Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism.

Previous studies have shown that the polyamine spermidine increased the maximum life span in C. elegans and the median life span in mice. Since spermidine increases autophagy, we asked if treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. Recently, chloroquine has intensively been discussed as a treatment option for COVID-19 patients. To rule out unfavorable long-term effects on longevity, we examined the effect of chronic treatment with chloroquine given in the drinking water on the lifespan and organ pathology of male middle-aged NMRI mice. We report that, surprisingly, daily treatment with chloroquine extended the median life span by 11.4% and the maximum life span of the middle-aged male NMRI mice by 11.8%. Subsequent experiments show that the chloroquine-induced lifespan elevation is associated with dose-dependent increase in LC3B-II, a marker of autophagosomes, in the liver and heart that was confirmed by transmission electron microscopy. Quite intriguingly, chloroquine treatment was also associated with a decrease in glycogenolysis in the liver suggesting a compensatory mechanism to provide energy to the cell. Accumulation of autophagosomes was paralleled by an inhibition of proteasome-dependent proteolysis in the liver and the heart as well as with decreased serum levels of insulin growth factor binding protein-3 (IGFBP3), a protein associated with longevity. We propose that inhibition of proteasome activity in conjunction with an increased number of autophagosomes and decreased levels of IGFBP3 might play a central role in lifespan extension by chloroquine in male NMRI mice.

PCB126 induced toxic actions on liver energy metabolism is mediated by AhR in rats.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to more planar aromatic hydrocarbons, like TCDD. We previously described the sequence of events following exposure of male rats to a dioxin-like polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), that binds avidly to the AhR and causes various types of toxicity including metabolic syndrome, fatty liver, and disruption of energy homeostasis. The purpose of this study was, to investigate the role of AhR to mediate those toxic manifestations following sub-acute exposure to PCB126 and to examine possible sex differences in effects. For this goal, we created an AhR knockout (AhR-KO) model using CRISPR/Cas9. Comparison was made to the wild type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected with a single IP dose of corn oil vehicle or 5 µmol/kg PCB126 in corn oil and necropsied after 28 days. PCB126 caused significant weight loss, reduced relative thymus weights, and increased relative liver weights in WT male and female rats, but not in AhR-KO rats. Similarly, significant pathologic changes were visible which included necrosis and regeneration in female rats, micro- and macro-vesicular hepatocellular vacuolation in males, and a paucity of glycogen in livers of both sexes in WT rats only. Hypoglycemia and lower IGF1, and reduced serum non-esterified fatty acids (NEFAs) were found in serum of both sexes of WT rats, low serum cholesterol levels only in the females, and no changes in AhR-KO rats. The expression of genes encoding enzymes related to xenobiotic metabolism (e.g. CYP1A1), gluconeogenesis, glycogenolysis, and fatty acid oxidation were unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT rats where expression was significantly upregulated (PPARα, females only) or downregulated suggesting a disrupted energy homeostasis. Interestingly, Acox2, Hmgcs, G6Pase and Pc were affected in both sexes, the gluconeogenesis and glucose transporter genes Pck1, Glut2, Sds, and Crem only in male WT-PCB rats. These results show the essential role of the AhR in glycogenolysis, gluconeogenesis, and fatty acid oxidation, i.e. in the regulation of energy production and homeostasis, but also demonstrate a significant difference in the effects of PCB126 in males verses females, suggesting higher vulnerability of glucose homeostasis in males and more changes in fatty acid/lipid homeostasis in females. These differences in effects, which may apply to more/all AhR agonists, should be further analyzed to identify health risks to specific groups of highly exposed human populations.

Recombinant Bovine Growth Hormone-Induced Metabolic Remodelling Enhances Growth of Gilthead Sea-Bream (Sparus aurata): Insights from Stable Isotopes Composition and Proteomics.

