Glycogen-Endoplasmic Reticulum Connection in the Liver.

Glycogen, the branched polymer of glucose is found mainly in the liver and muscle in mammals. Along with several other proteins, glycogen forms separate cellular organelles, and particles in cells. Glycogen particles in the liver have a special metabolic and also regulatory connection to the intracellular endomembrane system, particularly the endoplasmic reticulum. This connection is part of the organelle homeostasis in hepatocytes and forms a "glycogenoreticular system". The actual size of hepatic glycogen stores and the rate of glycogenolysis determines several essential liver-specific metabolic processes, such as glucose secretion for the maintenance of blood glucose levels or the glucuronidation of certain vital endo-, and xenobiotics, and are also related to liver antioxidant defense. In starvation, and in certain physiological and pathological states, where glycogen stores are depleted, functions of the glycogenoreticular system are altered. The starvation-induced depletion of hepatic glycogen content changes the biotransformation of various endo- and xenobiotics. This can be observed especially in acute DILI (drug-induced liver injury) due to paracetamol overdose, which is the most common cause of acute liver failure in the West.

Autophagy, Mitophagy and MicroRNA Expression in Chronic Hepatitis C and Autoimmune Hepatitis.

Although the role of autophagy has been implicated in several forms of chronic hepatitis, it is still not fully understood. Active autophagy eliminates damaged molecules and organelles (such as mitochondria) by lysosomal degradation. In the present study, we aimed to examine and compare autophagy activity in chronic hepatitis C (CHC) and autoimmune hepatitis (AIH) by detecting the expression of autophagy (LC3 and p62) and mitochondrium-related (TOMM20) proteins, as well as the levels of selected microRNAs (miR-101, -155, -204 and - 224) known to be involved in the regulation of autophagy. In addition, the expression levels were related to pathohistological parameters. Liver biopsy samples, including 45 CHC and 18 AIH cases, were immunohistochemically stained for LC3, p62 and TOMM20 and the expression of miRNAs was determined using real-time PCR. We found elevated LC3 and p62 in AIH samples as compared with CHC ones, indicating an activated autophagy that is impaired in AIH as no degradation of p62 seemed to occur. Moreover, p62 showed strong correlation with necroinflammatory grades in the AIH group. The observed elevated levels of TOMM20 and p62 suggest a less efficient elimination of damaged mitochondria in AIH as opposed to CHC, in which autophagy seems to have a more active function. The level of miR-101 was increased in case of CHC as compared with AIH, however, miR-155, -204 and 224 resulted in no expressional. Furthermore, miR-224 level correlated with steatosis and miR-155 expression with fibrosis stage in CHC. In conclusion, dissimilar autophagic activity was observed in CHC and AIH, suggesting a close association between impaired autophagy and severity of necroinflammation. This impairment may not be regulated by the analyzed miRNAs. Nevertheless, miR-224 and - 155 seem to be associated with CHC progression.

BGP-15 Protects Mitochondria in Acute, Acetaminophen Overdose Induced Liver Injury.

Acetaminophen (APAP) induced hepatotoxicity involves activation of c-Jun amino-terminal kinase (JNK), mitochondrial damage and ER stress. BGP-15, a hydroximic acid derivative, has been reported to have hepatoprotective effects in APAP overdose induced liver damage. Effect of BGP-15 was further investigated on mitochondria in APAP-overdose induced acute liver injury in mice. We found that BGP-15 efficiently preserved mitochondrial morphology, and it caused a marked decrease in the number of damaged mitochondria. Attenuation of mitochondrial damage by BGP-15 is supported by immunohistochemistry as the TOMM20 label and the co-localized autophagy markers detected in the livers of APAP-treated mice were markedly reduced upon BGP-15 administration. This effect, along with the observed prevention of JNK activation likely contribute to the mitochondrial protective action of BGP-15.

The ER - Glycogen Particle - Phagophore Triangle: A Hub Connecting Glycogenolysis and Glycophagy?

