| 1. |
- Borg, Jörgen, et al.
(författare)
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Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.
- 2004
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 114:1-2, s. 21-30
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Tidskriftsartikel (refereegranskat)abstract
- Methods currently used for surface display on insect cells and budded baculovirus, all utilize the sequences from class I transmembrane proteins. This gives rise to some problems when handling unknown genes or cDNAs encoding full-length proteins. First, the stop codon from the cloned gene will be located upstream of the sequence for the transmembrane region. Second, the chance of getting the sequences encoding the signal peptide and the transmembrane region in frame with the cloned gene is small. To minimize these problems, we here present a method by which cDNAs or genes of interest can be cloned and fused to the codons for the signal peptide and transmembrane region of neuraminidase (NA), a class II transmembrane protein of the influenza virus. By placing both the signal peptide and transmembrane region at the amino-terminal, potential problems regarding stop codons are eliminated and errors in frame-shift minimized. To obtain proof of principle, the gene encoding enhanced green fluorescent protein, EGFP, was subcloned into a shuttle vector downstream of the neuraminidase sequence and the fusion product was then transferred to a baculovirus vector and transfected into insect cells (Sf9). Using this method, EGFP was found to be expressed on the surface of both infected cells and budded virus in an accessible manner.
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| 2. |
- Borg, Jörgen, et al.
(författare)
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Perilipin is present in islets of Langerhans and protects against lipotoxicity when overexpressed in the beta-cell line INS-1.
- 2009
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Ingår i: Endocrinology. - : The Endocrine Society. - 0013-7227 .- 1945-7170. ; 150:7, s. 3049-3057
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Tidskriftsartikel (refereegranskat)abstract
- Lipids have been shown to play a dual role in pancreatic beta-cells - a lipid-derived signal appears to be necessary for glucose-stimulated insulin secretion, whereas lipid accumulation causes impaired insulin secretion and apoptosis. The ability of the protein perilipin to regulate lipolysis prompted an investigation of the presence of perilipin in the islets of Langerhans. In this study evidence is presented for perilipin expression in rat, mouse and human islets of Langerhans as well as in the rat clonal beta-cell line INS-1. In rat and mouse islets, perilipin was verified to be present in beta-cells. In order to examine if the development of lipotoxicity could be prevented by manipulating the conditions for lipid storage in the beta-cell, INS-1 cells with adenoviral-mediated overexpression of perilipin were exposed to lipotoxic conditions for 72 hours. In cells exposed to palmitate, perilipin overexpression caused increased accumulation of triacylglycerols and decreased lipolysis compared to control cells. Whereas glucose-stimulated insulin secretion was retained following palmitate exposure in cells overexpressing perilipin, it was completely abolished in control beta-cells. Thus, overexpression of perilipin appears to confer protection against the development of beta-cell dysfunction following prolonged exposure to palmitate by promoting lipid storage and limiting lipolysis.
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| 3. |
- Fernandez, Celine, et al.
(författare)
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Hormone-sensitive lipase is necessary for normal mobilization of lipids during sub-maximal exercise.
- 2008
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Ingår i: American Journal of Physiology: Endocrinology and Metabolism. - : American Physiological Society. - 1522-1555 .- 0193-1849. ; 295, s. 179-186
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Tidskriftsartikel (refereegranskat)abstract
- For the working muscle there are a number of fuels available for oxidative metabolism, including glycogen, glucose and non-esterified fatty acids. Non-esterified fatty acids originate from lipolysis in white adipose tissue, from hydrolysis of VLDL-triglycerides or from hydrolysis of intramyocellular triglyceride stores. A key enzyme in the mobilization of fatty acids from intracellular lipid stores is hormone-sensitive lipase (HSL). The aim of the present study was to investigate the metabolic response of HSL-null mice challenged with exercise or fasting and to examine if other lipases are able to fully compensate for the lack of HSL. The results showed that HSL-null mice have reduced capacity to perform aerobic exercise. The liver glycogen stores were more rapidly depleted in HSL-null mice during treadmill exercise and HSL-null mice had reduced plasma concentrations of both glycerol and non-esterified fatty acids after exercise and fasting, respectively. The data support the hypothesis that in the absence of HSL mice are not able to respond to an exercise challenge with increased mobilization of the lipid stores. Consequently, the impact of the lipid sparing effect on liver glycogen will be reduced in the HSL-null mice, resulting in faster depletion of this energy source, contributing to the decreased endurance during sub-maximal exercise. Key words: Treadmill exercise, lipid metabolism, glycogen, skeletal muscle, liver.
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| 4. |
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| 5. |
- Krintel, Christian, et al.
(författare)
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Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL) isoforms.
- 2010
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependent manner, where the predominant 84 kDa form in adipocytes is the most extensively studied. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed negative stain electron microscopy (EM) to analyze the quarternary structure of the different HSL isoforms. The results show that all three isoforms adopt a head-to-head homodimeric organization, where each monomer contains two structural domains. We also used enzymatic assays to show that despite the variation in the size of the N-terminal domain all three isoforms exhibit similar enzymological properties with regard to psychrotolerance and protein kinase A (PKA)-mediated phosphorylation and activation. CONCLUSIONS/SIGNIFICANCE: We present the first data on the quaternary structure and domain organization of the three HSL isoforms. We conclude that despite large differences in the size of the N-terminal, non-catalytic domain all three HSL isoforms exhibit the same three-dimensional architecture. Furthermore, the three HSL isoforms are very similar with regard to two unique enzymological characteristics of HSL, i.e., cold adaptation and PKA-mediated activation.
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| 6. |
- Mulder, Hindrik, et al.
(författare)
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Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact.
