High fat diet- induced obesity decreases ErbB receptors expression in liver and adipose tissue in C57BL/6 mice

Neuregulins (NRGs) are a family of signaling proteins that bind to receptor tyrosine kinases of the ErbB family (ErbB2 to ErbB4), which can homo- or heterodimerize depending on their structural features and cell type. Many studies have proposed that decreased NRG levels are a common characteristic of obesity. In liver and adipose tissue, the increase in NRG expression has protective effects against obesity. However, it is still unknown whether ErbBs expression is altered in this pathology. We hypothesized that high fat diet-induced obesity downregulates ErbB receptors expression in obese mice compared to normal weight mice. Males C57BL/6 mice (n=6-7 for each group) were fed for 12 weeks and divided into: (i) control diet (CD; 10%-kcal fat, 20%-kcal protein, 70%-kcal carbohydrates), and (ii) high fat diet (HFD; 60%-kcal fat, 20%-kcal protein, 20%-kcal carbohydrates). General parameters and ErbBs expression (qPCR, immunohistochemistry and Western blot) were evaluated. We observed a significant increase in final body weight (47%), adipose tissue to body weight ratio (244%) and HOMA-IR (69%), among other parameters, in obese mice. In HFD group significantly decreased ErbB2 (48%) and ErbB3 (66%) mRNA levels in liver (no change in ErbB4), and ErbB2 (43%), ErbB3 (76%) and ErbB4 (35%) in adipose tissue, compared to CD. Furthermore, ErbB2 and ErbB3 protein levels decreased significantly in HFD group compared to the CD in liver. Therefore, our results suggest that HFD-induced obesity significantly decreases ErbBs expression in liver and adipose tissue in this murine model, that may be associated with alterations in the NRG pathway in obese mice.

Las neuregulinas (NRGs) son una familia de proteínas de señalización que se unen a receptores tirosina quinasas de la familia ErbB (ErbB2 a ErbB4), que pueden homo- o heterodimerizar dependiendo de sus características estructurales y del tipo celular. Estudios han propuesto que la disminución de los niveles de NRG es una característica común de la obesidad. En el hígado y el tejido adiposo (TA), el aumento de la expresión de NRG tiene efectos protectores contra la obesidad. Sin embargo, aún se desconoce si la expresión de ErbBs está alterada en esta patología. Nuestra hipótesis es que la obesidad inducida por una dieta alta en grasas (DAG) disminuye la expresión de los ErbB en ratones obesos. Ratones machos C57BL/6 (n=6-7 para c/grupo) fueron alimentados durante 12 semanas y divididos en: (i) dieta control (DC; 10%-kcal grasa, 20%-kcal proteína, 70%-kcal carbohidratos), y (ii) DAG (60%-kcal grasa, 20%-kcal proteína, 20%-kcal carbohidratos). Se evaluaron los parámetros generales y la expresión de ErbBs (qPCR, inmunohistoquímica y Western blot). Observamos un aumento significativo del peso corporal final (47%), de la relación tejido adiposo/peso corporal (244%) y del HOMA-IR (69%), entre otros parámetros, en ratones obesos. En este grupo disminuyó significativamente los niveles de ARNm de ErbB2 (48%) y ErbB3 (66%) en el hígado (sin cambios en ErbB4), y de ErbB2 (43%), ErbB3 (76%) y ErbB4 (35%) en el TA. Además, los niveles de proteína ErbB2 y ErbB3 disminuyeron significativamente, en comparación con el grupo DC en el hígado. Nuestros resultados sugieren que la obesidad inducida por DAG disminuye significativamente la expresión de ErbBs en el hígado y el TA, que puede estar asociado con alteraciones en la vía NRG en ratones obesos.

CETP-derived Peptide Seq-1, the Key Component of HB-ATV-8 Vaccine Prevents Stress Responses, and Promotes Downregulation of Pro-Fibrotic Genes in Hepatocytes and Stellate Cells.