Glycogen particle is an intracellular organelle, which serves as a carbohydrate reserve in various cells. The function of glycogen is not entirely known in several cell types. Glycogen can be mobilized for different purposes, which can be related to cellular metabolic needs, intracellular redox state, metabolic state of the whole organism depending on regulatory aspects and also on cell functions. Essentially there are two different ways of glycogen degradation localized in different cellular organelles: glycogenolysis or lysosomal breakdown by acid alpha-glucosidase. While glycogenolysis occurs in glycogen particles connected to endoplasmic reticulum membrane, glycogen particles can be also combined with phagophores forming autophagosomes. A subdomain of the endoplasmic reticulum membrane - omegasomes - are the sites for phagophore formation. Thus, three organelles, the endoplasmic reticulum, the phagophore and the glycogen particle forms a triangle in which glycogen degradation occurs. The physiological significance, molecular logic and regulation of the two different catabolic paths are summarized and discussed with special aspect on the role of glycogen particles in intracellular organelle homeostasis and on molecular pathology of the cell. Pathological aspects and some diseases connected to the two different degradation pathways of glycogen particles are also detailed.

Activation of mitochondrial fusion provides a new treatment for mitochondria-related diseases.

Mitochondria fragmentation destabilizes mitochondrial membranes, promotes oxidative stress and facilitates cell death, thereby contributing to the development and the progression of several mitochondria-related diseases. Accordingly, compounds that reverse mitochondrial fragmentation could have therapeutic potential in treating such diseases. BGP-15, a hydroxylamine derivative, prevents insulin resistance in humans and protects against several oxidative stress-related diseases in animal models. Here we show that BGP-15 promotes mitochondrial fusion by activating optic atrophy 1 (OPA1), a GTPase dynamin protein that assist fusion of the inner mitochondrial membranes. Suppression of Mfn1, Mfn2 or OPA1 prevents BGP-15-induced mitochondrial fusion. BGP-15 activates Akt, S6K, mTOR, ERK1/2 and AS160, and reduces JNK phosphorylation which can contribute to its protective effects. Furthermore, BGP-15 protects lung structure, activates mitochondrial fusion, and stabilizes cristae membranes in vivo determined by electron microscopy in a model of pulmonary arterial hypertension. These data provide the first evidence that a drug promoting mitochondrial fusion in in vitro and in vivo systems can reduce or prevent the progression of mitochondria-related disorders.

The Level of ALR is Regulated by the Quantity of Mitochondrial DNA.

Augmenter of liver regeneration (ALR) contributes to mitochondrial biogenesis, maintenance and to the physiological operation of mitochondria. The depletion of ALR has been widely studied and had serious consequences on the mitochondrial functions. However the inverse direction, the effect of the depletion of mitochondrial electron transfer chain and mtDNA on ALR expression has not been investigated yet. Thus mtDNA depleted, ρ(0) cell line was prepared to investigate the role of mitochondrial electron transfer chain and mtDNA on ALR expression. The depletion of mtDNA has not caused any difference at mRNA level, but at protein level the expression of ALR has been markedly increased. The regulatory role of ATP and ROS levels could be ruled out because the treatment of the parental cell line with different respiratory inhibitors and uncoupling agent could not provoke any changes in the protein level of ALR. The effect of mtDNA depletion on the protein level of ALR has been proved not to be liver specific, since the phenomenon could be observed in the case of two other, non-hepatic cell lines. It seems the level of mtDNA and/or its products may have regulatory role on the protein level of ALR. The up-regulation of ALR can be a part of the adaptive response in ρ(0) cells that preserves the structural integrity and the transmembrane potential despite the absence of protein components encoded by the mtDNA.

Novel compounds reducing IRS-1 serine phosphorylation for treatment of diabetes.

Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK), and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a central role both in insulin resistance and in ß-cell dysfunction. IRS-1 phosphorylation is stimulated by elevated free fatty acid levels through different pathways in obesity. A series of novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and type 2 diabetes.

Ferroptosis is Involved in Acetaminophen Induced Cell Death.