Growth hormone and insulin-like growth factors (GH/IGF axis) regulate somatic growth in mammals and fish, although their action on metabolism is not fully understood in the latter. An intraperitoneal injection of extended-release recombinant bovine growth hormone (rbGH, Posilac®) was used in gilthead sea bream fingerlings and juveniles to analyse the metabolic response of liver and red and white muscles by enzymatic, isotopic and proteomic analyses. GH-induced lipolysis and glycogenolysis were reflected in liver composition, and metabolic and redox enzymes reported higher lipid use and lower protein oxidation. In white and red muscle reserves, rBGH increased glycogen while reducing lipid. The isotopic analysis of muscles showed a decrease in the recycling of proteins and a greater recycling of lipids and glycogen in the rBGH groups, which favoured a protein sparing effect. The protein synthesis capacity (RNA/protein) of white muscle increased, while cytochrome-c-oxidase (COX) protein expression decreased in rBGH group. Proteomic analysis of white muscle revealed only downregulation of 8 proteins, related to carbohydrate metabolic processes. The global results corroborated that GH acted by saving dietary proteins for muscle growth mainly by promoting the use of lipids as energy in the muscles of the gilthead sea bream. There was a fuel switch from carbohydrates to lipids with compensatory changes in antioxidant pathways that overall resulted in enhanced somatic growth.

Astrocyte-neuron lactate transport in the ACC contributes to the occurrence of long-lasting inflammatory pain in male mice.

Lactate transport is an important means of communication between astrocytes and neurons and is implicated in a variety of neurobiological processes. However, the connection between astrocyte-neuron lactate transport and nociceptive modulation has not been well established. Here, we found that Complete Freund's adjuvant (CFA)-induced inflammation pain leads to a significant increase in extracellular lactate levels in the anterior cingulate cortex (ACC). Inhibition of glycogenolysis and lactate release in the ACC disrupted the persistent, but not acute, inflammation pain induced by CFA, and this effect was reversed by exogenous L-lactate administration. Knocking down the expression of lactate transporters (MCT1, MCT4, or MCT2) also disrupted the long lasting inflammation pain induced by CFA. Moreover, glycogenolysis in the ACC is critical for the induction of molecular changes related to neuronal plasticity, including the induction of phospho- (p-) ERK, p-CREB, and Fos. Taken together, our findings indicate that astrocyte-neuron lactate transport in the ACC is critical for the occurrence of persistent inflammation pain, suggesting a novel mechanism underlying chronic pain.

Role of nitration in control of phosphorylase and glycogenolysis in mouse skeletal muscle.

Phosphorylase is one of the most carefully studied proteins in history, but knowledge of its regulation during intense muscle contraction is incomplete. Tyrosine nitration of purified preparations of skeletal muscle phosphorylase results in inactivation of the enzyme and this is prevented by antioxidants. Whether an altered redox state affects phosphorylase activity and glycogenolysis in contracting muscle is not known. Here, we investigate the role of the redox state in control of phosphorylase and glycogenolysis in isolated mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscle preparations during repeated contractions. Exposure of crude muscle extracts to H2O2 had little effect on phosphorylase activity. However, exposure of extracts to peroxynitrite (ONOO-), a nitrating/oxidizing agent, resulted in complete inactivation of phosphorylase (half-maximal inhibition at ∼200 µM ONOO-), which was fully reversed by the presence of an ONOO- scavanger, dithiothreitol (DTT). Incubation of isolated muscles with ONOO- resulted in nitration of phosphorylase and marked inhibition of glycogenolysis during repeated contractions. ONOO- also resulted in large decreases in high-energy phosphates (ATP and phosphocreatine) in the rested state and following repeated contractions. These metabolic changes were associated with decreased force production during repeated contractions (to ∼60% of control). In contrast, repeated contractions did not result in nitration of phosphorylase, nor did DTT or the general antioxidant N-acetylcysteine alter glycogenolysis during repeated contractions. These findings demonstrate that ONOO- inhibits phosphorylase and glycogenolysis in living muscle under extreme conditions. However, nitration does not play a significant role in control of phosphorylase and glycogenolysis during repeated contractions.NEW & NOTEWORTHY Here we show that exogenous peroxynitrite results in nitration of phosphorylase as well as inhibition of glycogenolysis in isolated intact mouse skeletal muscle during short-term repeated contractions. However, repeated contractions in the absence of exogenous peroxynitrite do not result in nitration of phosphorylase or affect glycogenolysis, nor does the addition of antioxidants alter glycogenolysis during repeated contractions. Thus phosphorylase is not subject to redox control during repeated contractions.

Metformin and Berberine suppress glycogenolysis by inhibiting glycogen phosphorylase and stabilizing the molecular structure of glycogen in db/db mice.