BACKGROUND: The nasal vaccine HB-ATV-8 has emerged as a promising approach for NAFLD (non-alcoholic fatty liver disease) and atherosclerosis prevention. HB-ATV-8 contains peptide seq-1 derived from the carboxy-end of the Cholesteryl Ester Transfer Protein (CETP), shown to reduce liver fibrosis, inflammation, and atherosclerotic plaque formation in animal models. Beyond the fact that this vaccine induces B-cell lymphocytes to code for antibodies against the seq-1 sequence, inhibiting CETP's cholesterol transfer activity, we have hypothesized that beyond the modulation of CETP activity carried out by neutralizing antibodies, the observed molecular effects may also correspond to the direct action of peptide seq-1 on diverse cellular systems and molecular features involved in the development of liver fibrosis. METHODS: The HepG2 hepatoma-derived cell line was employed to establish an in vitro steatosis model. To obtain a conditioned cell medium to be used with hepatic stellate cell (HSC) cultures, HepG2 cells were exposed to fatty acids or fatty acids plus peptide seq-1, and the culture medium was collected. Gene regulation of COL1A1, ACTA2, TGF-ß, and the expression of proteins COL1A1, MMP-2, and TIMP-2 were studied. AIM: To establish an in vitro steatosis model employing HepG2 cells that mimics molecular processes observed in vivo during the onset of liver fibrosis. To evaluate the effect of peptide Seq-1 on lipid accumulation and pro-fibrotic responses. To study the effect of Seq-1-treated steatotic HepG2 cell supernatants on lipid accumulation, oxidative stress, and pro-fibrotic responses in HSC. RESULTS AND CONCLUSION: Peptide seq-1-treated HepG2 cells show a downregulation of COLIA1, ACTA2, and TGF-ß genes, and a decreased expression of proteins such as COL1A1, MMP-2, and TIMP-2, associated with the remodeling of extracellular matrix components. The same results are observed when HSCs are incubated with peptide Seq-1-treated steatotic HepG2 cell supernatants. The present study consolidates the nasal vaccine HB-ATV-8 as a new prospect in the treatment of NASH directly associated with the development of cardiovascular disease.

In Vitro Lipid Overload Affects Cellular Proliferation, Apoptosis, and Senescence in a Time-Dependent Manner in HepG2 Hepatocytes and LX-2 Hepatic Stellate Cells.

Different cellular mechanisms influence steatotic liver disease (SLD) progression. The influence of different levels of steatogenic inputs has not been studied in hepatocytes and hepatic stellate cells (HSCs). METHODS: HepG2 hepatocytes and LX-2 HSCs were cultured in mild (MS) and severe (SS) steatogenic conditions. TGF-ß stimulation was also tested for HSCs in control (T) and steatogenic conditions (MS-T and SS-T). Steatosis was stained with Oil Red, and the proliferation was assayed via WST-8 reduction, apoptosis via flow cytometry, and senescence via SA-ß-galactosidase activity. RESULTS: Regarding hepatocytes, steatosis progressively increased; proliferation was lower in MS and SS; and the viability of both conditions significantly decreased at 72 h. Apoptosis increased in MS at 72 h, while it decreased in SS. Senescence increased in MS and diminished in SS. Regarding HSCs, the SS and SS-T groups showed no proliferation, and the viability was reduced in MS at 72 h and in SS and SS-T. The LX-2 cells showed increased apoptosis in SS and SS-T at 24 h, and in MS and MS-T at 72 h. Senescence decreased in MS, SS, and SS-T. CONCLUSIONS: Lipid overload induces differential effects depending on the cell type, the steatogenic input level, and the exposure time. Hepatocytes are resilient to mild steatosis but susceptible to high lipotoxicity. HSCs are sensitive to lipid overload, undergoing apoptosis and lowering senescence and proliferation. Collectively, these data may help explain the development of steatosis and fibrosis in SLD.

Mitochondrial sodium/calcium exchanger (NCLX) regulates basal and starvation-induced autophagy through calcium signaling.

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporters and efflux by Na+ /Ca2+ exchangers (NCLX). Here, we describe a novel relationship among NCLX, intracellular Ca2+ , and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

Mitofusin-2 induced by exercise modifies lipid droplet-mitochondria communication, promoting fatty acid oxidation in male mice with NAFLD.

