Intracellular Complement Component 3 Attenuated Ischemia-Reperfusion Injury in the Isolated Buffer-Perfused Mouse Heart and Is Associated With Improved Metabolic Homeostasis.

The innate immune system is rapidly activated during myocardial infarction and blockade of extracellular complement system reduces infarct size. Intracellular complement, however, appears to be closely linked to metabolic pathways and its role in ischemia-reperfusion injury is unknown and may be different from complement activation in the circulation. The purpose of the present study was to investigate the role of intracellular complement in isolated, retrogradely buffer-perfused hearts and cardiac cells from adult male wild type mice (WT) and from adult male mice with knockout of complement component 3 (C3KO). Main findings: (i) Intracellular C3 protein was expressed in isolated cardiomyocytes and in whole hearts, (ii) after ischemia-reperfusion injury, C3KO hearts had larger infarct size (32 ± 9% in C3KO vs. 22 ± 7% in WT; p=0.008) and impaired post-ischemic relaxation compared to WT hearts, (iii) C3KO cardiomyocytes had lower basal oxidative respiration compared to WT cardiomyocytes, (iv) blocking mTOR decreased Akt phosphorylation in WT, but not in C3KO cardiomyocytes, (v) after ischemia, WT hearts had higher levels of ATP, but lower levels of both reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) compared to C3KO hearts. Conclusion: intracellular C3 protected the heart against ischemia-reperfusion injury, possibly due to its role in metabolic pathways important for energy production and cell survival.

Critical COVID-19 is associated with distinct leukocyte phenotypes and transcriptome patterns.

BACKGROUND: Prognostic markers for disease severity and identification of therapeutic targets in COVID-19 are urgently needed. We have studied innate and adaptive immunity on protein and transcriptomic level in COVID-19 patients with different disease severity at admission and longitudinally during hospitalization. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected at three time points from 31 patients included in the Norwegian SARS-CoV-2 cohort study and analysed by flow cytometry and RNA sequencing. Patients were grouped as either mild/moderate (n = 14), severe (n = 11) or critical (n = 6) disease in accordance with WHO guidelines and compared with patients with SARS-CoV-2-negative bacterial sepsis (n = 5) and healthy controls (n = 10). RESULTS: COVID-19 severity was characterized by decreased interleukin 7 receptor alpha chain (CD127) expression in naïve CD4 and CD8 T cells. Activation (CD25 and HLA-DR) and exhaustion (PD-1) markers on T cells were increased compared with controls, but comparable between COVID-19 severity groups. Non-classical monocytes and monocytic HLA-DR expression decreased whereas monocytic PD-L1 and CD142 expression increased with COVID-19 severity. RNA sequencing exhibited increased plasma B-cell activity in critical COVID-19 and yet predominantly reduced transcripts related to immune response pathways compared with milder disease. CONCLUSION: Critical COVID-19 seems to be characterized by an immune profile of activated and exhausted T cells and monocytes. This immune phenotype may influence the capacity to mount an efficient T-cell immune response. Plasma B-cell activity and calprotectin were higher in critical COVID-19 while most transcripts related to immune functions were reduced, in particular affecting B cells. The potential of these cells as therapeutic targets in COVID-19 should be further explored.

Five-year mortality in patients treated for severe community-acquired pneumonia - a retrospective study.

BACKGROUND: The mortality rate in patients with severe community-acquired pneumonia (SCAP) is high. We investigated the 5-year mortality rate and causes of death in a patient population treated for SCAP in our intensive care unit (ICU), and compared the mortality rate in patients with or without chronic obstructive pulmonary disease (COPD) as comorbidity. METHODS: This retrospective study, which covers a period of 10 years, included patients aged > 18 years admitted to our ICU with SCAP as primary diagnosis and in need of mechanical ventilation for more than 24 h. Data were collected from the ICU internal database and the patients' medical records. The times of death were collected from the Norwegian National Registry, and the causes of death from the Norwegian Cause of Death Registry. RESULTS: Hundred and seventy three patients were included in the study. The 5-year mortality rate for the total study population was 57.2%. There were no significant differences in the mortality rate between the group with COPD and the group without COPD (61.2% vs. 54.7%, P = 0.43). There was a wide range of comorbidities. The most common were COPD, myocardial infarction and diabetes mellitus. The two main causes of death after discharge were COPD (17 deaths) and cardiovascular diseases (seven deaths). CONCLUSIONS: The 5-year mortality rate of the study population was high (57.2%). COPD did not seem to be a risk factor for mortality compared to non-COPD patients. The most common causes of death after discharge were COPD and cardiovascular diseases.

