PubMed Health⌕ Search

Biomedical subjects

Cecilia Holm

Publications and source records attributed to Cecilia Holm.

At least 19 recordsLinked to original sources
PubMed2006

Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine.

A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.

Animals↗
PubMed2006

Glucose-stimulated insulin secretion correlates with beta-cell lipolysis.

BACKGROUND AND AIMS: Lipids are needed for optimal glucose-stimulated insulin secretion (GSIS), and long-chain acyl-CoA (LC-CoA) has been suggested as one candidate molecule active as a lipidic coupling factor. LC-CoAs may be available to the beta-cell via uptake of circulating free fatty acids or from hydrolysis of intracellularly stored triglycerides. Inhibition of lipolysis in rat islets using a non-specific lipase inhibitor (orlistat) resulted in blunted GSIS. The aim of this study was to investigate the relationship between GSIS and lipolysis in clonal beta-cells and in mouse islets. METHODS AND RESULTS: INS-1 cells, cultured overnight at 3.3 mM or 11.1 mM glucose, or freshly isolated islets were incubated with 3.3 mM or 16.7 mM glucose for 1 h. Medium samples were collected and analyzed for insulin and glycerol. Triglycerides were measured in both INS-1 cells and islets. There was a dose-dependent glucose-stimulated lipolysis in INS-1 cells, which strongly correlated with insulin secretion (r=0.85, P<0.0001). The same phenomenon was observed in mouse islets (r=0.9, P=0.013). Low levels of triglycerides, which were observed in INS-1 cells pre-cultured at 3.3 mM glucose, were associated with reduced GSIS. CONCLUSIONS: This study suggests that lipids obtained from lipolysis of intracellular triglycerides are involved in GSIS.

Animals↗
PubMed2006

Inflammatory response in white adipose tissue in the non-obese hormone-sensitive lipase null mouse model.

In the present study, a local inflammatory response in white adipose tissue from the nonobese HSL-null mouse model is demonstrated. The protein levels of several well-known markers of inflammation, like TNFalpha and ferritin HC, were highly increased and accompanied by an activation of NFkappaB. A number of macrophage proteins, i.e., gal-3, Capg, and MCP-4, were expressed at increased levels and immunohistochemical analyses revealed an increased infiltration of F4/80+ cells.

Adipose Tissue↗
PubMed2006

Comparison of MS/MS methods for protein identification from 2D-PAGE.

We have compared the use of a low resolution MALDI-Ion Trap MS/MS and a high-resolution ESI-TOF-MS/MS for the analysis of spots from 2D gels. The main criteria were speed and accuracy of protein identification. The results obtained using the MALDI-MS/MS system are comparable to those from the LC-MS/MS system in terms of accuracy, but less low-level proteins are identified while the time required for the analysis is dramatically reduced.

Computational Biology↗
PubMed2005

Transcriptome and proteome analysis of soleus muscle of hormone-sensitive lipase-null mice.

Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization in adipocytes, has been demonstrated also in skeletal muscle. To gain further insight into the role and importance of HSL in skeletal muscle, a transcriptome analysis of soleus muscle of HSL-null mice was performed. A total of 161 transcripts were found to be differentially expressed. Increased mRNA levels of fructose-1,6-bisphosphatase, fructose-2,6-bisphosphatase, and phosphorylase kinase gamma1A suggest a higher glycogen flux in soleus muscle of HSL-null mice. An observed increase in the utilization of glycogen stores supports this finding. Moreover, an increased amount of intramyocellular lipid droplets, observed by transmission electron microscopy, suggests decreased mobilization of lipid stores in HSL-null mice. To complement the transcriptome data, protein expression analysis was performed. Five spots were found to be differentially expressed: pyruvate dehydrogenase E1alpha, creatine kinase (CK), ankyrin-repeat domain 2, glyceraldehyde-3-phosphate dehydrogenase, and one protein yet to be identified. The increased protein level of CK indicates creatine phosphate degradation to be of increased importance in HSL-null mice. The results of this study suggest that in the absence of HSL, a metabolic switch from reliance on lipid to carbohydrate energy substrates takes place, supporting an important role of HSL in soleus muscle lipid metabolism.