BACKGROUND AND AIM: The excessive accumulation of lipid droplets (LDs) is a defining characteristic of nonalcoholic fatty liver disease (NAFLD). The interaction between LDs and mitochondria is functionally important for lipid metabolism homeostasis. Exercise improves NAFLD, but it is not known if it has an effect on hepatic LD-mitochondria interactions. Here, we investigated the influence of exercise on LD-mitochondria interactions and its significance in the context of NAFLD. APPROACH AND RESULTS: Mice were fed high-fat diet (HFD) or HFD-0.1 % methionine and choline-deficient diet (MCD) to emulate simple hepatic steatosis or non-alcoholic steatohepatitis, respectively. In both models, aerobic exercise decreased the size of LDs bound to mitochondria and the number of LD-mitochondria contacts. Analysis showed that the effects of exercise on HOMA-IR and liver triglyceride levels were independent of changes in body weight, and a positive correlation was observed between the number of LD-mitochondria contacts and NAFLD severity and with the lipid droplet size bound to mitochondria. Cellular fractionation studies revealed that ATP-coupled respiration and fatty acid oxidation (FAO) were greater in hepatic peridroplet mitochondria (PDM) from HFD-fed exercised mice than from equivalent sedentary mice. Finally, exercise increased FAO and mitofusin-2 abundance exclusively in PDM through a mechanism involving the curvature of mitochondrial membranes and the abundance of saturated lipids. Accordingly, hepatic mitofusin-2 ablation prevented exercise-induced FAO in PDM. CONCLUSIONS: This study demonstrates that aerobic exercise has beneficial effects in murine NAFLD models by lessening the interactions between hepatic LDs and mitochondria, and by decreasing LD size, correlating with a reduced severity of NAFLD. Additionally, aerobic exercise increases FAO in PDM and this process is reliant on Mfn-2 enrichment, which modifies LD-mitochondria communication.

Efectos de fuentes de grasa sobre características del hígado y la morfometría intestinal en un modelo animal a temprana edad / Effects of fat sources on liver characteristics and intestinal morphometry in an early-life animal model

RESUMEN El objetivo fue determinar el efecto del consumo de tres fuentes de ácidos grasos sobre el peso relativo, caracterización macroscópica y microscópica del hígado, y la morfometría intestinal en un modelo animal a temprana edad. Se utilizaron 76 pollitos distribuidos al azar, que recibieron una de las dietas (T1: 97,0% de dieta basal (DB) + 3,0% de material inerte, T2: 97,0% de DB + 3,0% de manteca vegetal parcialmente hidrogenada, T3: 97,0% de DB + 3,0% de aceite de quinua, y T4: 97,0% de DB + 3,0% de aceite de pescado) hasta el séptimo día de vida; luego se extrajeron muestras para los análisis correspondientes. Se encontró que, los animales que consumieron aceite de quinua (T3) o aceite de pescado (T4) tuvieron resultados favorables asociados a un menor peso del hígado y, una mejor absorción de nutrientes a nivel intestinal debido a mayores valores en la relación longitud del vello y profundidad de cripta; en comparación a la manteca vegetal parcialmente hidrogenada (T2). En conclusión, el aceite de quinua constituye una opción saludable de consumo y fuente alternativa al aceite de pescado.

ABSTRACT We aimed to determine the effect of the consumption of three sources of fatty acids on the relative weight, macroscopic and microscopic characteristics of the liver, and intestinal morphometry in an early-life animal model. Seventy-six randomly distributed chicks received one of the diets (T1: 97.0% basal diet (BD) + 3.0% inert material, T2: 97.0% BD + 3.0% partially hydrogenated vegetable shortening, T3: 97.0% BD + 3.0% quinoa oil, and T4: 97.0% BD + 3.0% fish oil) until the seventh day of life; samples were then extracted in order to be analyzed. We found that the animals that consumed quinoa oil (T3) or fish oil (T4) had favorable results associated to lower liver weight and better absorption of nutrients at intestinal level due to higher values in the hair length and crypt depth ratio, in comparison to partially hydrogenated vegetable shortening (T2). In conclusion, quinoa oil constitutes a healthy option for consumption and an alternative source to fish oil.