The recently described form of programmed cell death, ferroptosis can be induced by agents causing GSH depletion or the inhibition of GPX4. Ferroptosis clearly shows distinct morphologic, biochemical and genetic features from apoptosis, necrosis and autophagy. Since NAPQI the highly reactive metabolite of the widely applied analgesic and antipyretic, acetaminophen induces a cell death which can be characterized by GSH depletion, GPX inhibition and caspase independency the involvement of ferroptosis in acetaminophen induced cell death has been investigated. The specific ferroptosis inhibitor ferrostatin-1 failed to elevate the viability of acetaminophen treated HepG2 cells. It should be noticed that these cells do not form NAPQI due to the lack of phase I enzyme expression therefore GSH depletion cannot be observed. However in the case of acetaminophen treated primary mouse hepatocytes the significant elevation of cell viability could be observed upon ferrostatin-1 treatment. Similar to ferrostatin-1 treatment, the addition of the RIP1 kinase inhibitor necrostatin-1 could also elevate the viability of acetaminophen treated primary hepatocytes. Ferrostatin-1 has no influence on the expression of CYP2E1 or on the cellular GSH level which suggest that the protective effect of ferrostatin-1 in APAP induced cell death is not based on the reduced metabolism of APAP to NAPQI or on altered NAPQI conjugation by cellular GSH. Our results suggest that beyond necroptosis and apoptosis a third programmed cell death, ferroptosis is also involved in acetaminophen induced cell death in primary hepatocytes.

On the role of 4-hydroxynonenal in health and disease.

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,ß-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

Metformin attenuates palmitate-induced endoplasmic reticulum stress, serine phosphorylation of IRS-1 and apoptosis in rat insulinoma cells.

Lipotoxicity refers to cellular dysfunctions caused by elevated free fatty acid levels playing a central role in the development and progression of obesity related diseases. Saturated fatty acids cause insulin resistance and reduce insulin production in the pancreatic islets, thereby generating a vicious cycle, which potentially culminates in type 2 diabetes. The underlying endoplasmic reticulum (ER) stress response can lead to even ß-cell death (lipoapoptosis). Since improvement of ß-cell viability is a promising anti-diabetic strategy, the protective effect of metformin, a known insulin sensitizer was studied in rat insulinoma cells. Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells. Attenuation of ß-cell lipotoxicity was also revealed by lower induction/activation of various ER stress markers, e.g. phosphorylation of eukaryotic initiation factor 2α (eIF2α), c-Jun N-terminal kinase (JNK), insulin receptor substrate-1 (IRS-1) and induction of CCAAT/enhancer binding protein homologous protein (CHOP). Our results indicate that the ß-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.

Synergic insulin sensitizing effect of rimonabant and BGP-15 in Zucker-obese rats.

Abdominal obesity is referred for as a common pathogenic root of multiple risk factors, which include insulin resistance, dyslipidemia, hypertension, and a pro-atherogenic and pro-inflammatory state. Irrespective of its psychiatric side effects, rimonabant through blocking cannabinoid-1 receptor (CB1R) induces an increase in whole body insulin sensitivity. The aim of this work was to study the effect of selected doses of another insulin sensitizer compound BGP-15, and rimonabant on insulin resistance in Zucker obese rats with a promise of inducing insulin sensitization together at lower doses than would have been expected by rimonabant alone. We found that BGP-15 potentiates the insulin sensitizing effect of rimonabant. The combination at doses, which do not induce insulin sensitization by themselves, improved insulin signaling. Furthermore our results suggest that capsaicin-induced signal may play a role in insulin sensitizing effect of both molecules. Our data might indicate that a lower dose of rimonabant in the treatment of insulin resistance and type 2 diabetes is sufficient to administer, thus a lower incidence of the unfavorable psychiatric side effects of rimonabant are to be expected.

Inhibition of microsomal cortisol production by (-)-epigallocatechin-3-gallate through a redox shift in the endoplasmic reticulum--a potential new target for treating obesity-related diseases.