NLRP3 inflammasome activation during myocardial ischemia reperfusion is cardioprotective.

BACKGROUND: The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1ß mediates release of bioactive IL-1ß. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. METHODS AND RESULTS: The left coronary artery (LCA) of WT, NLRP3(-/-) and ASC(-/-) mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. CONCLUSION: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

Twenty-two years of candidaemia surveillance: results from a Norwegian national study.

Several studies have reported an increased incidence of candidaemia and a redistribution of species, with a decrease in the number of Candida albicans isolates. In Norway, a prospective, national surveillance study of candidaemia has been ongoing since 1991. Data from the period 1991-2003 have been published previously. The aim of this study was to follow up the incidence, species distribution and antifungal susceptibility of Candida species isolates from blood cultures in the period 2004-2012, and compare them with the corresponding findings from the period 1991-2003. Blood culture isolates of Candida species from all medical microbiological laboratories in Norway were identified and susceptibility tested at the Norwegian Mycological Reference Laboratory. A total of 1724 isolates were recovered from 1653 patients in the period 2004-2012. Comparison of the two periods showed that the average incidence of candidaemia episodes per 100 000 inhabitants increased from 2.4 (1991-2003) to 3.9 (2004-2012). The increase in incidence in the latter period was significantly higher in patients aged >40 years (p 0.001), and a marked increase was observed in patients aged >60 years (p < 0.001). In conclusion, the average incidence in Norway over a period of 22 years modestly increased from 2.4 to 3.9 per 100,000 inhabitants, this being mainly accounted for by candidaemia in the elderly. The species distribution was stable, and the rate of acquired resistance was low.

Characterization of novel mutations in the catalytic domain of the PCSK9 gene.

OBJECTIVES: To expand our understanding of the structure and function of proprotein convertase subtilisin/kexin type 9 (PCSK9) by studying how naturally occurring mutations in PCSK9 disrupt the function of PCSK9. DESIGN: Mutations in PCSK9 were identified by sequencing of DNA from subjects with hypo- or hypercholesterolemia. The effect of the identified mutations on the autocatalytic cleavage and secretion of PCSK9, as well as the effect on PCSK9-mediated degradation of the low density lipoprotein receptors, were determined in HepG2 or HEK293 cells transiently transfected with mutant PCSK9-containing plasmids. The findings were collated to the clinical characteristics of the subjects possessing these mutations, and the phenotypic effects were analysed in terms of available structural data for PCSK9. RESULTS: Five novel mutations in PCSK9 were identified. Mutation R215H was a gain-of-function mutation which causes hypercholesterolemia. Mutation G236S and N354I were loss-of-function mutations due to failure to exit the endoplasmic reticulum or failure to undergo autocatalytic cleavage, respectively. Mutations A245T and R272Q were most likely normal genetic variants. By comparing the number of patients with gain-of-function mutations in PCSK9 with the number of familial hypercholesterolemia heterozygotes among subjects with hypercholesterolemia, the prevalence of subjects with gain-of-function mutations in PCSK9 in Norway can be estimated to one in 15,000. CONCLUSION: This study has provided novel information about the structural requirements for the normal function of PCSK9. However, more studies are needed to determine the mechanisms by which gain-of-function mutations in PCSK9 cause hypercholesterolemia.

Low-density lipoprotein receptor activity in Epstein-Barr virus-transformed lymphocytes from heterozygotes for the D374Y mutation in the PCSK9 gene.

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have been found to cause autosomal dominant hypercholesterolemia. The objective of this study was to investigate possible mechanisms by which mutation D374Y in the PCSK9 gene causes hypercholesterolemia. MATERIAL AND METHODS: Binding and internalization of low-density lipoprotein LDL in Epstein-Barr virus (EBV)-transformed lymphocytes from D374Y heterozygotes were examined. The autocatalytic activity of the D374Y mutant was studied in transiently transfected HEK293 cells. RESULTS: As determined by Western blot analysis of transiently transfected HEK293 cells, the autocatalytic activity of the D374Y mutant was approximately 95% of the wild-type. Levels of PCSK9 mRNA in EBV-transformed lymphocytes from D374Y heterozygotes and normal controls were similar and less than 1/1000 of the level in HepG2 cells. The amount of cell surface LDL receptors (LDLRs) in EBV-transformed lymphocytes from five D374Y heterozygotes was non-significantly increased by 17% compared with the amount in normal controls. LDLR-dependent binding and internalization of LDL in EBV-transformed lymphocytes from D374Y heterozygotes were non-significantly reduced by 11% and 12%, respectively, compared to the corresponding values in normal controls. CONCLUSIONS: LDLR-mediated endocytosis of LDL is not reduced in EBV-transformed lymphocytes from D374Y heterozygotes. Because of the extremely low levels of PCSK9 mRNA in EBV-transformed lymphocytes, it is possible that the LDLR-dependent endocytosis of LDL could be more severely affected in hepatocytes from D374Y heterozygotes than in EBV-transformed lymphocytes.