Histomorphological, Histochemical and Ultrastructural Studies on the Healthy Liver of Yemen Veiled Chameleon (Chamaeleo calyptratus) in Southern Saudi Arabia / Estudios Histomorfológicos, Histoquímicos y Ultraestructurales en el Hígado Sano del Camaleón Velado de Yemen (Chamaeleo calyptratus) en el Sur de Arabia Saudita

SUMMARY: The livers of reptiles are being studied as a model for the link between the environment and hepatic tissue. There have been few investigations on the histology of reptile livers, and very few or no studies have examined the histology of liver of veiled chameleon (Chamaeleo calyptratus). This paper describes the histomorphological, histochemical and ultrastructural characterization of the liver of veiled chameleons in southern Saudi Arabia. Seven Chamaeleo calyptratus were captured in the summer season in Abha City, Aseer region, southern Saudi Arabia. Chamaeleon liver samples were processed for histomorphology, histochemistry and ultrastructure analyses. Morphologically liver of Chamaeleo calyptratus was observed as a large dark brown organ with lighter speckles, which represent melanin deposits. It located at the ventral part of abdominal cavity forward of the stomach. Its dimensions approximately were 3.7 x 2 cm. The liver was a bilobed organ divided into two lobes, right and left lobes. The right one was bigger than the others. The gallbladder was well developed and had an elongated shape, situated between the two lobes and contained the bile for the digestion. Microscopically, the liver was found to be covered by a thick layer of connective tissue, which formed the hepatic capsule. Hepatic parenchyma probably appeared in cross sections as hepatic glandular-like alveoli "acini" or follicular structures with various diameters, each acinus contains approximately four to six hepatocytes, surrounded by sinusoidal capillaries filled with abundant melanomacrophages, which are absent in birds and mammals. Melanomacrophages are common in the hepatic parenchyma's perisinusoidal areas, particularly near portal spaces. Hepatocytes are polyhedral or pyramidal with and mostly contained large, rounded nuclei mostly peripherally located, with prominent dark oval nucleoli. Some of nuclei are eccentric or central position. The cytoplasm appeared spongy or vacuolated and more eosinophilic when stained by hematoxylin-eosin and strongly reactive to PAS staining technique, indicating abundant glycogen content. The reticular fibers that surround hepatocytes, blood arteries, and sinusoids supported the hepatic parenchyma. The blood sinusoids are seen interspersed among hepatocytes of varying sizes. The sinusoidal lumen was bordered by flattened endothelial cells and includes elliptical nucleated erythrocytes and liver macrophages as phagocytes, which are also known as Kupffer cells. Branches of the portal vein, hepatic artery, small bile duct, and lymph vessels were detected in the hepatic portal area "tract" or triad which made up of connective. Hematopoietic tissue was observed in subcapsular region and portal triads. Ultrastructurally, the hepatocyte appeared polyhedric containing a single large rounded basal or eccentric vesicular nucleus with prominent nucleolus. Extensive network of rough endoplasmic reticulum (RER) often arranged in an array parallel to the nuclear membrane with many mitochondria, and Golgi apparatus were described. The cytoplasm contained glycogen granules, vesicles or vacuoles scattered throughout the cytoplasm especially at the apical region were reported. The bile canaliculi and the hepatic "Kupffer" cells were also discussed. This is the first study on the histological characterization of the healthy liver of Yemen veiled chameleon in southern Saudi Arabia. The findings reported here should be used as a reference to compare with the pathological abnormalities of the liver in this animal.

está disponible en el texto completo

The harmful acute effects of clomipramine in the rat liver: Impairments in mitochondrial bioenergetics.