Glycogen is a branched glucose polymer involved in sustaining blood glucose homeostasis. Liver glycogen comprises α particles (up to 300 nm in diameter) made of joined ß particles (∼20 nm in diameter). Glycogen α particles in a mouse model for diabetes are molecularly fragile, breaking down into smaller ß particles more readily than in healthy mice. Glycogen phosphorylase (GP), a rate-limiting enzyme in glycogen degradation, is overexpressed in diabetic mice. This study shows that Metformin and Berberine, two common drugs, two common drugs used to treat diabetes, are able to revert the liver glycogen of diabetic mice to the stable structure seen in non-diabetic mice. It is also shown that these drugs reduce the GP level via the cAMP/PKA signaling pathway in diabetic livers and decrease the affinity of GP with the glycogen of db/db mice. These effects of these drugs may slow down the degradation of liver glycogen and improve glucose homeostasis.

ß1-adrenoceptor-stimulated lactate production in cultured astrocytes is predominantly glycogen-independent.

Noradrenaline (NA) promotes breakdown of the glucose-polymer, glycogen, and hence enhances glycolytic production of lactate in astrocytes. Here, in cultured rat cerebrocortical astrocytes, we examined the contributions of different adrenoceptor subtypes to NA-modulated glucose metabolism, and the relationship of NA-induced glycogenolysis to lactate production. Stimulation of astrocytic glucose metabolism by NA was mediated predominantly via ß1-adrenoceptors and cAMP. Constitutive ß 1-adrenoceptor activity - in the absence of exogenous NA - contributed to the basal rate of glycogen turnover. Although mRNAs encoding both ß 1- and ß 2-adrenoceptors were detected in these astrocytes, ß 2-adrenoceptors contributed little to NA-induced modulation of glucose metabolism. Activation of α2- and α 1-adrenoceptors in these cells decreased cAMP and increased cytosolic Ca2+, respectively, but did not modulate NA-induced glycogenolysis: α 2-adrenoceptors because glycogenolysis was induced maximally by NA concentrations that only began to inhibit cAMP production; and α 1-adrenoceptors possibly because of desensitisation and depletion of Ca2+ stores. Under basal conditions, astrocytes converted glucose to extracellular lactate in near stoichiometric manner. When glucose-starved astrocytes were given fresh glucose-containing medium, lactate accumulation displayed a brief lag period before attaining a steady-state rate. During this lag period NA, acting at ß 1-adrenoceptors, increased the rate of lactate accumulation both in the absence and presence of an inhibitor of glycogen turnover. At the steady-state, the rate of glucose incorporation into accumulated glycogen was ~ 5% of that into lactate, but NA enhanced lactate output by 20-50%: this further indicates that NA, via ß 1-adrenoceptors and cAMP, can enhance astrocytic lactate production independently of its effect on glycogen turnover.

Analysis of Direct Effects of the CB1 Receptor Antagonist Rimonabant on Fatty Acid Oxidation and Glycogenolysis in Liver and Muscle Cells in vitro.

Recent pharmacological findings regarding rimonabant, an anorectic and cannabinoid type 1 receptor (CB1R) antagonist, strongly suggest that some of its effects on the metabolic parameters and energy balance in rats are not related to the centrally mediated reduction in caloric intake. Instead, they may be associated with acute induction of glycogenolysis in the liver, in combination with transient increase in glucose oxidation and persistent increase in fat oxidation. It is possible that rimonabant produced direct short- or long-term stimulatory effect on these processes in primary and cultured rat cells. Rimonabant slightly stimulated ß-oxidation of long-chain fatty acids in cultured rat myocytes overexpressing glucose transporter isoform 4, as well as activated phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK) in primary rat hepatocytes upon long-term incubation. However, short-term action of rimonabant failed to stimulate ß-oxidation in myocytes, myotubes, and hepatocytes, as well as to upregulate AMPK phosphorylation, glycogenolysis, and cAMP levels in hepatocytes. As a consequence, the acute effects of rimonabant on hepatic glycogen content (reduction) and total energy expenditure (increase) in rats fed with a standard diet cannot be explained by direct stimulation of glycogenolysis and fatty acid oxidation in muscles and liver. Rather, these effects seem to be centrally mediated.

Identification of C-ß-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies.

