Glycogen Deposition Research Articles

Robust effect of metabolic syndrome on major metabolic pathways in the myocardium.

Although the high-fat-diet-induced metabolic syndrome (MetS) is a precursor of human cardiac pathology, the myocardial metabolic state in MetS is far from clear. The discrepancies in metabolite handling between human and small animal models and the difficulties inherent in obtaining human tissue complicate the identification of the myocardium-specific metabolic response in patients. Here we use the large animal model of swine that develops the hallmark criteria of human MetS. Our comparative metabolomics together with transcriptomics and computational nonnegative matrix factorization (NMF) interpretation of the data exposes significant decline in metabolites related to the fatty acid oxidation, glycolysis, and pentose phosphate pathway. Behind the reversal lies decreased expression of enzymes that operate in the pathways. We showed that diminished glycogen deposition is a metabolic signature of MetS in the pig myocardium. The depletion of glycogen arises from disbalance in expression of genes that break down and synthesize glycogen. We show robust acetoacetate accumulation and activated expression of key enzymes in ketone body formation, catabolism and transporters, suggesting a shift in fuel utilization in MetS. A contrasting enrichment in O-GlcNAcylated proteins uncovers hexosamine pathway and O-GlcNAcase (OGA) expression involvement in the myocardial response to MetS. Although the hexosamine biosynthetic pathway (HBP) activity and the availability of the UDP-GlcNAc substrate in the MetS myocardium is low, the level of O-GlcNacylated proteins is high as the O-GlcNacase is significantly diminished. Our data support the perception of transcriptionally driven myocardial alterations in expression of standard fatty acids, glucose metabolism, glycogen, and ketone body related enzymes and subsequent paucity of their metabolite products in MetS. This aberrant energy metabolism in the MetS myocardium provide insight into the pathogenesis of CVD in MetS.

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase, two proteins involved in Lafora disease

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.

Molecular adaptations of glucose and lipid metabolism to different levels of dietary carbohydrates in juvenile Japanese flounderParalichthys olivaceus

Japanese flounder juveniles (initial body weight: 7.12 ± 0.02 g) were fed three diets containing 0, 120 and 200 g/kg of carbohydrates (C0, C120 and C200, respectively) for 10 weeks. Results showed that higher dietary carbohydrate intake enabled further deposition of glycogen and lipids in liver and muscle. The mRNA levels of glucokinase (gck), phosphofructokinase (pfkl) and hexokinase (HK) activity involved in glycolysis were significantly up‐regulated in C120 (p .05) but significantly increased in C200 group (p < .05). In conclusion, Japanese flounder was able to use carbohydrates efficiently through regulation of glucose and lipid metabolism when dietary carbohydrate was not higher than 120 g/kg, while 200 g/kg of dietary carbohydrate caused the deregulation of glucose homoeostasis.

Anti-hyperglycemic Effect of Torbangun (Coleus amboinicus Lour) Leaves Extract Through Liver and Muscle Glycogen Deposits in Streptozotocin-induced Hyperglycmic Sprague-Dawley Rats Model

The association of liver and muscle glycogen deposits with serum insulin levels, β-cells pancreas, and fasting blood glucose (FBG) of streptozotocin (STZ)-induced hyperglycemic rats receiving Torbangun leaves extract (TE) investigated. The intervention performed on 25 8-week-old Sprague-Dawley rats divided into four groups. Seven rats separated as a normal group (N), and other rats injected with streptozotocin (STZ). Confirmation of hyperglycemic was characterized by fasting blood glucose &gt;126 mg/dl. Treatment group which is NG (hyperglycemic rats); N (normal rats); H-IM (62.5 mg/kg BW metformin); and H-IT (620 mg/kg BW TE) for 14 days. This study revealed that TE significantly decreased FBG levels, increased insulin production, and the amount of liver glycogen deposits (a=0.01). However, the intervention did not significantly increase the amount of muscle glycogen deposits. TE administration improves β-cells, increases the liver and muscle glycogen deposits. TE was shown to have antihyperglycemic activity by improving the β-cell, increasing blood serum insulin levels, decreasing blood glucose levels, and increasing the liver glycogen deposits.