Conversion of cortisone to cortisol by 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) in the endoplasmic reticulum (ER) of the target cells is a major determinant of glucocorticoid action, and plays an important role in the development of obesity-related diseases. Inhibition of 11ßHSD1 activity is, therefore, considered as a promising novel strategy for the treatment of metabolic syndrome and diabetes. Tea flavanols and their major representative, epigallocatechin gallate are known as antiobesity and antidiabetic agents. Their impacts on blood glucose level, hepatic glucose production, and insulin responsiveness resemble those observed on inhibition or depletion of 11ßHSD1. We aimed to study the effect of epigallocatechin gallate on 11ßHSD1 activity in ER-derived rat liver microsomes by measuring cortisone and cortisol with HPLC. Cortisol production was efficiently suppressed in a concentration dependent manner in intact microsomal vesicles. However, this effect was abolished by membrane permeabilization; and the three proteins involved in the overall process (11ßHSD1, hexose 6-phosphate dehydrogenase, and glucose 6-phosphate transporter) were not or only mildly affected. Further investigation revealed the oxidation of luminal NADPH to NADP⁺, which attenuates cortisone reduction and favors cortisol oxidation in this compartment. Such a redox shift in the ER lumen might contribute to the beneficial health effects of tea flavanols and should be regarded as a promising strategy for the development of novel selective 11ßHSD1 inhibitors to treat obesity-related diseases.

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase.

NADH cytochrome b5 oxidoreductase (Ncb5or) protects ß-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to ß-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.

Minireview: endoplasmic reticulum stress: control in protein, lipid, and signal homeostasis.

Endoplasmic reticulum (ER) stress is a regulatory mechanism that allows cells to adapt to a series of metabolic, redox, and other environmental changes. The role of ER stress was first identified in the maintenance of proteostasis. It has since been shown that ER stress is also critical to the regulation of lipid homeostasis, membrane turnover, and autophagy. ER stress initiates an intrinsic signaling network, the unfolded protein response, one component of the multifold and complex cellular signaling process system, which leads to major changes in the profiles of transcription factors. The unfolded protein response affects several other signaling routes through direct connections and also by indirect means. It directly influences hormone formation and life/death decisions at a cellular level; this relationship also involves connections to nutrient and environmental sensing-biotransformation processes. In conclusion, ER stress represents an integrated complex organelle response that makes an essential contribution to the maintenance of intracellular homeostasis.

Luminal accumulation of newly synthesized morphine-3-glucuronide in rat liver microsomal vesicles.

Morphine is converted to morphine 3-ß-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 µM). Therefore, the build-up of high (about 20 µM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 µM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion.

Hydroximic acid derivatives: pleiotropic HSP co-inducers restoring homeostasis and robustness.

According to the "membrane sensor" hypothesis, the membrane's physical properties and microdomain organization play an initiating role in the heat shock response. Clinical conditions such as cancer, diabetes and neurodegenerative diseases are all coupled with specific changes in the physical state and lipid composition of cellular membranes and characterized by altered heat shock protein levels in cells suggesting that these "membrane defects" can cause suboptimal hsp-gene expression. Such observations provide a new rationale for the introduction of novel, heat shock protein modulating drug candidates. Intercalating compounds can be used to alter membrane properties and by doing so normalize dysregulated expression of heat shock proteins, resulting in a beneficial therapeutic effect for reversing the pathological impact of disease. The membrane (and lipid) interacting hydroximic acid (HA) derivatives discussed in this review physiologically restore the heat shock protein stress response, creating a new class of "membrane-lipid therapy" pharmaceuticals. The diseases that HA derivatives potentially target are diverse and include, among others, insulin resistance and diabetes, neuropathy, atrial fibrillation, and amyotrophic lateral sclerosis. At a molecular level HA derivatives are broad spectrum, multi-target compounds as they fluidize yet stabilize membranes and remodel their lipid rafts while otherwise acting as PARP inhibitors. The HA derivatives have the potential to ameliorate disparate conditions, whether of acute or chronic nature. Many of these diseases presently are either untreatable or inadequately treated with currently available pharmaceuticals. Ultimately, the HA derivatives promise to play a major role in future pharmacotherapy.

A cellular stress-directed bistable switch controls the crosstalk between autophagy and apoptosis.

