubiquitylation active site. The data reveal evolutionarily conserved GID complexes as a family of multisubunit E3 ubiquitin ligases responsive to extracellular stimuli.

Metabolic Rewiring and Communication: An Integrative View of Kidney Proximal Tubule Function.

The kidney proximal tubule is a key organ for human metabolism. The kidney responds to stress with altered metabolite transformation and perturbed metabolic pathways, an ultimate cause for kidney disease. Here, we review the proximal tubule's metabolic function through an integrative view of transport, metabolism, and function, and embed it in the context of metabolome-wide data-driven research. Function (filtration, transport, secretion, and reabsorption), metabolite transformation, and metabolite signaling determine kidney metabolic rewiring in disease. Energy metabolism and substrates for key metabolic pathways are orchestrated by metabolite sensors. Given the importance of renal function for the inner milieu, we also review metabolic communication routes with other organs. Exciting research opportunities exist to understand metabolic perturbation of kidney and proximal tubule function, for example, in hypertension-associated kidney disease. We argue that, based on the integrative view outlined here, kidney diseases without genetic cause should be approached scientifically as metabolic diseases.

Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is considered a gluconeogenic enzyme; however, its metabolic functions and regulatory mechanisms beyond gluconeogenesis are poorly understood. Here, we describe that dynamic acetylation of PCK1 interconverts the enzyme between gluconeogenic and anaplerotic activities. Under high glucose, p300-dependent hyperacetylation of PCK1 did not lead to protein degradation but instead increased the ability of PCK1 to perform the anaplerotic reaction, converting phosphoenolpyruvate to oxaloacetate. Lys91 acetylation destabilizes the active site of PCK1 and favors the reverse reaction. At low energy input, we demonstrate that SIRT1 deacetylates PCK1 and fully restores the gluconeogenic ability of PCK1. Additionally, we found that GSK3ß-mediated phosphorylation of PCK1 decreases acetylation and increases ubiquitination. Biochemical evidence suggests that serine phosphorylation adjacent to Lys91 stimulates SIRT1-dependent deacetylation of PCK1. This work reveals an unexpected capacity of hyperacetylated PCK1 to promote anaplerotic activity, and the intersection of post-translational control of PCK1 involving acetylation, phosphorylation, and ubiquitination.

Proline hydroxylation of CREB-regulated transcriptional coactivator 2 controls hepatic glucose metabolism.

Prolyl hydroxylase domain (PHD) enzymes change HIF activity according to oxygen signal; whether it is regulated by other physiological conditions remains largely unknown. Here, we report that PHD3 is induced by fasting and regulates hepatic gluconeogenesis through interaction and hydroxylation of CRTC2. Pro129 and Pro615 hydroxylation of CRTC2 following PHD3 activation is necessary for its association with cAMP-response element binding protein (CREB) and nuclear translocation, and enhanced binding to promoters of gluconeogenic genes by fasting or forskolin. CRTC2 hydroxylation-stimulated gluconeogenic gene expression is independent of SIK-mediated phosphorylation of CRTC2. Liver-specific knockout of PHD3 (PHD3 LKO) or prolyl hydroxylase-deficient knockin mice (PHD3 KI) show attenuated fasting gluconeogenic genes, glycemia, and hepatic capacity to produce glucose during fasting or fed with high-fat, high-sucrose diet. Importantly, Pro615 hydroxylation of CRTC2 by PHD3 is increased in livers of fasted mice, diet-induced insulin resistance or genetically obese ob/ob mice, and humans with diabetes. These findings increase our understanding of molecular mechanisms linking protein hydroxylation to gluconeogenesis and may offer therapeutic potential for treating excessive gluconeogenesis, hyperglycemia, and type 2 diabetes.

A crucial role of adenosine deaminase in regulating gluconeogenesis in mice.

Adenosine deaminase (ADA) catalyzes the irreversible deamination of adenosine (ADO) to inosine and regulates ADO concentration. ADA ubiquitously expresses in various tissues to mediate ADO-receptor signaling. A significant increase in plasma ADA activity has been shown to be associated with the pathogenesis of type 2 diabetes mellitus. Here, we show that elevated plasma ADA activity is a compensated response to high level of ADO in type 2 diabetes mellitus and plays an essential role in the regulation of glucose homeostasis. Supplementing with more ADA, instead of inhibiting ADA, can reduce ADO levels and decrease hepatic gluconeogenesis. ADA restores a euglycemic state and recovers functional islets in db/db and high-fat streptozotocin diabetic mice. Mechanistically, ADA catabolizes ADO and increases Akt and FoxO1 phosphorylation independent of insulin action. ADA lowers blood glucose at a slower rate and longer duration compared to insulin, delaying or blocking the incidence of insulinogenic hypoglycemia shock. Finally, ADA suppresses gluconeogenesis in fasted mice and insulin-deficient diabetic mice, indicating the ADA regulating gluconeogenesis is a universal biological mechanism. Overall, these results suggest that ADA is expected to be a new therapeutic target for diabetes.