Clomipramine, a tricyclic antidepressant used to treat depression and obsessive-compulsive disorder, has been linked to a few cases of acute hepatotoxicity. It is also recognized as a compound that hinders the functioning of mitochondria. Hence, the effects of clomipramine on mitochondria should endanger processes that are somewhat connected to energy metabolism in the liver. For this reason, the primary aim of this study was to examine how the effects of clomipramine on mitochondrial functions manifest in the intact liver. For this purpose, we used the isolated perfused rat liver, but also isolated hepatocytes and isolated mitochondria as experimental systems. According to the findings, clomipramine harmed metabolic processes and the cellular structure of the liver, especially the membrane structure. The considerable decrease in oxygen consumption in perfused livers strongly suggested that the mechanism of clomipramine toxicity involves the disruption of mitochondrial functions. Coherently, it could be observed that clomipramine inhibited both gluconeogenesis and ureagenesis, two processes that rely on ATP production within the mitochondria. Half-maximal inhibitory concentrations for gluconeogenesis and ureagenesis ranged from 36.87 µM to 59.64 µM. The levels of ATP as well as the ATP/ADP and ATP/AMP ratios were reduced, but distinctly, between the livers of fasted and fed rats. The results obtained from experiments conducted on isolated hepatocytes and isolated mitochondria unambiguously confirmed previous propositions about the effects of clomipramine on mitochondrial functions. These findings revealed at least three distinct mechanisms of action, including uncoupling of oxidative phosphorylation, inhibition of the FoF1-ATP synthase complex, and inhibition of mitochondrial electron flow. The elevation in activity of cytosolic and mitochondrial enzymes detected in the effluent perfusate from perfused livers, coupled with the increase in aminotransferase release and trypan blue uptake observed in isolated hepatocytes, provided further evidence of the hepatotoxicity of clomipramine. It can be concluded that impaired mitochondrial bioenergetics and cellular damage are important factors underlying the hepatotoxicity of clomipramine and that taking excessive amounts of clomipramine can lead to several risks including decreased ATP production, severe hypoglycemia, and potentially fatal outcomes.

BMAL1 Regulates Glucokinase Expression Through E-Box Elements In Vitro.

The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.

Mechanism of VKORC1 and VKORC1L1 signaling in the effects of sodium dehydroacetate on coagulation factors in rat hepatocytes.

Sodium dehydroacetate (Na-DHA) is widely used as an antibacterial and preservative additive in food and cosmetics. Previously, we reported that repeated oral administration of Na-DHA induces coagulation disorders, and inhibited liver vitamin K epoxide reductase complex subunit 1 (VKORC1) and VKORC1-like protein 1 (VKORC1L1) in rats. However, the effects of Na-DHA on coagulation factors in rat hepatocytes and the mechanism of VKORC1 and VKORC1L1 signaling in that process are unclear. Here, we constructed stable Vkorc1 and Vkorc1l1 overexpressing cell lines using lentiviruses and transfected small interfering RNAs into buffalo rat liver BRL3A cells for Vkorc1 and Vkorc1l1 overexpression and silencing, respectively. After treatment with 5 mmol/L Na-DHA for 24 h, VKORC1 and VKORC1L1 expression levels were detected by real-time PCR and western blotting. Vitamin K (VK) and factor IX (FIX) contents were detected using enzyme linked immunosorbent assays. We observed that Na-DHA inhibited VKORC1 and VKORC1L1 expression levels and reduced VK and FIX levels in rat hepatocytes. Overexpression or silencing of Vkorc1 and Vkorc1l1 increased or decreased, respectively, the production and secretion of VK and FIX in rat hepatocytes, and alleviated or aggravated the inhibitory effects of Na-DHA on VKORC1 and VKORC1L1 expression levels. Taken together, the results indicated that both VKORC1 and VKORC1L1 signaling play regulatory roles in the effects of Na-DHA on coagulation factors in rat hepatocytes.

Stem Cell Technology in Organ Transplantation: A Novel Method for 3D Bioprinting Functional and Stable Liver Grafts Using Human iPS Cells Derived Cells.

For decades, scientists and physicians aim to generate functional human tissues in the laboratory using stem cells. In this section, we will describe a novel method that combines state-of-the-art stem cell culture with 3D bioprinting to generate potentially transplantable tissue grafts. For this purpose, we will use the liver as a model. The liver is an important metabolic hub and responsible to perform many endocrine and exocrine vital functions. It is estimated that two million deaths/year are associated with liver disease. This illustrates the need for developing new efficient alternatives for liver transplantation. Modern 3D bioprinting technologies in combination with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end-stage liver disease. Here, we described a novel method for 3D bioprinting functional and stable liver grafts using human iPS-derived cells. The novel method described in this section uses the hepatocyte-like cells in a spheroid culture format (i.e., obtained from three-dimensional cell culture), in combination or not with non-parenchymal cells (e.g., mesenchymal and endothelial cells), which generates the final liver graft. This method has proved to sustain epithelial phenotype and to increase the stability and functionality of the constructs for prolonged periods.

Lieber-DeCarli Diet Promotes Different Hepatic Histological Changes During Early and Late Pregnancy: An Example of Maternal Resilience.