Decision-making between life and death is one of the most important tasks of cells to maintain their genetic integrity. While the surviving mechanism is driven by Beclin1-dependent autophagy, the suicide processes are controlled by caspases-mediated apoptosis. Interestingly, both these processes share regulators such as Bcl2 and influence each other through feedback loops. The physiological relevance of the crosstalk between autophagy and apoptosis is still unclear. To gain system level insights, we have developed a mathematical model of the autophagy-apoptosis crosstalk. Our analysis reveals that a combination of Bcl2-dependent regulation and feedback loops between Beclin1 and caspases robustly enforces a sequential activation of cellular responses depending upon the intensity and duration of stress levels. The amplifying loops for caspases activation involving Beclin1-dependent inhibition of caspases and cleavage of Beclin1 by caspases (Beclin1 ┤ caspases ┤ Beclin1; caspases → cleaved Beclin1 → caspases) not only make the system bistable but also help to switch off autophagy at high stress levels. The presence of an additional positive feedback loop between Bcl2 and caspases helps to maintain the caspases activation by making the switch irreversible. Our results provide a framework for further experiments and modelling.

Keratinocyte ATP binding cassette transporter expression is regulated by ultraviolet light.

Many ATP binding cassette (ABC) transporters are important regulators of lipid homeostasis and have been implicated in keratinocyte lipid transport. Ultraviolet (UV) light exposure is a known epidermal stressor, which amongst other effects causes lipid alterations and defective lamellar body biogenesis. To elucidate the background of these lipid changes we studied the effect of UVB light on ABC transporter expression. The effect of UVB treatment on the levels of 47 known human ABC transporter mRNAs was analyzed in normal human epidermal keratinocytes. Immunoblots and promoter assays were carried out for ABCA1 and ABCG1. The mRNA levels of cholesterol transport regulators ABCA1 and ABCG1 were markedly downregulated by UVB, parallel to the lamellar ichthyosis related glucosylceramide transporter ABCA12 and the suspected sphingosine-1-phosphate and cholesterol sulfate transporter ABCC1. The long but not the short alternative splice variant of the ABCF2 was found to be markedly upregulated rapidly after UVB irradiation. Immunoblot confirmed ABCA1 and ABCG1 protein downregulation, and luciferase assays showed suppression of their promoters by UVB. These proteins mostly transport lipids, which account for the integrity of the epidermal barrier; therefore our findings on the UVB regulation of ABC transporters may explain the appearance of barrier dysfunction after UVB exposure.

A novel insulin sensitizer drug candidate-BGP-15-can prevent metabolic side effects of atypical antipsychotics.

Atypical antipsychotic drugs (AAPD) are widely used to treat severe psychiatric disorders, have well documented metabolic side effects such as disturbances in glucose metabolism, insulin resistance and weight gain. It has been shown that BGP-15, a hydroxylamine derivative with insulin sensitizing activity can prevent AAPD provoked fat accumulation in adipocyte cultures, and insulin resistance in animal experiments and in healthy volunteers. The aim of this study was to compare the preventive effect of BGP-15 with conventional oral antidiabetics on metabolic side effects of AAPDs. We found that BGP-15 that does not belong to either conventional insulin sensitizers or oral antidiabetics, is able to counteract insulin resistance and weight gain provoked by antipsychotic agents in rats while rosiglitazone and metformin were not effective in the applied doses. Our results confirm that BGP-15 is a promising new drug candidate to control the metabolic side effects of atypical antipsychotics. Data indicate that this rat model is suitable to analyze the metabolic side effects of AAPDs and the protective mechanism of BGP-15.

G6PT-H6PDH-11ßHSD1 triad in the liver and its implication in the pathomechanism of the metabolic syndrome.

The metabolic syndrome, one of the most common clinical conditions in recent times, represents a combination of cardiometabolic risk determinants, including central obesity, glucose intolerance, insulin resistance, dyslipidemia, non-alcoholic fatty liver disease and hypertension. Prevalence of the metabolic syndrome is rapidly increasing worldwide as a consequence of common overnutrition and consequent obesity. Although a unifying picture of the pathomechanism is still missing, the key role of the pre-receptor glucocorticoid activation has emerged recently. Local glucocorticoid activation is catalyzed by a triad composed of glucose-6-phosphate-transporter, hexose-6-phosphate dehydrogenase and 11ß-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum. The elements of this system can be found in various cell types, including adipocytes and hepatocytes. While the contribution of glucocorticoid activation in adipose tissue to the pathomechanism of the metabolic syndrome has been well established, the relative importance of the hepatic process is less understood. This review summarizes the available data on the role of the hepatic triad and its role in the metabolic syndrome, by confronting experimental findings with clinical observations.