Cacao extract enriched in polyphenols prevents endocrine-metabolic disturbances in a rat model of prediabetes triggered by a sucrose rich diet

Cocoa extracts rich in polyphenols are used as potential agent for treating diabetes. Cocoa polyphenols have been proved to ameliorate important hallmarks of type-2 diabetes (T2D). They can regulate glucose levels by increasing insulin secretion, promoting β-cell proliferation and a reduction of insulin resistance. In addition, epidemiological evidence indicates that consumption of flavonoid decreases the incidence of T2D. T2D is preceded by a prediabetic state in which the endocrine-metabolic changes described in T2D are already present. Since epidemiological evidence indicates that consumption of flavonoid decreases its incidence, we evaluated possible preventive effects of polyphenol-enriched cocoa extract on a model of prediabetes induced by sucrose. We determined circulating parameters and insulin sensitivity indexes, liver protein carbonyl groups and reduced glutathione, liver mRNA expression levels of lipogenic enzymes, expression of different pro-inflammatory mediators, fructokinase activity and liver glycogen content. For that, radioimmunoassay, real-time polymerase chain reaction, Western blot, spectrophotometry, and immunohistochemistry were used. We demonstrated that sucrose administration triggered hypertriglyceridemia, insulin-resistance, and liver increased oxidative stress and inflammation markers compared to control rats. Additionally, we found an increase in glycogen deposit, fructokinase activity, and lipogenic genes expression (SREBP-1c, FAS and GPAT) together with a decrease in P-Akt and P-eNOS protein content (P < 0.05). Sucrose-induced insulin resistance, hepatic carbohydrate and lipid dysmetabolism, oxidative stress, and inflammation were effectively disrupted by polyphenol-enriched cocoa extract (PECE) co-administration (P < 0.05). Dietary administration of cocoa flavanols may be an effective and complementary tool for preventing or reverting T2D at an early stage of its development (prediabetes).

E-POSTERS – MITOCHONDRIAL / METABOLIC DISEASES –CASE REPORTS: EP.01Becker muscular dystrophy with acid α-glucosidase pseudodeficiency

Diagnosis of Pompe disease is sometimes challenging because it exhibits clinical similarities to muscular dystrophy. We describe a case of Becker muscular dystrophy (BMD) with a remarkable reduction in activity of the acid α-glucosidase (GAA) enzyme, caused by a combination of pathogenic mutation and polymorphism variants resulting in pseudodeficiency in GAA. The three-year-old boy demonstrated asymptomatic creatinine kinase elevation. Neither exon deletion nor duplication was detected on multiplex ligation-dependent probe amplification (MLPA) of dystrophin. GAA enzyme activity in both dried blood spots and lymphocytes was low, at 11.7% and 7.7% of normal, respectively. However, genetic analysis of GAA detected only heterozygosity for a nonsense mutation (c.118C>T, p.Arg40*). Muscle pathology showed no glycogen deposits and no high acid phosphatase activity. Hematoxylin-eosin staining detected scattered regenerating fibers; the fibers were faint and patchy on immunochemistry staining of dystrophin. The amount of dystrophin protein was reduced to 11.8% of normal, on Western blotting analysis. Direct sequencing analysis of dystrophin revealed hemizygosity for a nonsense mutation (c.72G>A, p.Trp24*). The boy was diagnosed with BMD, despite remarkable reduction in GAA activity; further, he demonstrated heterozygosity for [p.Gly576Ser; p.Glu689Lys] polymorphism variants that indicated pseudodeficiency on another allele in GAA. Pseudodeficiency alleles are detected in approximately 4% of the Asian population; these demonstrate low activity of acid α-glucosidase (GAA), similar to levels found in Pompe disease. Clinicians should be careful in their interpretations of pseudodeficiency alleles that complicate diagnosis in cases of elevated creatinine kinase.