Several C-ß-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(ß-d-glucopyranosyl)-imidazoles and 2-aryl-4-(ß-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-ß-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.

Mild Physiologic Hyperglycemia Induces Hepatic Insulin Resistance in Healthy Normal Glucose-Tolerant Participants.

CONTEXT: Chronic hyperglycemia worsens skeletal muscle insulin resistance and ß-cell function. However, the effect of sustained physiologic hyperglycemia on hepatic insulin sensitivity is not clear. OBJECTIVE: To examine the effect of sustained physiologic hyperglycemia (similar to that observed in patients with type 2 diabetes) on endogenous (primarily reflecting hepatic) glucose production (EGP) in healthy individuals. DESIGN: Volunteers participated in a three-step hyperinsulinemic (10, 20, 40 mU/m2 per minute) euglycemic clamp before and after a 48-hour glucose infusion to increase plasma glucose concentration by ∼40 mg/dL above baseline. EGP was measured with 3-3H-glucose before and after chronic glucose infusion. PARTICIPANTS: Sixteen persons with normal glucose tolerance [eight with and eight without a family history (FH) of diabetes] participated in the study. MAIN OUTCOME MEASURE: EGP. RESULTS: Basal EGP increased following 48 hours of glucose infusion (from a mean ± SEM of 2.04 ± 0.08 to 3.06 ± 0.29 mg/kgffm⋅ min; P < 0.005). The hepatic insulin resistance index (basal EGP × fasting plasma insulin) markedly increased following glucose infusion (20.1 ± 1.8 to 51.7 ± 6.6; P < 0.005) in both FH+ and FH- subjects. CONCLUSION: Sustained physiologic hyperglycemia for as little as 48 hours increased the rate of basal hepatic glucose production and induced hepatic insulin resistance in health persons with normal glucose tolerance, providing evidence for the role of glucotoxicity in the increase in hepatic glucose production in type 2 diabetes.

A Potential Role for α-Amylase in Amyloid-ß-Induced Astrocytic Glycogenolysis and Activation.

BACKGROUND: Astrocytes produce and store the energy reserve glycogen. However, abnormal large glycogen units accumulate if the production or degradation of glycogen is disturbed, a finding often seen in patients with Alzheimer's disease (AD). We have shown increased activity of glycogen degrading α-amylase in AD patients and α-amylase positive glial cells adjacent to AD characteristic amyloid-ß (Aß) plaques. OBJECTIVES: Investigate the role of α-amylase in astrocytic glycogenolysis in presence of Aß. METHODS: Presence of α-amylase and large glycogen units in postmortem entorhinal cortex from AD patients and non-demented controls were analyzed by immunohistological stainings. Impact of different Aß42 aggregation forms on enzymatic activity (α-amylase, pyruvate kinase, and lactate dehydrogenase), lactate secretion, and accumulation of large glycogen units in cultured astrocytes were analyzed by activity assays, ELISA, and immunocytochemistry, respectively. RESULTS: AD patients showed increased number of α-amylase positive glial cells. The glial cells co-expressed the astrocytic marker glial fibrillary acidic protein, displayed hypertrophic features, and increased amount of large glycogen units. We further found increased load of large glycogen units, α-amylase immunoreactivity and α-amylase activity in cultured astrocytes stimulated with fibril Aß42, with increased pyruvate kinase activity, but unaltered lactate release as downstream events. The fibril Aß42-induced α-amylase activity was attenuated by ß-adrenergic receptor antagonist propranolol. DISCUSSION: We hypothesize that astrocytes respond to fibril Aß42 in Aß plaques by increasing their α-amylase production to either liberate energy or regulate functions needed in reactive processes. These findings indicate α-amylase as an important actor involved in AD associated neuroinflammation.

Enhanced hepatic glycogen synthesis and suppressed adenosine deaminase activity by lithium attenuates hepatic triglyceride accumulation in nicotine-exposed rats.