Animals↗
PubMed2005

Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting.

Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phosphoenolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was approximately 30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism.

Adaptation, Physiological↗
PubMed2005

Hormone-sensitive lipase as mediator of lipolysis in contracting skeletal muscle.

The authors propose that the enzyme hormone-sensitive lipase (HSL), which is the rate-limiting enzyme for hydrolysis of triacylglycerol in adipocytes, also regulates the intramyocellular triacylglycerol mobilization and is controlled by mechanisms similar to those regulating glycogen phosphorylase. From an exercise perspective, it is fascinating that the primary enzymatic setting allows simultaneous mobilization of all major extramuscular and intramuscular energy stores.

Animals↗
PubMed2005

Adipocyte lipases and defect of lipolysis in human obesity.

The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine- and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine- and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity.

Adipocytes↗
PubMed2004

Marked reutilization of free fatty acids during activated lipolysis in human skeletal muscle.

Release of glycerol and free fatty acids (FFA) was investigated in human skeletal muscle strips. In the basal state, glycerol and FFA were released at almost equimolar rates (0.3 nmol/ng tissue.90 min). A nonselective beta-adrenoceptor agonist, isoprenaline, caused a concentration-dependent stimulation of glycerol release, whereas FFA release was unaffected. Basal and isoprenaline-induced glycerol release correlated positively with the age of the donors (r = 0.5, P < 0.005) but not with their body mass index (P > or = 0.4). Biochemical experiments with hormone-sensitive lipase (HSL) showed that most enzyme activity was both in the cytosol and mitochondrial fraction and that it constituted the common long and active form of the protein. Electron microscopy studies in rat skeletal muscle using labeled highly specific HSL antibodies verified the cytosolic location of HSL and, furthermore, indicated an accumulation of HSL-adjoining mitochondria. These results suggest that FFA produced in myocytes during catecholamine-induced lipolysis are retained by the muscle and, therefore by inference, reused. It is conceivable that efficient hydrolysis of acylglycerol by HSL located in the cytosol as well as near the mitochondria may facilitate mitochondrial FFA oxidation. In addition, muscle lipolysis activity increases during aging and may be independent of total body fat.

Adult↗
PubMed2004

Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.

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.

Amino Acids↗
PubMed2004

Separation of lipids from blood utilizing ultrasonic standing waves in microfluidic channels.

A method to continuously separate different particle types in a suspension is reported. Acoustic forces in a standing wave field were utilized to discriminate lipid particles from erythrocytes in whole blood. The presented technology proposes a new method of cleaning, i.e. removing lipid emboli from, shed blood recovered during cardiac surgery. Blood contaminated with lipid particles enter a laminar flow micro channel. Erythrocytes and lipid particles suspended in blood plasma are exposed to a half wavelength standing wave field orthogonal to the direction of flow as they pass through the channel. Because of differences in compressibility and density the two particle types move in different directions, the erythrocytes towards the centre of the channel and the lipid particles towards the side walls. The end of the channel is split into three outlet channels conducting the erythrocytes to the centre outlet and the lipid particles to the side outlets due to the laminar flow profile. The separation channel was evaluated in vitro using polyamide spheres suspended in water, showing separation efficiencies approaching 100%. The system was also evaluated on whole blood using tritium labelled lipid particles added to bovine blood. More than 80% of the lipid particles could be removed while approximately 70% of the erythrocytes were collected in one third of the original fluid volume. The study showed that the further reduced micro channel dimensions provided improved performance with respect to; (i) separation efficiency, (ii) actuation voltage, and (iii) volumetric throughput as compared to earlier work.

Acoustics↗
PubMed2004

The testicular form of hormone-sensitive lipase HSLtes confers rescue of male infertility in HSL-deficient mice.

Inactivation of the hormone-sensitive lipase gene (HSL) confers male sterility with a major defect in spermatogenesis. Several forms of HSL are expressed in testis. HSLtes mRNA and protein are found in early and elongated spermatids, respectively. The other forms are expressed in diploid germ cells and interstitial cells of the testis. To determine whether the absence of the testis-specific form of HSL, HSLtes, was responsible for the infertility in HSL-null mice, we generated transgenic mice expressing HSLtes under the control of its own promoter. The transgenic animals were crossed with HSL-null mice to produce mice deficient in HSL in nongonadal tissues but expressing HSLtes in haploid germ cells. Cholesteryl ester hydrolase activity was almost completely blunted in HSL-deficient testis. Mice with one allele of the transgene showed an increase in enzymatic activity and a small elevation in the production of spermatozoa. The few fertile hemizygous male mice produced litters of very small to small size. The presence of the two alleles led to a doubling in cholesteryl ester hydrolase activity, which represented 25% of the wild type values associated with a qualitatively normal spermatogenesis and a partial restoration of sperm reserves. The fertility of these mice was totally restored with normal litter sizes. In line with the importance of the esterase activity, HSLtes transgene expression reversed the cholesteryl ester accumulation observed in HSL-null mice. Therefore, expression of HSLtes and cognate cholesteryl ester hydrolase activity leads to a rescue of the infertility observed in HSL-deficient male mice.

Alleles↗
PubMed2004

Hormone-sensitive lipase deficiency in mouse islets abolishes neutral cholesterol ester hydrolase activity but leaves lipolysis, acylglycerides, fat oxidation, and insulin secretion intact.

Lipids are thought to serve as coupling factors in insulin secretion. Hormone-sensitive lipase (HSL) is expressed in pancreatic beta-cells and could potentially regulate insulin secretion via mobilization of stored triglycerides. Here, we examined the impact of HSL deficiency on fuel metabolism and insulin secretion in mouse islets. Lack of HSL resulted in abrogation of neutral cholesterol ester hydrolase activity, whereas diglyceride lipase activity remained intact. Although glucose stimulates lipolysis in rat islets, elevation of glucose with or without addition of cAMP failed to increase lipolysis in mouse islets regardless of genotype, as indicated by release of glycerol from islets. Storage of lipids, assayed as total acylglycerides, was unaltered in HSL null islets, and oxidation of fatty acids or glucose was not different. The intracellular rise in Ca(2+) triggered by glucose and its subsequent oscillations was unaffected in HSL null islets. Accordingly, insulin secretion in static incubations of islets, in response to fuel- and nonfuel secretagogues, was in no instance significantly different between wild-type and HSL null mice. The lacking impact of HSL deficiency on insulin secretion may be attributed to the failure of insulin secretagogues to stimulate lipolysis. Consequently, a regulatory function of lipid mobilization in insulin secretion in the mouse appears unlikely.

Animals↗
PubMed2004

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic beta-cells.

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic beta-cells by a combination of electrophysiological recordings, intracellular Ca(2+) ([Ca(2+)](i)) microfluorimetry and insulin release measurements. Addition of palmitate (1 mm, bound to fatty acid-free albumin) to intact islets exposed to 15 mm glucose increased the [Ca(2+)](i) by approximately 30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca(2+)](i) and insulin release in the presence of tolbutamide and in islets depolarized by high K(+) in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K(+) channels (K(ATP) channels). Palmitate (0.5 mm) augmented exocytosis (measured as an increase in cell capacitance) in single beta-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca(2+) currents rose by approximately 25% following addition of 0.5 mm palmitate, an effect that was abolished in the presence of 10 microm isradipine indicating that the free fatty acid specifically acts on L-type Ca(2+) channels. The actions of palmitate on exocytosis and Ca(2+) currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K(ATP) channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca(2+) currents and an increased size of the RRP.

ATP-Binding Cassette Transporters↗
PubMed2004

Contractions induce phosphorylation of the AMPK site Ser565 in hormone-sensitive lipase in muscle.

Intramyocellular triglyceride is an important energy store which is related to insulin resistance. Mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by epinephrine via PKA and by contractions via PKC and ERK. 5' AMP-activated protein kinase (AMPK) is an intracellular fuel gauge which regulates metabolism. In this study we incubated rat soleus muscle to investigate if AMPK influences HSL during 5min of repeated tetanic contractions. An eightfold increase in AMPK activity was accompanied by a 2.5-fold increase in phosphorylation of the AMPK-site Ser(565) in HSL (p<0.05). Inhibition of PKC by Calphostin C abolished the contraction-mediated HSL activation while HSL-Ser(565) phosphorylation was not reduced. The study indicates that during contractions AMPK phosphorylates HSL in Ser(565), but this phosphorylation is not directly responsible for the contraction-induced activation of HSL.

AMP-Activated Protein Kinases↗
PubMed2004

Particle separation using ultrasound can radically reduce embolic load to brain after cardiac surgery.

BACKGROUND: Microembolism during cardiopulmonary bypass has been suggested as being the predominant cause of neurocognitive disorders after cardiac surgery. Shed blood, normally retransfused into the patient during cardiopulmonary bypass, is a major source of lipid microemboli in the brain capillaries. A newly developed technique based on acoustic standing-wave separation of particles in fluid in microchannels, with the capacity to remove lipid particles in blood, is presented. METHODS: A separator consisting of eight parallel, high-fidelity microfabricated channels was actuated with an ultrasound field to create a standing wave. Three different concentrations of lipid particles (diameter, 0.3 microm) were added to blood samples with increasing hematocrits and introduced into the separator channels to separate lipid particles and erythrocytes. RESULTS: The mean separation rates for lipid particles were 81.9% +/- 7.6% and for erythrocytes 79.8% +/- 9.9%, and both were related to the hematocrit level of the incoming blood sample. The procedure was atraumatic and did not cause hemolysis. CONCLUSIONS: Particle separation by means of an acoustic standing-wave technique can be used for atraumatic and effective removal of lipid particles from blood, with the possible clinical implication of reducing neurocognitive complications after cardiopulmonary bypass.

Animals↗
PubMed2004

Effects of growth hormone on the function of beta-adrenoceptor subtypes in rat adipocytes.

OBJECTIVE: The influence of growth hormone (GH) on the regulation of lipolytic response to specific agonists to beta-adrenoceptors and several post-receptor steps in the lipolytic cascade were investigated. RESEARCH METHODS AND PROCEDURES: Adipose tissues from rats were incubated with or without GH (1.38 nM). After a 24-hour incubation, isolated adipocytes were prepared for different assays. Rats were hypophysectomized. One week after operation, L-thyroxine and hydrocortisone acetate was given to hypophysectomized rats. One group of rats was treated with GH (1.33 mg/kg, daily). After 1 week of hormonal treatment, adipose tissues were removed for different studies. RESULTS: GH treatment increased both basal lipolysis and lipolytic sensitivity to dobutamine and CGP 12177 in adipocytes. The lipolytic sensitivity to terbutaline was not influenced by GH treatment. GH treatment increased the maximal lipolytic response to dobutamine and CGP 12177, but not to terbutaline as determined with absolute values of lipolysis. Forskolin-induced lipolysis was increased by addition of GH to tissues. Moreover, GH treatment resulted in enhanced expression of hormone-sensitive lipase. GH treatment in hypophysectomized rats influenced neither the expressions of G alpha s protein and cholera toxin-catalyzed adenosine diphosphate-ribosylation of G alpha s protein, nor cholera toxin-induced 3',5'-cyclic adenosine monophosphate accumulation. However, the expression of G alpha i protein was decreased after GH treatment. DISCUSSION: These and previous results suggest that GH increases lipolysis in rat adipocytes partly through the beta-adrenergic system, including increases in both beta(1)- and beta(3)-adrenergic receptor function, and partly through enhanced adenylate cyclase function, and expression of hormone-sensitive lipase, perhaps via a decrease in G alpha i protein expression.

Adipocytes↗