Genotoxic, cytotoxic, and cytopathological effects in rats exposed for 18 months to a mixture of 13 chemicals in doses below NOAEL levels

The present study investigates the genotoxic and cytotoxic effects of long term exposure to low doses of a mixture consisting of methomyl, triadimefon, dimethoate, glyphosate, carbaryl, methyl parathion, aspartame, sodium benzoate, EDTA, ethylparaben, buthylparaben, bisphenol A and acacia gum in rats. Four groups of ten Sprangue Dawley rats (5 males and 5 females per group) were exposed for 18 months to the mixture in doses of 0xNOAEL, 0.0025xNOAEL, 0.01xNOAEL and 0.05xNOAEL (mg/kg bw/day). After 18 months of exposure, the rats were sacrificed and their organs were harvested. Micronuclei frequency was evaluated in bone marrow erythrocytes whereas the organs were cytopathologically examined by the touch preparation technique. The exposure to the mixture caused a genotoxic effect identified only in females. Cytopathological examination showed specific alterations of tissue organization in a tissue-type dependent manner. The observed effects were dose-dependent and correlated to various tissue parameters. Specifically, testes samples revealed degenerative and cellularity disorders, liver hepatocytes exhibited decreased glycogen deposition whereas degenerative changes were present in gastric cells. Lung tissue presented increased inflammatory cells infiltration and alveolar macrophages with enhanced phagocytic activity, whereas brain tissue exhibited changes in glial and astrocyte cells’ numbers. In conclusion, exposure to very low doses of the tested mixture for 18 months induces genotoxic effects as well as monotonic cytotoxic effects in a tissue-dependent manner.

Glucogenosis Hepática. Características clínicas, laboratoriales e imagenológicas en pacientes pediátricos menores de 15 años

Antecedentes: La glucogenosis (GSD) hepática es una enfermedad hereditaria autosómica recesiva caracterizada por la alteración del depósito de glucógeno en los tejidos. La enfermedad se presenta con hepatomegalia, debilidad muscular y retraso del crecimiento. Esta patología usualmente se diagnostica clínicamente a partir de los 6 meses de edad cuando la ingesta de alimentos del lactante es más espaciada y puede debutar con sintomatología de hipoglicemia. Debido a la inespecificidad de la presentación clínica de la enfermedad es muy importante la sospecha diagnóstica desde los centros de primer nivel de atención y su derivación oportuna a centros de especialidad. Objetivo: Evaluar y describir el perfil nutricional y clínico en pacientes menores de 15 años con Glucogenosis Hepática. Método: Se describe una serie de casos de 14 pacientes menores de 15 años con diagnóstico clínico de GSD hepática, atendidos en la consulta de Gastroenterología y Nutrición Pediátrica del Hospital Carlos Andrade Marín entre 2016 y 2018. El diagnóstico se lo realizó de acuerdo a la clínica que presentó cada paciente como la presencia de distensión abdominal, hepatomegalia, adinamia, retraso en el crecimiento y datos laboratoriales como niveles de glicemia en sangre periférica, transaminasas, y realización de elastografía entre los principales. Se analizaron datos sociodemográficos, antropométricos, de laboratorio (transaminasas, glicemia periférica) y elastografía hepática. Para el análisis de datos se creó una base de datos en Microsoft Excel 2013 y se procesó con el programa Epi Info 7. Resultados: En este grupo de casos, los tipos específicos de GSD hepática fueron tipo IX, 57,14% (8), tipo III, 28,57% (4) y tipo Ia-b, 14,29% (2) pacientes. La prevalencia de características clínicas ante la sospecha de la GSD hepática fueron: hepatomegalia 100% (14), y retraso en el crecimiento el 64,3% (9). De acuerdo a los exámenes de sangre periférica los valores promedio de transaminasas hepáticas (AST/TGO U/L) (ALT/TGP U/L) y glucosa, fueron de 364±384, 302±255 y 61±15 mg/dL, respectivamente. La elastografía con la que se evaluó el nivel de fibrosis hepática al momento del diagnóstico arrojó los siguientes resultados: F0 (no fibrosis hepática) en el 28,57% (4), F1 con el 28,57% (4), F2-F3 con el 35,71% (5), y F4 7,14%. Conclusión: La Glucogenosis es una patología que debería ser sospechada a tiempo en centros del primer nivel de salud para luego referir oportunamente los casos a los centros de referencia. La hepatomegalia y el retardo en el crecimiento son signos cardinales de alerta para la sospecha de esta patología.