Reduced liver glycogen synthesis might signify increased glucose flux towards fat synthesis and triggers hepatic triglyceride accumulation and dysmetabolism. Adenosine deaminase (ADA) reduces adenosine content which increases glycogenolysis. In the present study, we evaluate the effect of modulating glycogen synthesis and ADA by lithium chloride (LiCl) on nicotine-induced dysmetabolism. Twenty four male Wistar rats (n = 6/group) were allotted into four groups namely; vehicle-treated (po), nicotine-treated (1.0 mg/kg; po), LiCl-treated (5.0 mg/kg; po) and nicotine + LiCl-treated groups. The treatments lasted for 8 weeks. Nicotine exposure resulted in reduced body weight gain, liver weight, visceral adiposity, glycogen content and synthase. Along with increased insulin resistance (IR), fasting plasma glucose, lactate, plasma and hepatic ADA, XO, UA, and triglyceride (TG), total cholesterol (TC), free fatty acid, lipid peroxidation and liver injury markers. However, plasma and hepatic glucose-6-phosphate dehydrogenase-dependent antioxidant defenses were not affected by nicotine exposure. Concurrent treatment with LiCl normalizes all alterations with exception of hepatic TC. This result shows that enhancement of hepatic glycogen synthesis and suppression of ADA/XO/uric acid pathway by lithium can salvage the liver from nicotine-induced TG accumulation.

Gintonin, a Ginseng-Derived Exogenous Lysophosphatidic Acid Receptor Ligand, Protects Astrocytes from Hypoxic and Re-oxygenation Stresses Through Stimulation of Astrocytic Glycogenolysis.

Astrocytes are a unique brain cell-storing glycogen and express lysophosphatidic acid (LPA) receptors. Gintonin is a ginseng-derived exogenous G protein-coupled LPA receptor ligand. Accumulating evidence shows that astrocytes serve as an energy supplier to neurons through astrocytic glycogenolysis under physiological and pathophysiological conditions. However, little is known about the relationships between LPA receptors and astrocytic glycogenolysis or about the roles of LPA receptors in hypoxia and re-oxygenation stresses. In the present study, we examined the functions of gintonin-mediated astrocytic glycogenolysis in adenosine triphosphate (ATP) production, glutamate uptake, and cell viability under normoxic, hypoxic, and re-oxygenation conditions. The application of gintonin or LPA to astrocytes induced glycogenolysis in concentration- and time-dependent manners. The stimulation of gintonin-mediated astrocytic glycogenolysis was achieved through the LPA receptor-Gαq/11 protein-phospholipase C-inositol 1,4,5-trisphosphate receptor-intracellular calcium ([Ca2+]i) transient pathway. Gintonin treatment to astrocytes increased the phosphorylation of brain phosphorylase kinase, with sensitive manner to K252a, an inhibitor of phosphorylase kinase. Gintonin-mediated astrocytic glycogenolysis was blocked by isofagomine, a glycogen phosphorylase inhibitor. Gintonin additionally increased astrocytic glycogenolysis under hypoxic and re-oxygenation conditions. Moreover, gintonin increased ATP production, glutamate uptake, and cell viability under the hypoxic and re-oxygenation conditions. Collectively, we found that the gintonin-mediated [Ca2+]i transients regulated by LPA receptors were coupled to astrocytic glycogenolysis and that stimulation of gintonin-mediated astrocytic glycogenolysis was coupled to ATP production and glutamate uptake under hypoxic and re-oxygenation conditions, ultimately protecting astrocytes. Hence, the gintonin-mediated astrocytic energy that is modulated via LPA receptors helps to protect astrocytes under hypoxia and re-oxygenation stresses.

Norepinephrine stimulates glycogenolysis in astrocytes to fuel neurons with lactate.

The mechanism of rapid energy supply to the brain, especially to accommodate the heightened metabolic activity of excited states, is not well-understood. We explored the role of glycogen as a fuel source for neuromodulation using the noradrenergic stimulation of glia in a computational model of the neural-glial-vasculature ensemble (NGV). The detection of norepinephrine (NE) by the astrocyte and the coupled cAMP signal are rapid and largely insensitive to the distance of the locus coeruleus projection release sites from the glia, implying a diminished impact for volume transmission in high affinity receptor transduction systems. Glucosyl-conjugated units liberated from glial glycogen by NE-elicited cAMP second messenger transduction winds sequentially through the glycolytic cascade, generating robust increases in NADH and ATP before pyruvate is finally transformed into lactate. This astrocytic lactate is rapidly exported by monocarboxylate transporters to the associated neuron, demonstrating that the astrocyte-to-neuron lactate shuttle activated by glycogenolysis is a likely fuel source for neuromodulation and enhanced neural activity. Altogether, the energy supply for both astrocytes and neurons can be supplied rapidly by glycogenolysis upon neuromodulatory stimulus.