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 278:38, s. 36380-36388
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Tidskriftsartikel (refereegranskat)abstract
- Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic beta-cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of beta-cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.
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| 7. |
- Skoug, Cecilia, et al.
(författare)
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Genetic deletion of hormone-sensitive lipase in mice reduces cerebral blood flow but does not aggravate the impact of diet-induced obesity on memory
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Ingår i: Journal of Neurochemistry. - 0022-3042.
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL) is active throughout the brain and its genetic ablation impacts brain function. Its activity in the brain was proposed to regulate bioactive lipid availability, namely eicosanoids that are inflammatory mediators and regulate cerebral blood flow (CBF). We aimed at testing whether HSL deletion increases susceptibility to neuroinflammation and impaired brain perfusion upon diet-induced obesity. HSL−/−, HSL+/−, and HSL+/+ mice of either sex were fed high-fat diet (HFD) or control diet for 8 weeks, and then assessed in behavior tests (object recognition, open field, and elevated plus maze), metabolic tests (insulin and glucose tolerance tests and indirect calorimetry in metabolic cages), and CBF determination by arterial spin labeling (ASL) magnetic resonance imaging (MRI). Immunofluorescence microscopy was used to determine coverage of blood vessels, and morphology of astrocytes and microglia in brain slices. HSL deletion reduced CBF, most prominently in cortex and hippocampus, while HFD feeding only lowered CBF in the hippocampus of wild-type mice. CBF was positively correlated with lectin-stained vessel density. HSL deletion did not exacerbate HFD-induced microgliosis in the hippocampus and hypothalamus. HSL−/− mice showed preserved memory performance when compared to wild-type mice, and HSL deletion did not significantly aggravate HFD-induced memory impairment in object recognition tests. In contrast, HSL deletion conferred protection against HFD-induced obesity, glucose intolerance, and insulin resistance. Altogether, this study points to distinct roles of HSL in periphery and brain during diet-induced obesity. While HSL−/− mice were protected against metabolic syndrome development, HSL deletion reduced brain perfusion without leading to aggravated HFD-induced neuroinflammation and memory dysfunction.
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| 8. |
- Skoug, Cecilia, et al.
(författare)
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Hormone-sensitive lipase is localized at synapses and is necessary for normal memory functioning in mice
- 2022
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Ingår i: Journal of Lipid Research. - : Elsevier BV. - 1539-7262 .- 0022-2275. ; 63:5
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL) is mainly present in adipose tissue where it hydrolyses diacylglycerol. Although expression of HSL has also been reported in the brain, its presence in different cellular compartments is uncertain, and its role in regulating brain lipid metabolism remains hitherto unexplored. We hypothesized HSL might play a role in regulating the availability of bioactive lipids necessary for neuronal function, and therefore investigated whether dampening HSL activity could lead to brain dysfunction. In mice, we found HSL protein and enzymatic activity throughout the brain, both localized within neurons and enriched in synapses. HSL-null mice were then analyzed using a battery of behavioral tests. Relative to wild-type littermates, HSL-null mice showed impaired short- and long-term memory, yet preserved exploratory behaviurs. Molecular analysis of the cortex and hippocampus showed increased expression of genes involved in glucose utilization in the hippocampus, but not cortex, of HSL-null mice compared to controls. Furthermore, lipidomics analyses indicated an impact of HSL deletion on the profile of bioactive lipids, including a decrease in endocannabinoids and eicosanoids that are known to modulate neuronal activity, cerebral blood flow, and inflammation processes. Accordingly, mild increases in the expression of pro-inflammatory cytokines in HSL mice compared to littermates were suggestive of low-grade inflammation. We conclude that HSL has a homeostatic role in maintaining pools of lipids required for normal brain function. It remains to be tested, however, whether the recruitment of HSL for the synthesis of these lipids occurs during increased neuronal activity, or whether HSL participates in neuroinflammatory responses.
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| 9. |
- Ström, Kristoffer, et al.
(författare)
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Attainment of brown adipocyte features in white adipocytes of hormone-sensitive lipase null mice.
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:3
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored tri- and diglycerides, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. METHODOLOGY/PRINCIPAL FINDINGS: Following a high-fat diet (HFD) regimen, energy expenditure, measured using indirect calorimetry, was increased in HSL null mice. White adipose tissue of HSL null mice was characterized by reduced mass and reduced protein expression of PPARgamma, a key transcription factor in adipogenesis, and stearoyl-CoA desaturase 1, the expression of which is known to be positively correlated to the differentiation state of the adipocyte. The protein expression of uncoupling protein-1 (UCP-1), the highly specific marker of brown adipocytes, was increased 7-fold in white adipose tissue of HSL null mice compared to wildtype littermates. Transmission electron microscopy revealed an increase in the size of mitochondria of white adipocytes of HSL null mice. The mRNA expression of pRb and RIP140 was decreased in isolated white adipocytes, while the expression of UCP-1 and CPT1 was increased in HSL null mice compared to wildtype littermates. Basal oxygen consumption was increased almost 3-fold in white adipose tissue of HSL null mice and was accompanied by increased uncoupling activity. CONCLUSIONS: These data suggest that HSL is involved in the determination of white versus brown adipocytes during adipocyte differentiation The exact mechanism(s) underlying this novel role of HSL remains to be elucidated, but it seems clear that HSL is required to sustain normal expression levels of pRb and RIP140, which both promote differentiation into the white, rather than the brown, adipocyte lineage.
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| 10. |
- Alsted, Thomas J., et al.
(författare)
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Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle
- 2013
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Ingår i: Journal of Physiology. - : Wiley. - 1469-7793 .- 0022-3751. ; 591:20, s. 5141-5155
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Tidskriftsartikel (refereegranskat)abstract
- In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for approximate to 98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.
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