BACKGROUND/AIM: During pregnancy, maternal liver can be affected by ethanol (ETOH) intake, whose effects depend on concentration levels ingested. This study aims to describe histological and serum marker characteristics of maternal liver during two metabolic conditions: gestation (G), and sustained ETOH intake, in early and late pregnancy. MATERIALS AND METHODS: Wistar rats were fed with Lieber-DeCarli diet during pregnancy, following an experimental protocol that allows a semi-chronic intake of ETOH (5%). Liver and serum samples were processed for histological characterization and biochemical profiling. Hematoxylin/eosin and Schiff's Periodic Acid staining were used. RESULTS: During pregnancy, a significant elevation in ballooned and edamatous hepatocytes, and a significant increase in micro and macrovesicular deposits were observed in rats fed with the ETOH diet at gestation days 3G, 8G and 15G. These changes were reverted by 20G. Liver glycogen content increased significantly at 15G. Serum metabolites in pregnant rats fed with the ETOH diet showed a significant reduction in urea (from 3G to 15G), an increase in albumin and uric acid at 20G, and a reduction in creatinine. Number of offsprings and weight of male newborns were reduced by 20% and 14%, respectively. Liver function markers in serum showed no significant changes. CONCLUSION: ETOH diet intake promotes hepatic histological changes and histological modifications during pregnancy. These results support the assumption that pregnancy is an adaptive procedure that is associated with nutritional conditions and has a strong influence on hepatic histology. They suggest that pregnancy promotes a state of resilience to the liver function during the sustained intake of 5% ETOH.

Efecto del extracto hidroalcohólico de la planta Lampaya medicinalis Phil . sobre marcadores inflamación inducidos por ácidos grasos en hepatocitos humanos HepG2

RESUMEN Introducción: la enfermedad de hígado graso no alcohólico (EHGNA) se caracteriza por la acumulación de gotas lipídicas (GL) y sobre expresión de la proteína de GL Perilipina 1 (PLIN1) en los hepatocitos. En su patogénesis y progresión participan NF-ĸB, caspasa-1 y citoquinas proinflamatorias como IL-1β. La medicina popular del norte de Chile utiliza la planta Lampaya medicinalis Phil. (Verbenaceae) contra enfermedades. Objetivo: evaluar el efecto de un extracto hidroalcohólico de lampaya (EHL) sobre la expresión de marcadores inflamatorios y proteínas asociadas a las GL en hepatocitos tratados con ácidos grasos. Métodos: se incubó hepatocitos HepG2 con 0,66 mM de ácido oleico (AO) y 0,33 mM de ácido palmítico (AP) por 24 o 48 horas en presencia o no de EHL. Se evaluó la expresión proteica de NF-ĸB, PLIN1 y caspasa-1 por Western blot y la expresión de ARNm de IL-1β por qPCR. Resultados: los hepatocitos tratados por 48 h con AO/AP mostraron un aumento en la expresión de IL-1β que fue revertido por la co-incubación con EHL. Conclusión: estos antecedentes aportan nueva evidencia respecto a la actividad biológica del EHL en un modelo de alteraciones metabólicas e inflamatorias, asociadas a la EHGNA, inducidas por AO/AP en hepatocitos humanos.

ABSTRACT Introduction: Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets (LD) and overexpression of the LD-associated protein Perilipin 1 (PLIN1). NF-ĸB, caspase-1 and proinflammatory cytokine such as IL-1β participate in the pathogenesis and progression of NAFLD. Traditional medicine in northern Chile uses the plant Lampaya medicinalis Phil. (Verbenaceae) against diseases. Objective: To evaluate the effect of a hydroalcoholic extract of lampaya (HEL) on the expression of inflammatory markers and LD-associated proteins in hepatocytes treated with fatty acids. Methods: HepG2 hepatocytes were incubated with 0.66 mM oleic acid (OA) and 0.33 mM palmitic acid (PA) for 24 or 48 h in the presence or not of HEL. The protein expression of NF-ĸB, PLIN1 and caspase-1 was evaluated by Western blot while the mRNA expression of IL-1β was assessed by qPCR. Results: hepatocytes treated for 48 h with OA/AP showed an increase in IL-1β expression that was reversed by co-incubation with HEL. Conclusion: These antecedents provide new evidence regarding the biological activity of HEL in a model of metabolic and inflammatory alterations, associated with NAFLD, induced by OA/PA in human hepatocytes.