Metabolic network-based predictions of toxicant-induced metabolite changes in the laboratory rat.

In order to provide timely treatment for organ damage initiated by therapeutic drugs or exposure to environmental toxicants, we first need to identify markers that provide an early diagnosis of potential adverse effects before permanent damage occurs. Specifically, the liver, as a primary organ prone to toxicants-induced injuries, lacks diagnostic markers that are specific and sensitive to the early onset of injury. Here, to identify plasma metabolites as markers of early toxicant-induced injury, we used a constraint-based modeling approach with a genome-scale network reconstruction of rat liver metabolism to incorporate perturbations of gene expression induced by acetaminophen, a known hepatotoxicant. A comparison of the model results against the global metabolic profiling data revealed that our approach satisfactorily predicted altered plasma metabolite levels as early as 5 h after exposure to 2 g/kg of acetaminophen, and that 10 h after treatment the predictions significantly improved when we integrated measured central carbon fluxes. Our approach is solely driven by gene expression and physiological boundary conditions, and does not rely on any toxicant-specific model component. As such, it provides a mechanistic model that serves as a first step in identifying a list of putative plasma metabolites that could change due to toxicant-induced perturbations.

Insulin Regulates Glycogen Synthesis in Human Endometrial Glands Through Increased GYS2.

Context: Glycogen synthesis is a critical metabolic function of the endometrium to prepare for successful implantation and sustain embryo development. Yet, regulation of endometrial carbohydrate metabolism is poorly characterized. Whereas glycogen synthesis is attributed to progesterone, we previously found that the metabolic B isoform of the insulin receptor is maximally expressed in secretory-phase endometrium, indicating a potential role of insulin in glucose metabolism. Objective: We sought to determine whether insulin or progesterone regulates glycogen synthesis in human endometrium. Design, Participants, Outcome Measurements: Endometrial epithelial cells were isolated from 28 healthy women and treated with insulin, medroxyprogesterone (MPA), or vehicle. Intracellular glycogen and the activation of key enzymes were quantified. Results: In epithelia, insulin induced a 4.4-fold increase in glycogen, whereas MPA did not alter glycogen content. Insulin inactivated glycogen synthase (GS) kinase 3α/ß (GSK3α/ß), relieving inhibition of GS. In a regulatory mechanism, distinct from liver and muscle, insulin also increased GS by 3.7-fold through increased GS 2 (GYS2) gene expression. Conclusions: We demonstrate that insulin, not progesterone, directly regulates glycogen synthesis through canonical acute inactivation of GSK3α/ß and noncanonical stimulation of GYS2 transcription. Persistently elevated GS enables endometrium to synthesize glycogen constitutively, independent of short-term nutrient flux, during implantation and early pregnancy. This suggests that insulin plays a key, physiological role in endometrial glucose metabolism and underlines the need to delineate the effect of maternal obesity and hyperinsulinemia on fertility and fetal development.

The acute effects of citrus flavanones on the metabolism of glycogen and monosaccharides in the isolated perfused rat liver.

Citrus flavanones are often linked to their antihyperglycemic properties. This effect may be in part due to the inhibition of hepatic gluconeogenesis through different mechanisms. One of the possible mechanisms appears to be impairment of oxidative phosphorylation, which may also interfere with glycogen metabolism. Based on these facts, the purpose of the present study was to investigate the effects of three citrus flavanones on glycogenolysis in the isolated perfused rat liver. Hesperidin, hesperetin, and naringenin stimulated glycogenolysis and glycolysis from glycogen with concomitant changes in oxygen uptake. At higher concentrations (300 µM), hesperetin and naringenin clearly altered fructose and glucose metabolism, whereas hesperidin exerted little to no effects. In subcellular fractions hesperetin and naringenin inhibited the activity of glucose 6-phosphatase and glucokinase and the mitochondrial respiration linked to ADP phosphorylation. Hesperetin and naringenin also inhibited the transport of glucose into the cell. At a concentration of 300 µM, the glucose influx rate inhibition was 83% and 43% for hesperetin and naringenin, respectively. Hesperidin was the less active among the assayed citrus flavanones, indicating that the rutinoside moiety noticeably decrease the activity of these compounds. The effects on glycogenolysis and fructolysis were mainly consequence of an impairment on mitochondrial energy metabolism. The increased glucose release, due to the higher glycogenolysis, together with glucose transport inhibition is the opposite of what is expected for antihyperglycemic agents.