Augmented PLA2 activity in pre-eclamptic decidual tissue--a key player in the pathophysiology of 'acute atherosis' in pre-eclampsia?

Decidual acute atherosis is associated with pre-eclampsia, but the underlying mechanism is still unclear. We have previously demonstrated elevated level of the oxidative stress marker 8-isoprostaglandin F(2 alpha)(8-isoprostane) and lipids in pre-eclamptic decidual tissue. Arachidonic acid (AA) in tissue phospholipids is a source for 8-isoprostane generation, and 8-isoprostane is liberated from tissue phospholipids by phospholipase A(2)(PLA(2)). The aims of this study were to explore whether AA content or PLA(2)expression in pre-eclamptic decidual tissue differed from controls. Decidua basalis tissues were obtained by vacuum aspiration at Caesarean delivery in pre-eclamptic and control pregnancies. We demonstrated a statistically significantly higher total PLA(2)activity in pre-eclamptic decidua compared to control tissue. On the other hand, no differences in AA content of tissue phospholipids or protein expression of secretory and cytosolic PLA(2)between pre-eclamptic and control decidual tissue were found. In conclusion, the elevated level of free 8-isoprostane in pre-eclamptic decidual tissue could be caused by augmented PLA(2)activity. We speculate that an elevated PLA(2)enzyme activity in pre-eclamptic decidual tissue could be of importance in the pathogenesis of 'acute atherosis', comparable to the atherogenesis in cardiovascular diseases.

Experimental infection of Atlantic halibut Hippoglossus hippoglossus with nodavirus: tissue distribution and immune response.

Atlantic halibut Hippoglossus hippoglossus, age 8 mo and weighing 20 g, were challenged by either intraperitoneal injection (i.p.) or by bath exposure using nodavirus isolated from Atlantic halibut. Fish were sampled at intervals over a 41 d period, starting on Day 5 post-challenge. Although no clinical disease or mortality was recorded, the data show that nodavirus did successfully propagate in i.p.-challenged fish. Using conventional end-point reverse transcription (RT)-PCR, nodavirus was detected in the kidney of all examined i.p.-challenged fish, and further in the head, heart, liver and posterior intestine of most of these individuals. Quantitative real-time RT-PCR revealed that the amount of virus in head samples from the i.p.-challenged group increased during the experiment. The presence of nodavirus in nervous tissue of i.p.-challenged fish was detected by immunohistochemistry from Day 13 post-challenge. In the retina, virus positive cells were found adjacent to the circumferential germinal zone at the ciliary margin towards the iris. In the brain, a few positive cells were detected in the tectum opticum. An ELISA was developed to detect anti-nodavirus activity in plasma. The method included an optimized coating procedure, which allowed the use of non-purified nodavirus as the coating antigen in a simple indirect ELISA. An anti-nodavirus antibody response was detected from Day 19 post-challenge in i.p.-challenged fish, while a response was not detected in the bath-challenged or control fish. This experiment demonstrates a subclinical nodavirus infection in Atlantic halibut at a post-juvenile stage induced by i.p. injection of virus.

Pathological changes in juvenile Atlantic halibut Hippoglossus hippoglossus persistently infected with nodavirus.