Sympathetic nerve-adipocyte interactions in response to acute stress.

Psychological stress predisposes our body to several disorders. Understanding the cellular and molecular mechanisms involved in the physiological responses to psychological stress is essential for the success of therapeutic applications. New studies show, by using in vivo inducible Cre/loxP-mediated approaches in combination with pharmacological blockage, that sympathetic nerves, activated by psychological stress, induce brown adipocytes to produce IL-6. Strikingly, this cytokine promotes gluconeogenesis in hepatocytes, that results in the decline of tolerance to inflammatory organ damage. The comprehension arising from this research will be crucial for the handling of many inflammatory diseases. Here, we review recent advances in our comprehension of the sympathetic nerve-adipocyte axis in the tissue microenvironment.

Differential Effects of Oleic and Palmitic Acids on Lipid Droplet-Mitochondria Interaction in the Hepatic Cell Line HepG2.

Fatty acid overload, either of the saturated palmitic acid (PA) or the unsaturated oleic acid (OA), causes triglyceride accumulation into specialized organelles termed lipid droplets (LD). However, only PA overload leads to liver damage mediated by mitochondrial dysfunction. Whether these divergent outcomes stem from differential effects of PA and OA on LD and mitochondria joint dynamics remains to be uncovered. Here, we contrast how both fatty acids impact the morphology and interaction between both organelles and mitochondrial bioenergetics in HepG2 cells. Using confocal microscopy, we showed that short-term (2-24 h) OA overload promotes more and bigger LD accumulation than PA. Oxygen polarography indicated that both treatments stimulated mitochondrial respiration; however, OA favored an overall build-up of the mitochondrial potential, and PA evoked mitochondrial fragmentation, concomitant with an ATP-oriented metabolism. Even though PA-induced a lesser increase in LD-mitochondria proximity than OA, those LD associated with highly active mitochondria suggest that they interact mainly to fuel fatty acid oxidation and ATP synthesis (that is, metabolically "active" LD). On the contrary, OA overload seemingly stimulated LD-mitochondria interaction mainly for LD growth (thus metabolically "passive" LDs). In sum, these differences point out that OA readily accumulates in LD, likely reducing their toxicity, while PA preferably stimulates mitochondrial oxidative metabolism, which may contribute to liver damage progression.

Dose- and Time-Dependent Effects of Oleate on Mitochondrial Fusion/Fission Proteins and Cell Viability in HepG2 Cells: Comparison with Palmitate Effects.

Mitochondrial impairments in dynamic behavior (fusion/fission balance) associated with mitochondrial dysfunction play a key role in cell lipotoxicity and lipid-induced metabolic diseases. The present work aimed to evaluate dose- and time-dependent effects of the monounsaturated fatty acid oleate on mitochondrial fusion/fission proteins in comparison with the saturated fatty acid palmitate in hepatic cells. To this end, HepG-2 cells were treated with 0, 10 µM, 50 µM, 100 µM, 250 µM or 500 µM of either oleate or palmitate for 8 or 24 h. Cell viability and lipid accumulation were evaluated to assess lipotoxicity. Mitochondrial markers of fusion (mitofusin 2, MFN2) and fission (dynamin-related protein 1, DRP1) processes were evaluated by Western blot analysis. After 8 h, the highest dose of oleate induced a decrease in DRP1 content without changes in MFN2 content in association with cell viability maintenance, whereas palmitate induced a decrease in cell viability associated with a decrease mainly in MFN2 content. After 24 h, oleate induced MFN2 increase, whereas palmitate induced DRP1 increase associated with a higher decrease in cell viability with high doses compared to oleate. This finding could be useful to understand the role of mitochondria in the protective effects of oleate as a bioactive compound.

Histomorphometric analysis of liver biopsies of treated patients with Gaucher disease type 1.

Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by a disturbance in the metabolism of glucocerebroside in the macrophages. Most of its manifestations - hepatosplenomegaly, anemia, thrombocytopenia, and bone pain - are amenable to a macrophage-target therapy such as enzyme replacement. However, there is increasing evidence that abnormalities of the liver persist despite the specific GD treatment. In this work, we adapted histomorphometry techniques to the study of hepatocytes in GD using liver tissue of treated patients, developing the first morphometrical method for canalicular quantification in immunohistochemistry-stained liver biopsies, and exploring histomorphometric characteristics of GD. This is the first histomorphometric technique developed for canalicular analysis on histological liver biopsy samples.