CCL2 Induces the Production of ß2 Adrenergic Receptors and Modifies Astrocytic Responses to Noradrenaline.

The decline in brain noradrenaline levels is associated with the progression of certain neurodegenerative diseases. This seems to be due, at least in part, to the ability of noradrenaline to limit glial activation and to reduce the damage associated with it. Our previous studies of the mechanisms involved in this process indicate that noradrenaline induces the production of the chemokine CCL2 in astrocytes. While CCL2 can protect neurons against certain injuries, its overproduction has also proven to be harmful and to prevent noradrenaline neuroprotective effects. Therefore, in this study, we analyze if the modifications caused to astrocytes by an excessive production of CCL2 may alter their response to noradrenaline. Using primary cultures of rat cortical astrocytes, we observed that CCL2 enhances the production of beta 2 adrenergic receptors in these cells. While this potentiates noradrenaline signaling through cAMP, the activation of the transcription factor CREB is inhibited by CCL2. Furthermore, although CCL2 potentiates noradrenaline induction of glycogenolysis, this does not translate into an augmented release of lactate, one of the processes through which astrocytes help support neurons. Additionally, other neuroprotective actions of noradrenaline, such as the production of brain derived neurotrophic factor and the inhibition of the inducible nitric oxide synthase in astrocytes were modified by CCL2. These data suggest that some of the central nervous system alterations related to CCL2 could be due to its effects on adrenergic receptors and its interference with noradrenaline signaling.

Refeeding after caloric restriction reverses altered liver glucose release.

CONTEXT: Caloric restriction increases liver glucose release (LGR), but it is not known if this is a permanent condition. OBJECTIVE: To investigate if refeeding after caloric restriction reverses the high LGR. MATERIALS AND METHODS: Rats were organised in six-pups litters (GC); 12-pups litters with either 50% caloric restriction from 21 to 80 days of age (GR) or fed at will from 50 to 80 days of age (GRL). Liver perfusion was made at the age of 80 days. RESULTS: LGR was higher in the GR both during basal and adrenaline-stimulated conditions. Refeeding after caloric restriction decreased it to values close to those of GC rats. DISCUSSION: The altered LGR of GR rats was reversed by refeeding (group GRL). The influence of hypothalamic neuropetides on these hepatic changes is suggested. CONCLUSIONS: Enhanced LGR under caloric restriction is not programmed by early feeding; instead, it is determined by the current nutritional conditions.

From the Cover: Metabolism Modulation in Different Organs by Silver Nanoparticles: An NMR Metabolomics Study of a Mouse Model.

Although silver nanoparticles (AgNPs) are widely disseminated and show great potential in the biomedical field, there is a recognized need to better understand their action at the metabolic and functional levels. In this work, we have used NMR metabolomics, together with conventional clinical chemistry and histological examination, to characterize multi-organ and systemic metabolic responses to AgNPs intravenously administered to mice at 8 mg/kg body weight (a dose not eliciting overt toxicity). The major target organs of AgNPs accumulation, liver and spleen, showed the greatest metabolic changes, in a clear 2-stage response. In particular, the liver of dosed mice was found to switch from glycogenolysis and lipid storage, at 6 h postinjection, to glycogenesis and lipolysis, at subsequent times up to 48 h. Moreover, metabolites related to antioxidative defense, immunoregulation and detoxification seemed to play a crucial role in avoiding major hepatic damage. The spleen showed several early changes, including depletion of several amino acids, possibly reflecting impairment of hemoglobin recycling, while only a few differences remained at 48 h postinjection. In the heart, the metabolic shift towards TCA cycle intensification and increased ATP production possibly reflected a beneficial adaptation to the presence of AgNPs. On the other hand, the TCA cycle appeared to be down regulated in the lungs of injected mice, which showed signs of inflammation. Thekidneys showed the mildest metabolic response to AgNPs. Overall, this study has shown that NMR metabolomics is a powerful tool to monitor invivo metabolic responses to nanoparticles, revealing unforeseen effects.