Hilkka Reunanen

Baculovirus Entry into Human Hepatoma Cells

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV- and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron microscopy studies revealed that virions were located on the cell surface in pits with an electron-dense coating resembling clathrin. In addition, virus particles were found in larger noncoated plasma membrane invaginations and in intracellular vesicles resembling macropinosomes. In double-labeling experiments, virus particles were detected by confocal microscopy in early endosomes at 30 min and in late endosomes starting at 45 min posttransduction. Viruses were also seen in structures specific for early endosomal as well as late endosomal/lysosomal markers by nanogold preembedding immunoelectron microscopy. No indication of viral entry into recycling endosomes or the Golgi complex was observed by confocal microscopy. In conclusion, these results suggest that AcMNPV enters mammalian cells via clathrin-mediated endocytosis and possibly via macropinocytosis. Thus, the data presented here should enable future design of baculovirus vectors suitable for more specific and enhanced delivery of genetic material into mammalian cells.

Gene expression centroids that link with low intrinsic aerobic exercise capacity and complex disease risk

A strong link exists between low aerobic exercise capacity and complex metabolic diseases. To probe this linkage, we utilized rat models of low and high intrinsic aerobic endurance running capacity that differ also in the risk for metabolic syndrome. We investigated in skeletal muscle gene‐phenotype relationships that connect aerobic endurance capacity with metabolic disease risk factors. The study compared 12 high capacity runners (HCRs) and 12 low capacity runners (LCRs) from generation 18 of selection that differed by 615% for maximal treadmill endurance running capacity. On average, LCRs were heavier and had increased blood glucose, insulin, and triglycerides compared with HCRs. HCRs were higher for resting metabolic rate, voluntary activity, serum high density lipoproteins, muscle capillarity, and mitochondrial area. Bioinformatic analysis of skeletal muscle gene expression data revealed that many genes up‐regulated in HCRs were related to oxidative energy metabolism. Seven mean mRNA expression centroids, including oxidative phosphorylation and fatty acid metabolism, correlated significantly with several exercise capacity and disease risk phenotypes. These expression‐phenotype correlations, together with diminished skeletal muscle capillarity and mitochondrial area in LCR rats, support the general hypothesis that an inherited intrinsic aerobic capacity can underlie disease risks.—Kivelä, R., Silvennoinen, M., Lehti, M., Rinnankoski‐Tuikka, R., Purhonen, T., Ketola, T., Pullinen, K., Vuento, M., Mutanen, N., Sartor, M. A., Reunanen, H., Koch, L. G., Britton, S. L., Kainulainen, H. Gene expression centroids that link with low intrinsic aerobic exercise capacity and complex disease risk. FASEB J. 24, 4565–4574 (2010). www.fasebj.org

Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in ehrlich ascites cells

The microtubule inhibitor vinblastine causes accumulation of autophagic vacuoles in many cell types. In hepatocytes, many of the accumulated vacuoles are nascent, which has been interpreted to suggest that vinblastine acts by inhibiting the fusion of hydrolase-containing lysosomes with early autophagic vacuoles. However, our previous results suggested that, in Ehrlich ascites cells, vinblastine causes accumulation mainly of older autophagic vacuoles (AVs). This study was undertaken to further characterize the mode of action of vinblastine in these cells. The vinblastine-accumulated AVs were quantified by electron-microscopic morphometry. In addition, the effects of inhibitors of autophagic segregation (leucine, histidine, and 3-methyladenine) on the vinblastine-induced accumulation of autophagic vacuoles were studied. Protein degradation was measured using [14C]valine. Vinblastine caused accumulation of advanced autophagic vacuoles but did not increase the rate of protein degradation. The volume density of early vacuoles remained at the control level. The amino acids retarded but did not prevent the accumulation of autophagic vacuoles, whereas 3-methyladenine almost completely prevented the accumulation. The results suggest that in Ehrlich ascites cells vinblastine acts by inhibiting the maturation of advanced autophagic vacuoles into residual bodies and by stimulating the formation of new autophagic vacuoles. However, 3-methyladenine almost completely prevents the formation of new autophagic vacuoles in the presence of vinblastine. In conclusion, in Ehrlich ascites cells, vinblastine does not prevent the entry of hydrolases into autophagic vacuoles. This calls into question the importance of microtubules in the transport of lysosomal enzymes into autophagic vacuoles.

Effects of griseofulvin and nocodazole on the accumulation of autophagic vacuoles in Ehrlich ascites tumor cells.

Accumulation of autophagic vacuoles (AVs) is a phenomenon observed in various cells treated with the microtubule inhibitor vinblastine. In order to test whether the accumulation of AVs is a result of retarded fusion of autophagosomes and lysosomes an investigation was carried out to ascertain whether other antimicrotubular drugs, e.g., nocodazole and griseofulvin, also induce accumulation of AVs. Ehrlich ascites tumor cells were incubated with nocodazole (20 micrograms/ml) or griseofulvin (50 micrograms/ml). Morphometric analyses were performed after incubation periods of 3, 30, 60, and 120 min. The volume densities of autophagic vacuoles did not differ significantly from the control values after the various incubation periods tested. It was concluded that intact microtubules are not needed in the fusion of autophagosomes and lysosomes and that vinblastine accelerates the rate of AV formation.

Effects of high-fat diet and physical activity on pyruvate dehydrogenase kinase-4 in mouse skeletal muscle

BackgroundThe expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4) gene expression via the estrogen-related receptor α (ERRα). We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle.MethodsInsulin resistance was induced by a high-fat (HF) diet for 19 weeks in C57BL/6 J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed.ResultsThe HF mice were more insulin-resistant than the low-fat (LF) -fed mice. Upregulation of PDK4 and ERRα mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria.ConclusionsOur results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

Filipin labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

SummaryCholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, doublemembrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazolebuffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic vacuoles were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.

Lipid droplet-associated proteins in high-fat fed mice with the effects of voluntary running and diet change

OBJECTIVE The relation between lipid accumulation and influence of exercise on insulin sensitivity is not straightforward. A proper balance between lipid droplet synthesis, lipolysis, and oxidative metabolism would ensure low local intramyocellular fatty acid levels, thereby possibly protecting against lipotoxicity-associated insulin resistance. This study investigated whether the accumulation of triglycerides and lipid droplets in response to high availability of fatty acids after high-fat feeding would parallel the abundance of intramyocellular perilipin proteins, especially PLIN5. The effects on these variables after diet change or voluntary running exercise intervention in skeletal muscle were also investigated. METHODS During a 19-week experiment, C57BL/6J mice were studied in six different groups: low-fat diet sedentary, low-fat diet active, high-fat diet sedentary, high-fat diet active and two groups which were high-fat sedentary for nine weeks, after which divided into low-fat sedentary or low-fat active groups. Myocellular triglyceride concentration and perilipin protein expression levels were assessed. RESULTS We show that, concurrently with impaired insulin sensitivity, the expression level of PLIN5 and muscular triglyceride concentration increased dramatically after high-fat diet. These adaptations were reversible after the diet change intervention with no additional effect of exercise. CONCLUSIONS After high-fat diet, lipid droplets become larger providing more surface area for PLIN5. We suggest that PLIN5 is an important regulator of lipid droplet turnover in altered conditions of fatty acid supply and consumption. Imbalances in lipid droplet metabolism and turnover might lead to lipotoxicity-related insulin resistance.

VINBLASTINE-INDUCED AUTOPHAGOCYTOSIS IN CULTURED FIBROBLASTS

1. Balb/c 3T3 fibroblasts were incubated in a medium containing 10(-5) M vinblastine for 1, 2 and 3 hr. Morphometric analyses were performed after an incubation period of 2 hr. 2. The volume fraction of advanced autophagic vacuoles increased tenfold (P less than 0.05) concomitantly with a sixfold decrease in round lysosomes (P less than 0.01). 3. The volume fractions of pleomorphic lysosomes, nascent autophagic vacuoles and residual bodies did not differ significantly from the control values. 4. In many cells, advanced autophagic vacuoles resembled multivesicular bodies, which may indicate that the type of autophagocytosis occurring in cultured fibroblasts is microautophagy.

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

SummaryThe effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-d-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes.Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-d-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AVs in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-d-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AVs. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AVs resembled endoplasmic reticulum and still had attached ribosomes.It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.

Cytochemical studies on induced autophagocytosis in mouse exocrine pancreas.

1. The origin of the limiting membranes of autophagic vacuoles (AVs) in mouse pancreatic acinar cells was studied in vinblastine-induced autophagocytosis. 2. The marker enzymes used were adenosine triphosphatase, lipase, inosine diphosphatase and thiamine pyrophosphatase. The following impregnation techniques were used: unbuffered osmium tetroxide impregnation, imidazole-buffered osmium tetroxide impregnation and uranyl-lead-copper impregnation. 3. Only a weak lipase activity was observed between the limiting membranes of a few AVs. The AV membranes were stained heavily with all impregnation techniques used. 4. The origin of AV membranes seems to be same in mouse liver and exocrine pancreas in vinblastine-induced autophagocytosis.