Enhanced ammonia detoxification to urea in hepatocytes transduced with human aquaporin-8 gene.

Hepatic ammonia detoxification to urea is critical for the prevention of hyperammonemia and neurological damage. Hepatocyte mitochondrial aquaporin-8 (AQP8) channels have been involved in ammonia-derived ureagenesis. Herein, we studied whether the adenoviral gene transfer of human AQP8 (hAQP8) to hepatocyte mitochondria enhances ammonia conversion to urea. Using primary cultured rat hepatocytes, we first confirmed the mitochondrial expression of hAQP8 and then, using unlabeled or 15 N-labeled ammonia, we demonstrated that the urea synthesis was significantly enhanced in hAQP8-transduced hepatocytes. Studies using isolated hAQP8-expressing mitochondria also showed an increased ammonia metabolism. hAQP8 transduction was able to recover the impaired ammonia-derived ureagenesis in hepatotoxin-treated hepatocytes. Our data suggest that mitochondrially-expressed hAQP8 enhances and improves hepatocyte ammonia conversion to urea, a finding with potential therapeutic implications for liver disease with impaired ammonia detoxification.

Modulation of SCD1 activity in hepatocyte cell lines: evaluation of genomic stability and proliferation.

Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme for the synthesis of monounsaturated fatty acids (MUFA). SCD1 overexpression is associated with a genetic predisposition to hepatocarcinogenesis in mice and rats. This work hypothesized possible roles of SCD1 to genomic stability, lipogenesis, cell proliferation, and survival that contribute to the malignant transformation of non-tumorigenic liver cells. Therefore, HepG2 tumor cells were treated with the SCD1 inhibitor (CAY10566) to ensure a decrease in proliferation/survival, as confirmed by a lipidomic analysis that detected an efficient decrease in the concentration of MUFA. According to that, we switched to a model of normal hepatocytes, the HepaRG cell line, where we: (i) overexpressed SCD1 (HepaRG-SCD1 clones), (ii) inhibited the endogenous SCD1 activity with CAY10566, or (iii) treated with two monounsaturated (oleic OA and/or palmitoleic PA) fatty acids. SCD1 overexpression or MUFA stimulation increased cell proliferation, survival, and the levels of AKT, phospho-AKT(Ser473), and proliferating cell nuclear antigen (PCNA) proteins. By contrast, opposite molecular and cellular responses were observed in HepaRG cells treated with CAY10566. To assess genomic stability, HepaRG-SCD1 clones were treated with ionizing radiation (IR) and presented reduced levels of DNA damage and higher survival at doses of 5 Gy and 10 Gy compared to parental cells. In sum, this work suggests that modulation of SCD1 activity not only plays a role in cell proliferation and survival, but also in maintaining genomic stability, and therefore, contributes to a better understanding of this enzyme in molecular mechanisms of hepatocarcinogenesis projecting SCD1 as a potential translational target.

Ursolic Acid Lactone Obtained from Eucalyptus tereticornis Increases Glucose Uptake and Reduces Inflammatory Activity and Intracellular Neutral Fat: An In Vitro Study.

Obesity has a strong relationship to insulin resistance and diabetes mellitus, a chronic metabolic disease that alters many physiological functions. Naturally derived drugs have aroused great interest in treating obesity, and triterpenoids are natural compounds with multiple biological activities and antidiabetic mechanisms. Here, we evaluated the bioactivity of ursolic acid lactone (UAL), a lesser-known triterpenoid, obtained from Eucalyptus tereticornis. We used different cell lines to show for the first time that this molecule exhibits anti-inflammatory properties in a macrophage model, increases glucose uptake in insulin-resistant muscle cells, and reduces triglyceride content in hepatocytes and adipocytes. In 3T3-L1 adipocytes, UAL inhibited the expression of genes involved in adipogenesis and lipogenesis, enhanced the expression of genes involved in fat oxidation, and increased AMP-activated protein kinase phosphorylation. The range of biological activities demonstrated in vitro indicates that UAL is a promising molecule for fighting diabetes.