Glycerol inoculation in eggs of young broiler breeders at different embryonic periods.

Small eggs have lesser amounts of nutrients to be used by the embryo, and the yolk glycerol is the main substrate for glycogen production, which is the main energy source in the last days of incubation. Thus, the present study aimed to assess the effect of a glycerol injection in light weight eggs at 2 different days of incubation. To this end, 336 light eggs (55.6 to 58.6g) from 32-wk-old broiler breeders were incubated. The eggs were divided into 3 treatment groups: 1 group inoculated with saline solution on the 17th d of embryonic development (E17) (control group), the second group injected with a 6mg glycerol/mL solution at E17, and the third group injected with 6mg glycerol/mL on the 18th d of incubation (E18). Incubation parameters, liver and muscle glycogen, and broilers performance at 7 d of age were evaluated. Glycerol administration in ovo did not influence hatchability, period of embryonic death or early hatching. Chicks exposed to glycerol in ovo feeding (IOF) used more yolk than birds inoculated with saline solution. Glycerol inoculation at E18 enhanced liver glycogen deposition (P = 0.001) and also improved broilers performance at 7 d, although this improvement in performance and glycogen reserves was not observed when eggs were inoculated at 17 d of incubation. Birds receiving glycerol IOF at E18 showed higher feed intake and body weight gain when compared to the control group and the group inoculated at E17. It was found that glycerol inoculation in light eggs at the 18th d of incubation contributed to raise liver glycerol levels and also to improve broilers performance at 7 d.

The Protein Phosphatase 1 Complex Is a Direct Target of AKT that Links Insulin Signaling to Hepatic Glycogen Deposition

Insulin-stimulated hepatic glycogen synthesis is central to glucose homeostasis. Here, we show that PPP1R3G, a regulatory subunit of protein phosphatase 1 (PP1), is directly phosphorylated by AKT. PPP1R3G phosphorylation fluctuates with fasting-refeeding cycle and is required for insulin-stimulated dephosphorylation, i.e., activation of glycogen synthase (GS) in hepatocytes. In this study, we demonstrate that knockdown of PPP1R3G significantly inhibits insulin response. The introduction of wild-type PPP1R3G, and not phosphorylation-defective mutants, increases hepatic glycogen deposition, blood glucose clearance, and insulin sensitivity invivo. Mechanistically, phosphorylated PPP1R3G displays increased binding for, and promotes dephosphorylation of, phospho-GS. Furthermore, PPP1R3B, another regulatory subunit of PP1, binds to the dephosphorylated GS, thereby relaying insulin stimulation to hepatic glycogen deposition. Importantly, this PP1-mediated signaling cascade is independent of GSK3. Therefore, we reveal a regulatory axis consisting of insulin/AKT/PPP1R3G/PPP1R3B that operates in parallel to the GSK3-dependent pathway, controlling glycogen synthesis and glucose homeostasis in insulin signaling.

The impacts of dietary carbohydrate levels on growth performance, feed utilization, glycogen accumulation and hepatic glucose metabolism in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

The present study was conducted to investigate the effects of dietary carbohydrate on growth performance, feed utilization, glycogen deposition and hepatic glucose metabolism in hybrid grouper. Triplicate groups of grouper (9.16 ± 0.18 g) were fed diets with grade levels of carbohydrate (5.27, 8.95, 11.49, 14.37, 17.78, 20.82 and 23.65%) twice daily for 10 weeks. Results showed that specific growth rate were quadratically and linearly decreased with the increase of dietary carbohydrate, which was related to reduced feed intake and apparent digestibility coefficient of nutrients. Meanwhile, the increase of dietary carbohydrate resulted in both quadratic and linear increase of hepatic glycogen accumulation, which further resulted in the elevated viscerosomatic index. The specific activity of glycolysis and gluconeogenesis related enzymes were both quadratic and linear enhanced as dietary carbohydrate increased, mainly due to the deceased hepatic protein content. The highest expressions of glucokinase, liver-type 6-phosphofructokinase, pyruvate kinase were obtained in the 8.95, 8.95 and 11.49% carbohydrate group, respectively. However, any significant differences were not observed in the expressions of gluconeogenesis related genes as dietary carbohydrate increased. The expression of insulin receptor, insulin receptor substrate 1 and serine/threonine kinase 1 was quadratic and linear elevated in the grouper fed the diet with 8.95% carbohydrate in accordance with the variation of serum insulin content, indicating the activation of insulin pathway at least at the transcription level. These results suggested optimal dietary carbohydrate would benefit for improving growth performance and feed utilization, and activating the insulin pathway along with the promotion of glycolysis in this grouper.

Late-onset Pompe disease: preliminary results of enzyme replacement therapy

Pompe disease is an orphan hereditary accumulation disease associated with a deficiency of the lysosomal enzyme alglucosidase alpha. Manifestations of the disease are associated with pathological deposition of glycogen in body tissues as a result of GAA gene mutation and subsequent reduction in the activity of the enzyme alglucosidase alpha or acid maltase. The variety of phenotypic forms and varying degrees of damage to the skeletal and respiratory muscles, cardiomyocytes and internal organs greatly complicates the diagnosis and treatment of patients with Pompe»s disease. This article describes the clinical case of late-onset Pompe disease, which was followed by a course of enzyme replacement therapy, as well as an assessment of the condition before and after treatment and preliminary results.

Metabolic Effects of Metformin in Humans.

Both insulin deficiency and insulin resistance due to glucagon secretion cause fasting and postprandial hyperglycemia in patients with diabetes. Metformin enhances insulin sensitivity, being used to prevent and treat diabetes, although its mechanism of action remains elusive. Patients with diabetes fail to store glucose as hepatic glycogen via the direct pathway (glycogen synthesis from dietary glucose during the post-prandial period) and via the indirect pathway (glycogen synthesis from "de novo" synthesized glucose) owing to insulin deficiency and glucagoninduced insulin resistance. Depletion of the hepatic glycogen deposit activates gluconeogenesis to replenish the storage via the indirect pathway. Unlike healthy subjects, patients with diabetes experience glycogen cycling due to enhanced gluconeogenesis and failure to store glucose as glycogen. These defects raise hepatic glucose output causing both fasting and post-prandial hyperglycemia. Metformin reduces post-prandial plasma glucose, suggesting that the drug facilitates glucose storage as hepatic glycogen after meals. Replenishment of glycogen store attenuates the accelerated rate of gluconeogenesis and reduces both glycogen cycling and hepatic glucose output. Metformin also reduces fasting hyperglycemia due to declining hepatic glucose production. In addition, metformin reduces plasma insulin concentration in subjects with impaired glucose tolerance and diabetes and decreases the amount of insulin required for metabolic control in patients with diabetes, reflecting improvement of insulin activity. Accordingly, metformin preserves β-cell function in patients with type 2 diabetes. Several mechanisms have been proposed to explain the metabolic effects of metformin, but evidence is not conclusive and the molecular basis of metformin action remains unknown.

371-P: Hypoglycemia in the Brain Is Required to Augment Counterregulatory Responses to Hypoglycemia when Liver Glycogen Levels Are Enhanced

In a previous study, we showed that elevated hepatic glycogen levels can generate a signal in the liver during insulin (INS)-induced hypoglycemia that is relayed to the brain, resulting in enhanced counterregulatory (CR) hormone secretion and net hepatic glucose output (NHGO). The purpose of this study was to determine whether hypoglycemia in the brain is required for this augmentation to occur. Fasted (18 hour) dogs were studied and each experiment began with a 4h glycogen deposition period in which each animal received somatostatin and basal intraportal infusions of both INS and glucagon (GGN). Plasma glucose (PG) was doubled by an intravenous (IV) glucose infusion and liver glycogen content was enhanced in all animals (76 ± 6 and 68 ± 4 mg/g in Brain EU and HYPO, respectively) by an intraportal infusion of fructose. A 2h control period followed, during which hyperglycemia and basal replacement of hormones were continued but fructose infusion was stopped. In the final 2h hypoglycemic period, somatostatin and GGN infusions were stopped and the plasma INS level was increased 16-fold by an IV infusion of INS. During the final period, whole body PG was clamped at 52 ± 1 mg/dL in one group (HYPO; n=6). In a second group, PG in the head was clamped at 92 ± 3 mg/dL by infusing glucose into the carotid and vertebral arteries (Brain EU; n=8) while the glucose level in the rest of the body remained at 51 ± 1 mg/dL. During the final hour of the hypoglycemic period, NHGO was lower in Brain EU compared to HYPO (0.8 ± 0.2 vs. 2.2 ± 0.4 mg/kg/minute, p&amp;lt;0.01). This decrease in NHGO was associated with a decrease in CR hormone secretion, as GGN (27 ± 6 vs. 53 ± 8 pg/mL, p&amp;lt;0.01) and epinephrine (451 ± 103 vs. 1106 ± 143 pg/mL, p&amp;lt;0.01) were lower when the brain was euglycemic (Brain EU vs. HYPO, respectively). These data suggest that the augmented CR responses that occur during INS-induced hypoglycemia when liver glycogen content is enhanced, require hypoglycemia in the brain. Disclosure S.O. Warner: None. M.S. Smith: None. B. Farmer: None. J.J. Winnick: None. Funding National Institutes of Health

Features of the visualization of the liver in children with glycogen storage disease according to data of computed tomography

Diagnostic value of computed tomography (CT) of the liver imaging in children with glycogen storage disease (GSD) is still poorly understood, in this connection, there were examined 59 patients with essential hypertension (study group) and 27 - with Wilson's disease (WD) (control group). In dependence on the type of GSD patients of the main group were divided into 3 groups: 1st with GSD Type I (n = 18), 2nd - with GSD type III (n = 21), the third - with the GSD type VI/IX (n = 20). Reference group was consisted of 107 children without liver disease. CT of the abdominal cavity on multislice computed tomography Light Speed 16 and 750 HD (GE, USA) was performed in all cases. The pronounced hepatomegaly with predominant increase in the left lobe and the index of I segment was established to be the common diagnostic CT sign, typical for all types of GSD. The change in the X-ray density of the liver parenchyma, dependent on the type of disease is also the feature of the CT-picture of liver parenchyma. In GD type I there was often noted the decline in the X-ray density of the liver parenchyma, while in GSD types III and VI there was identified a significant increase in the density of the organ tissue, which was related with morphological changes in the liver parenchyma, caused by the deposition of glycogen in hepatocytes, fat and protein dystrophy. The authors believe that CT of the abdominal cavity can be an alternative, non-invasive, relatively inexpensive and accessible way to diagnose GD, which is especially important in pediatric practice.

MON-133 Exacerbation of Lactic Acidosis During DKA in Adolescents with Type 1 Diabetes Mellitus with Mauriac Syndrome

Background: Glycogenic hepatopathy (GH: deposition of glycogen in the liver) and lactic acidosis (LA) in poorly controlled type 1 diabetes (T1D) can be the only presentation of Mauriac syndrome (hypercholesterolemia, growth retardation, delayed puberty, and cushingoid features). LA in the background of DKA is the result of poor tissue perfusion due to dehydration and hypophosphatemia. Intensive insulin therapy during management of DKA inhibits glycogenolysis and gluconeogenesis leading to the anaerobic conversion of pyruvate to lactate, thereby worsening the LA. Case series: Patient #1 had ten DKA admissions and had LA in two admissions nine months apart [mean + SD-age: 12.5+/-0.7 years; BMI-28.7+/-0.33 kg/m2, HbA1c:10.5 +/- 0.4%; duration of diabetes: 6.5+/-0.7 years]. Patient #2 had five DKA admissions (age:14 years; BMI: 23.7 kg/m2, HbA1c: 11.7; duration of diabetes: 6 years). Clinical and biochemical characteristics:- initial presentation- pH:7.11+/- 0.06; HCO3: 6.5+/- 0.6 mmol/l, glucose: 807+/-50.4 mg/dl, phos: 5.3+/-0.4, lactate: 3.11+/-1.33 with hepatomegaly on exam. DKA resolved in 27.6 +/- 2.5 hours. Lactate levels peaked to 7.8+/-0.3mmol/l at 13+/-11.5 hours after resolution of DKA and the anion gap at the time of the peak was 13.6 +/- 7.5mmol/l. Liver enzymes were within normal limits on admission and after [AST-21.6 +/-4.04; (5-34 U/L); ALT- 23.6 +/- 9.6 (0-55 U/L)]. Liver ultrasound showed hepatomegaly (18.65 +/-2.33 cm; Normal:8.5-14 cm) suggestive of glycogen versus fat deposition. Basal bolus insulin was continued and the lactate levels declined in 29+/-6.02 hours after the peak to 2.7 +/-1.18mmol/l at the time of discharge. Since the liver enzymes were normal and the LA was improving, no liver biopsy was performed in either of our patients. Conclusions: The lactate levels were high on admission and the levels continued to rise during and after resolution of the DKAin both the patients. Since the patients have a history of poorly controlled diabetes and had hepatomegaly on ultrasound, GH was considered the most likely cause of the hepatomegaly. Our cases illustrate the following points: a. hepatomegaly in poorly controlled T1DM is likely GH. b. elevated lactate levels favor GH as elevated lactate levels are not seen in steatohepatitis, the caveat being that ultrasound cannot discriminate between the two conditions c. DKA can unmask this lactic acidemia which can be present in half of the patients with Mauriac’s syndrome d. liver biopsy is the gold standard for diagnosis of GH though gradient-dual-echo liver MRI can be non-invasive alternative for diagnosis of GH.

A "Hibernating-Like" Viable State Induced by Lentiviral Vector-Mediated Pigment Epithelium-Derived Factor Overexpression in Rat Acute Ischemic Myocardium.

The failure to maintain the viability of ischemic myocardium is one of the mechanisms that causes ischemic heart dysfunction after revascularization. Hibernating myocardium is considered to be able to maintain long-term viability during chronic hypoperfusion. Pigment epithelium-derived factor (PEDF) decreases the contractility of hypoxic cardiomyocytes and protects cardiomyocytes against ischemic injury, which is strikingly similar to the pathophysiologic characteristics of hibernating myocardium. It was therefore postulated that PEDF may induce acute ischemic myocardium into a "hibernating-like" state to maintain its viability. Adult Sprague-Dawley rat models of acute myocardial infarction were surgically established. Lentiviral vectors carrying the PEDF gene (PEDF-LVs) were delivered into myocardium with infarction to overexpress PEDF locally. It was found that PEDF local overexpression significantly reduced myocardial infarct size and cardiomyocytes necrosis but did not improve cardiac function at rest. The contractile reserve assessed by low-dose dobutamine stress echocardiography and "perfusion-metabolism mismatch" assessed by positron emission tomography, which are the characteristics of viable myocardium in hibernation, were observed in the PEDF overexpressed ischemic heart. Ultrastructural changes observed by electron microscopy and glycogen deposition explored by Periodic acid-Schiff staining were similar to the histological characteristics of hibernating myocardium. Moreover, PEDF overexpression protected the cardiomyocytes against anoxic injury and retained their functional recovery potential after reoxygenation in vitro. PEDF local overexpression may induce acute ischemic myocardium into a "hibernating-like" state and maintain its viability. This novel effect of PEDF presents an important clinical approach to enhance functional recovery after revascularization therapy in acute myocardial infarction.

Insulin-like growth factor-I promotes hepatic differentiation of human adipose tissue-derived stem cells.

There are numerous studies which provide support for the use of human adipose tissue-derived stem cells (hASCs) to generate hepatocyte-like cells. However, the produced cells exhibit only a certain level of differentiation, mainly due to inefficient induction conditions. Therefore, based on the important role of insulin-like growth factor (IGF-I) in hepatic function and development, in the current study we evaluated the differentiation efficacy of the mentioned factor to induce hASCs into functional hepatocyte-like cells. To investigate this, using a two-step protocol, hASCs were treated with a combination of HGF, Dex, and OSM in the presence or absence of IGF-I up to 21 days. Hepatic differentiation was evaluated by analyzing specific hepatocyte markers at different time points of differentiation induction. Increased expression of hepatocyte-specific genes including ALB, AFP, CK18, and HNF4a, downregulation of bile duct cells marker (CK19), the higher number of ALB positive cells, increased urea production together with higher glycogen deposit was observed upon the treatment of hASCs with the induction medium containing IGF-I compared to the other treatment. In conclusion, our findings suggest IGF-I as a potent inducer of hepatic differentiation of hASCs and its potential to generate more functional hepatocyte-like cells.

Development of Simple Protocol for Generation of Functionally Active Hepatocyte-like Cells from Human Adipose Tissue-derived Stem Cells

Background and Aims: Human adipose tissue-derived stem cells (hASCs) are considered as an attractive source of regenerative stem cells, mainly because of their higher proliferation rate, more accessibility and hepatocyte like properties as compared to mesenchymal stem cells isolated from other tissues. Numerous studies have described the beneficial use of adipose tissue-derived stem cells for generating hepatocyte-like cells. However, due to the lack of appropriate culture conditions, most of the produced cells exhibit poor functionality. The aim of the present study was to establish a new protocol for the efficient hepatic differentiation of hASCs. Materials and Methods: hASCs were cultured in hepatic differentiation medium containing fibroblast growth factor 4, hepatocyte growth factor, dexamethasone and oncostatin M using a three-step protocol up to 21 days. Then, the functionality of the treated cells was evaluated by analyzing specific hepatocyte genes and biochemical markers at various time points. Results: A significant upregulation in albumin, alpha-fetoprotein, cytokeratin 18 and hepatocyte nuclear factor-4α expressions was observed in differentiated cells relative to day 1 of differentiation protocol. Moreover, the finding of glycogen deposits increased urea production and positive immunofluorescence staining for albumin and alpha-fetoprotein in hepatocyte-like cells suggesting that most of the cells differentiate into hepatocyte-like cells. Conclusions: Our report has provided a simple protocol for differentiation of hASCs into more functional hepatocyte-like cells.

Aldosterone stimulation mediates cardiac metabolism remodeling via Sirt1/AMPK signaling in canine model.

Aldosterone (Aldo), a pivotal hormone that is ubiquitously expressed in systemic tissues of mammals, is a crucial factor in the pathogenesis of cardiac disease. Accumulating evidence suggests that disturbances in cell energy metabolism are involved in increasing aldosterone levels. However, the precise mechanism underlying the impact of cardiac metabolic remodeling underlying aldosterone stimulation remains limited. In this work, we evaluated the underlying effect of aldosterone on regulating cardiac metabolism remodeling in a canine model. Fifteen beagle dogs were divided into a control group (n = 5), Aldo group (n = 5), and a group treated with spironolactone (SP), a mineralocorticoid receptor antagonist (n = 5), for 4weeks. Blood pressure, electrocardiogram and respiratory parameters, H&E, Masson staining, ultrastructural changes, the adenosine triphosphate (ATP) and free fatty acid (FFA) levels of ventricular tissues, the level of mRNA, and the protein expression of key metabolic factors and regulators were assessed. The Sirt1/AMPK signaling pathway was significantly inhibited in the canine model of aldosterone stimulation, resulting in a reduction of the key downstream metabolic factors involved in glucose and fatty acid oxidation. The dysregulation of expression of key factors in glycogen metabolism led to glycogen deposition, an increase in FFA levels, a reduction in ATP levels, apoptosis, inflammatory cell infiltration, and mitochondrial damage in the ventricular myocardium. These effects were significantly restored by spironolactone. Aldosterone stimulation induced cardiac metabolic remodeling in ventricular cardiomyocytes possibly through the Sirt1/AMPK signaling pathway, implying that this pathway may provide a novel therapeutic target for cardiac metabolic remodeling.