Dietary protein load affects the energy and nitrogen balance requiring liver glutamate dehydrogenase to maintain physical activity.

Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids toward ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH KO mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e., suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH KO mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Hepatic ZBTB22 promotes hyperglycemia and insulin resistance via PEPCK1-driven gluconeogenesis.

Excessive gluconeogenesis can lead to hyperglycemia and diabetes through as yet incompletely understood mechanisms. Herein, we show that hepatic ZBTB22 expression is increased in both diabetic clinical samples and mice, being affected by nutritional status and hormones. Hepatic ZBTB22 overexpression increases the expression of gluconeogenic and lipogenic genes, heightening glucose output and lipids accumulation in mouse primary hepatocytes (MPHs), while ZBTB22 knockdown elicits opposite effects. Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance, accompanied by moderate hepatosteatosis, while ZBTB22-deficient mice display improved energy expenditure, glucose tolerance, and insulin sensitivity, and reduced hepatic steatosis. Moreover, hepatic ZBTB22 knockout beneficially regulates gluconeogenic and lipogenic genes, thereby alleviating glucose intolerance, insulin resistance, and liver steatosis in db/db mice. ZBTB22 directly binds to the promoter region of PCK1 to enhance its expression and increase gluconeogenesis. PCK1 silencing markedly abolishes the effects of ZBTB22 overexpression on glucose and lipid metabolism in both MPHs and mice, along with the corresponding changes in gene expression. In conclusion, targeting hepatic ZBTB22/PEPCK1 provides a potential therapeutic approach for diabetes.

Gluconeogenesis unraveled: A proteomic Odyssey with machine learning.

The metabolic pathway known as gluconeogenesis, which produces glucose from non-carbohydrate substrates, is essential for maintaining balanced blood sugar levels while fasting. It's extremely important to anticipate gluconeogenesis rates accurately to recognize metabolic disorders and create efficient treatment strategies. The implementation of deep learning and machine learning methods to forecast complex biological processes has been gaining popularity in recent years. The recognition of both the regulation of the pathway and possible therapeutic applications of proteins depends on accurate identification associated with their gluconeogenesis patterns. This article analyzes the uses of machine learning and deep learning models, to predict gluconeogenesis efficiency. The study also discusses the challenges that come with restricted data availability and model interpretability, as well as possible applications in personalized healthcare, metabolic disease treatment, and the discovery of drugs. The predictor utilizes statistics moments on the structures of gluconeogenesis and their enzymes, while Random Forest is utilized as a classifier to ensure the accuracy of this model in identifying the best outcomes. The method was validated utilizing the independent test, self-consistency, 10 k fold cross-validations, and jackknife test which achieved 92.33 %, 91.87 %, 87.88 %, and 87.02 %. An accurate prediction of gluconeogenesis has significant implications for understanding metabolic disorders and developing targeted therapies. This study contributes to the rising field of predictive biology by mixing algorithms for deep learning, and machine learning, with metabolic pathways.

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

The global nitrogen regulator GlnR is a direct transcriptional repressor of the key gluconeogenic gene pckA in actinomycetes.

In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei. Next, we proved that the pckA gene was essential for A. mediterranei gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes (Streptomyces coelicolor and Mycobacterium smegmatis). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses. IMPORTANCE: The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA, the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.

SHP-1 phosphatase acts as a coactivator of PCK1 transcription to control gluconeogenesis.

We previously reported that the protein-tyrosine phosphatase SHP-1 (PTPN6) negatively regulates insulin signaling, but its impact on hepatic glucose metabolism and systemic glucose control remains poorly understood. Here, we use co-immunoprecipitation assays, chromatin immunoprecipitation sequencing, in silico methods, and gluconeogenesis assay, and found a new mechanism whereby SHP-1 acts as a coactivator for transcription of the phosphoenolpyruvate carboxykinase 1 (PCK1) gene to increase liver gluconeogenesis. SHP-1 is recruited to the regulatory regions of the PCK1 gene and interacts with RNA polymerase II. The recruitment of SHP-1 to chromatin is dependent on its association with the transcription factor signal transducer and activator of transcription 5 (STAT5). Loss of SHP-1 as well as STAT5 decrease RNA polymerase II recruitment to the PCK1 promoter and consequently PCK1 mRNA levels leading to blunted gluconeogenesis. This work highlights a novel nuclear role of SHP-1 as a key transcriptional regulator of hepatic gluconeogenesis adding a new mechanism to the repertoire of SHP-1 functions in metabolic control.

An insulin-regulated arrestin domain protein controls hepatic glucagon action.

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver.