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C Holm

Publications and source records attributed to C Holm.

At least 73 records · Page 4Linked to original sources
PubMed1999

Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.

Decreased lipolytic effect of catecholamines in adipose tissue has repeatedly been demonstrated in obesity and may be a cause of excess accumulation of body fat. However, the mechanisms behind this lipolysis defect are unclear. The role of hormone-sensitive lipase was examined using abdominal subcutaneous adipocytes from 34 obese drug-free and otherwise healthy males or females and 14 non-obese control subjects. The enzyme catalyzes the rate-limiting step of the lipolysis pathway. The maximum lipolytic capacity of fat cells was significantly decreased in obesity when measured using either a non-selective beta-adrenergic receptor agonist (isoprenaline) or a phosphodiesterase resistant cyclic AMP analogue (dibutyryl cyclic AMP). Likewise, enzyme activity, protein expression, and mRNA of hormone-sensitive lipase were significantly decreased in adipocytes of obese subjects. The findings were not influenced by age or gender. The data suggest that a decreased expression of hormone-sensitive lipase in subcutaneous fat cells, which in turn causes decreased enzyme function and impaired lipolytic capacity of adipocytes, is present in obesity. Impaired expression of the hormone-sensitive lipase gene might at least in part explain the enzyme defect.

Adipose Tissue↗
PubMed1998

Protein kinase B is expressed in pancreatic beta cells and activated upon stimulation with insulin-like growth factor I.

Protein kinase B (PKB) is involved in signaling to a multitude of important cellular events and is activated by insulin and growth factors, including insulin-like growth factor I (IGF-I). We show here expression of PKB in pancreatic islets and in the beta cell lines HIT-T15, INS-1, and RINm5F. Expression of PKB mRNA and the presence of PKB isoforms (alpha, beta, and gamma) were assessed by Northern blot analysis and RT-PCR, respectively. Antibodies recognizing different parts of PKB isoforms were employed to demonstrate PKB protein expression by immunoblot analysis. By use of immunohistochemistry in rat and mouse pancreatic tissue sections, PKB was localized to predominantly beta cells. Regulation of PKB was examined in INS-1 and RINm5F cells; upon stimulation with IGF-I (5-10 min), PKB was phosphorylated and activated (approximately 3-fold) by a wortmannin-sensitive mechanism, indicating involvement of phosphatidylinositol-3 kinase. The possible participation of PKB in signal transduction pathways modulating cAMP-dependent insulin secretion and in proliferation of beta cells is discussed.

Animals↗
PubMed1998

Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro.

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser --> Ala mutations in residues 563 and 565 (S563A, S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Adipocytes↗
PubMed1998

Human hormone-sensitive lipase: expression and large-scale purification from a baculovirus/insect cell system.

Hormone-sensitive lipase (HSL) is a key enzyme in lipid metabolism and overall energy homeostasis in mammals. It catalyzes the rate-limiting step in the hydrolysis of triglyceride stores in the adipocytes, delivering free fatty acids for their use as energy substrates. HSL activity is under acute hormonal and neural control, mediated through reversible phosphorylation of the enzyme. Emerging data from clinical studies indicate that HSL deficiency or malfunction is associated with several pathological situations in humans. In order to perform a biochemical characterization of human HSL, and to elucidate its molecular properties, purification of homogeneous protein in large amounts is required. Here, we describe the expression and purification of a catalytically active recombinant human HSL. The process allows the purification of milligram amounts of homogeneous protein, and should provide a valuable tool for a thorough molecular characterization of the enzyme.

Amino Acid Sequence↗
PubMed1998

Hormone-sensitive lipase (HSL) expression and regulation in skeletal muscle.

Because the enzymatic regulation of muscle triglyceride metabolism is poorly understood we explored the character and activation of neutral lipase in muscle. Western blotting of isolated rat muscle fibers demonstrated expression of hormone-sensitive lipase (HSL). In incubated soleus muscle epinephrine increased neutral lipase activity by beta-adrenergic mechanisms involving cyclic AMP-dependent protein kinase (PKA). The increase was paralleled by an increase in glycogen phosphorylase activity and could be abolished by antiserum against HSL. Electrical stimulation caused a transient increase in activity of both neutral lipase and glycogen phosphorylase. The increase in lipase activity during contractions was not influenced by sympathectomy or propranolol. Training diminished the epinephrine induced lipase activation in muscle but enhanced the activation as well as the overall concentration of lipase in adipose tissue. In agreement with the in vitro findings, in adrenalectomized patients an increase in muscle neutral lipase activity was found at the end of prolonged exercise only if epinephrine was infused. In accordance with feedforward regulation of substrate mobilization in exercise, our studies have shown that HSL is present in skeletal muscle cells and is stimulated in parallel with glycogen phosphorylase by both epinephrine and contractions. HSL adapts differently to training in muscle compared with adipose tissue.

Animals↗
PubMed1998

The putative role of the hormone-sensitive lipase gene in the pathogenesis of Type II diabetes mellitus and abdominal obesity.

Impaired lipolysis has been proposed as a pathogenic factor contributing to clustering of abdominal obesity and dyslipidaemia in Type II (non-insulin-dependent) diabetes mellitus--that is, the metabolic syndrome (MSDR). As this syndrome clusters in families, alterations in the hormone-sensitive lipase (HSL) gene could contribute to the genetic predisposition to MSDR. To test this hypothesis we carried out population and intrafamily association studies in individuals with MSDR, using a polymorphic marker (LIPE) in the HSL gene. There was a significant difference in allele frequency distribution between 235 Type II diabetic patients and 146 control subjects (p = 0.002), particularly between 78 abdominally obese Type II diabetic patients with MSDR and the control group (p = 0.010). An extended transmission disequilibrium test (TDT) showed transmission disequilibrium of 66 alleles to 42 nondiabetic, abdominally obese offspring in families with Type II diabetes (p < 0.05). A slight difference in allele frequency distribution was seen between 71 individuals from the lowest and 71 from the highest tertile of isoprenaline-induced lipolysis in fat tissue (p = 0.07). No missense mutations were found with single-strand conformational polymorphism (SSCP) in 20 abdominally obese subjects with MSDR. In conclusion, our population and intrafamily association studies suggest that the LIPE marker in the HSL gene is in linkage disequilibrium with an allele and/or gene which increases susceptibility to abdominal obesity and thereby possibly to Type II diabetes.

Abdomen↗
PubMed1998

Hypoxaemia and myocardial ischaemia during colonoscopy.

BACKGROUND: Myocardial ischaemia (defined as ST-segment deviation on electrocardiogram (ECG)) may occur during colonoscopy, but the pathogenic mechanisms are unknown. We have evaluated the occurrence of arterial hypoxaemia, tachycardia, and myocardial ischaemia during routine colonoscopy. METHODS: Eighteen patients underwent colonoscopy under conscious sedation and without supplementary oxygen. Arterial oxygen saturation was measured by continuous pulse oximetry, and ECG was monitored continuously with a Holter tape recorder during the procedure. RESULTS: Arterial oxygen desaturation and tachycardia were common during colonoscopy and occurred in 45% and 35% of patients, respectively. Two patients developed signs of myocardial ischaemia during the colonoscopy: one case of ST depression (1.7 mV) and one case of ST elevation (4.3 mV). In both patients the ST deviation disappeared when the colonoscope was retracted. Myocardial ischaemia occurred in both patients simultaneously with tachycardia, and in one of these arterial hypoxaemia was also present. CONCLUSIONS: Myocardial ischaemia occurs during routine colonoscopy, but with a lower incidence than previously reported during upper endoscopy. Myocardial ischaemia during colonoscopy may be associated with tachycardia and/or hypoxaemia. Further studies should clarify the relative role of tachycardia, hypoxaemia, and viscerocardiac reflexes in the pathogenesis of myocardial ischaemia during colonoscopy.

Adult↗
PubMed1998

Effects of occlusive and conventional gauze dressings on incisional healing after abdominal operations.

OBJECTIVE: To compare the effect of occlusive (Comfeel) and conventional (Mepore) dressings on the healing of incisional wounds after abdominal operations. DESIGN: Prospective randomised study. SETTING: Laboratory and teaching hospital, Denmark. SUBJECTS: 73 patients who underwent clean operations requiring incisions longer than 5 cm between August 1993 and August 1995 were randomised to have their wounds dressed with either Comfeel or Mepore. INTERVENTIONS: Comfeel was left on until the sutures were removed, and Mepore was removed 2 days postoperatively. MAIN OUTCOME MEASURES: Infection, adherence, leakage, and cosmetic appearance three months later. RESULTS: 36 patients were randomised to have Comfeel and 37 to have Mepore. 29 patients were withdrawn from the study (20 having Mepore and 9 having Comfeel) leaving 26, and 17 for analysis, respectively. Wound infections developed in 1 patient in the Comfeel group and 5 in the Mepores group (p = 0.2). There were no differences between the groups regarding the need for dressings to be changed, the incidence of leakage, or loosening of the dressing from the skin. Comfeel adhered securely to the skin and remained more or less transparent until sutures were removed. It remained totally transparent in 23 (64%), and no dressing became totally opaque. There were no differences in cosmetic appearance after three months. We had the impression that patients who had Comfeel were more comfortable and found it easier to mobilise and carry out their daily activities. CONCLUSION: Occlusive dressings stay in place and stay transparent, and do not increase the risk of wound infection. They may even be more comfortable. they are a reasonable alternative to conventional dressings.

Abdomen↗
PubMed1998

The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumulate small single-stranded DNA fragments during DNA synthesis.

To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.

Cell Division↗
PubMed1998

Ribotype diversity of Actinomyces with similar intraoral tropism but different types of N-acetyl-beta-D-galactosamine binding specificity.

Sixty-three isolates of Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus from three subjects clustered into 22 ribotypes. Unique ribotypes were found in the subjects and within individual tissue sites (bucca, tooth and tongue). A odontolyticus ribotypes shared tongue-specific binding properties, while those of genospecies 1 and 2 from buccal and tooth surfaces shared different types of N-acetyl-beta-D-galactosamine binding specificity.

Acetylgalactosamine↗
PubMed1998

Actinomyces naeslundii displays variant fimP and fimA fimbrial subunit genes corresponding to different types of acidic proline-rich protein and beta-linked galactosamine binding specificity.

Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to beta-linked galactosamine (GalNAcbeta) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcbeta, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) and fimA (type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral Actinomyces isolates. Isolates of genospecies 1 and 2 and A. odontolyticus, but no other Actinomyces species, were positive for hybridization with fimP and fimA full-length probes irrespective of binding to APRPs and statherin, GalNAcbeta, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcbeta1-3Galalpha-O-ethyl-inhibitable coaggregation with Streptococcus oralis Ss34 and MPB1, were distinguished by a fimA central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a fimP central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of fimP and fimA central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion, A. naeslundii exhibits variant fimP and fimA genes corresponding to diverse APRP and GalNAcbeta specificities, respectively, while A. odontolyticus has a genetically related but distinct adhesin binding specificity.

Actinomyces↗
PubMed1998

Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells.

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. The activity of HSL is thought to be primarily regulated by reversible phosphorylation. However, the regulation of HSL activity by pre-translational mechanisms has been poorly studied. The present studies were undertaken to explore the relationship between the levels of HSL protein and mRNA expressions and the lipolytic capacity. The study was performed in human abdominal subcutaneous adipocytes with identical sizes but having either a high (HL) or low (LL) lipolytic capacity (n = 16). Basal and maximal lipolysis induced by catecholamines, an adenylyl cyclase activator forskolin, and a cyclic AMP analogue dibutyryl cAMP were 50% lower in LL- in comparison with HL-fat cells (P < 0.05 or better). No differences in drug sensitivity were found. HSL activity and quantity were about 50% lower in LL- compared with HL-fat cells (P < 0.05). Moreover, the mRNA ratio between HSL and gamma-actin was 35% lower in LL- compared with HL-fat cells (P < 0.05). There was a strong linear correlation between the protein and enzymatic HSL measurements (r2 = 0.91). In addition, the maximum lipolytic capacity was significantly correlated with HSL activity (r2 = 0.75) and HSL protein amount (r2 = 0.64). It is concluded that hormone-sensitive lipase (HSL) expression, measured either as total HSL protein by Western blot analysis or as total amount of activatable HSL enzyme, is a major determinant of the maximum lipolytic capacity of human fat cells. In addition, HSL protein expression is at least, in part, determined by HSL mRNA expression.

Adipocytes↗
PubMed1997

Characterization of the promoter of human adipocyte hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

Adipocytes↗
PubMed1997

Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.

Alternative Splicing↗
PubMed1997

cDNA cloning, tissue distribution, and identification of the catalytic triad of monoglyceride lipase. Evolutionary relationship to esterases, lysophospholipases, and haloperoxidases.

Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.

Adipocytes↗
PubMed1997

Partial purification and identification of hormone-sensitive lipase from chicken adipose tissue.

HSL from chicken adipose tissue exhibits remarkable activation upon phosphorylation with cAMP-dependent protein kinase (cAMP-PK) compared to HSL from rat and human adipose tissue. In order to characterize the chicken HSL enzyme, it was purified 3500 fold from a chicken adipose tissue homogenate using pH 5.2 precipitation and anion-exchange chromatography. The purified chicken HSL was identified as an 86 kDa protein using Western blot analysis. The HSL diacylglycerol lipase activity was inhibited by 98% upon incubation with anti-rat HSL antiserum, and the specific activity of chicken HSL was estimated to be approximately the same as for the rat enzyme. Furthermore, the 86 kDa polypeptide was phosphorylated by cAMP-PK to about the same stoichiometry as for the recombinant rat enzyme. Hence, our results demonstrate that HSL from chicken adipose tissue is comparable in size and specific activity to HSL from mammalian species, and not a smaller 42 kDa polypeptide with 1000-fold lower specific activity as previously reported (Berglund, L., Khoo, J. C., Jensen, D., and Steinberg, D., 1980 J. Biol. Chem. 255, 5420-5428).

Adipose Tissue↗
PubMed1997

Identification of essential aspartic acid and histidine residues of hormone-sensitive lipase: apparent residues of the catalytic triad.

It is expected that hormone-sensitive lipase (HSL), like most other lipases and esterases, adopts an alpha/beta-hydrolase fold and has a catalytic triad of serine, aspartic or glutamic acid, and histidine. Recently, we have published a three-dimensional model for the C-terminal catalytic domain of HSL, having an alpha/beta-hydrolase fold and with Ser-423(1), Asp-703 and His-733 in the catalytic triad (Contreras et al. (1996) J. Biol. Chem. 271, 31426-31430). It has been shown that Ser-423, situated in the motif GXSXG, is essential for catalysis (Holm et al. (1994) FEBS Lett. 344, 234-238). The suggested aspartic acid and histidine were here probed by site-directed mutagenesis. Mutants of residues Asp-703 and His-733 are devoid of both lipase and esterase activity, which is not the case for mutants of other tested aspartic acid and histidine residues. Thus, the presented data support the three-dimensional model structure with Asp-703 and His-733 as part of the traid.

Animals↗