This is the first description of a persistent subclinical nodavirus infection in the Atlantic halibut Hippoglossus hippoglossus. Juvenile fish (1 to 5 g) were sampled at 4, 5 and 8 mo of age at a fish farm in Norway during and after weaning. None showed clinical signs of viral encephalopathy and retinopathy (VER) or other disease. Pathological changes and/or nodavirus were detected by light microscopy, immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy in all fish examined. High numbers of virus particles were found in macrophage-like cells in the central nervous system, including brain and retina (CNS). The virus particles displayed the icosahedral shape and size (approximately 25 nm) characteristic of nodaviruses. The virus-infected cells formed focal cell aggregates and were seen in all regions of the brain and all nuclear cell layers of the retina. The cytoplasm of the infected cells was filled with membrane-enclosed inclusions packed with virus particles. Some virus particles lay along membranes and formed membrane-bound necklace-like arrangements. The virus-infected cells of the retina also contained pigment granula located generally inside virus inclusions and sometimes forming a coating around the virus particles. All frontal parts with the eyes and brain and 50% of the mid-parts, which included the abdominal organs, were found positive for nodavirus with RT-PCR. Pathological changes in these persistently nodavirus-infected fish differ from earlier descriptions in Atlantic halibut during outbreaks of VER. Vertical transmission from infected spawners is believed to be a major route for nodavirus infection. Detection of nodavirus in subclinical infected fish and a better understanding of its pathogenesis are important in order to prevent the spread of nodavirus in the fish-farming industry.

8-iso-prostaglandin F(2alpha) increases expression of LOX-1 in JAR cells.

Lectinlike oxidized LDL receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL (ox-LDL), is proposed to be involved in endothelial dysfunction and in the pathogenesis of atherosclerosis. Preeclampsia is a pregnancy complication diagnosed by hypertension and proteinuria, characterized by endothelial dysfunction, and supposedly caused by compounds from hypoxic uteroplacental tissues. A feature of preeclampsia is formation of foam cells in maternal arterial walls of gestational tissue ("acute atherosis"). Oxidative stress is believed to play a role in the pathophysiology of preeclampsia. 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) is a marker of oxidative stress in vivo, is biologically active in vitro, and is elevated in preeclamptic plasma and gestational tissue. In the present article, we hypothesized that 8-iso-PGF(2alpha) could induce the expression of LOX-1 in trophoblastic cells (JAR). We demonstrated augmented cellular uptake of (125)I-tyraminylcellobiose ox-LDL in JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L) versus control cells. Ligand blots revealed an increased binding of ox-LDL to LOX-1 in JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L). Incubation with 8-iso-PGF(2alpha) (10 micromol/L) also resulted in augmented LOX-1 protein levels (Western blots) and mRNA levels (Northern blots). JAR cells transfected with 3 copies of a nuclear factor-kappaB binding site demonstrated dose-dependent activation of the reporter gene luciferase after incubation with 8-iso-PGF(2alpha) (0 to 10 micromol/L). We also demonstrated increased accumulation of neutral fats in JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L) and ox-LDL compared with controls by oil red O staining. We speculate a potential role of isoprostanes and LOX-1 in preeclampsia in the development of "acute atherosis" of gestational spiral arteries.

VEGF mRNA is unaltered in decidual and placental tissues in preeclampsia at delivery.

BACKGROUND: VEGF (vascular endothelial growth factor) is a growth factor which is central in blood vessel formation. We wanted to determine whether the mRNA levels of VEGF are altered in preeclampsia in decidual and placental tissues compared to control pregnancies. METHODS: Decidua basalis was obtained by vacuum aspiration during cesarean section in 25 preeclamptic and 19 uneventful pregnancies. Placental biopsies were taken immediately after delivery. Relative mRNA levels of VEGF were quantified and compared to those of the housekeeping gene beta-actin. RESULTS: No statistically significant differences in VEGF mRNA levels were found between the preeclampsia and the control group for either the decidual or placental tissues. Furthermore, there were no significant differences between VEGF mRNA levels in the preeclamptic and control group with respect to gestational age of the women. CONCLUSIONS: This study provides no evidence of abnormal VEGF expression at delivery in decidua basalis or placenta in pregnancies complicated by preeclampsia.

8-Iso-prostaglandin f(2alpha) reduces trophoblast invasion and matrix metalloproteinase activity.

Preeclampsia is a common pregnancy complication in the latter half of gestation diagnosed by hypertension and proteinuria. A key feature of preeclampsia is an altered placentation with reduced trophoblast invasion. Normal placentation requires controlled invasion of trophoblasts into the maternal uterine wall, with secretion of specific proteolytic enzymes able to degrade basement membranes and extracellular matrix, such as the matrix metalloproteinases (MMPs). 8-Iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) is a marker of oxidative stress in vivo and is biologically active. We have recently reported an elevated content of free 8-iso-PGF(2alpha) in preeclamptic gestational tissue at delivery. Assuming an elevated level of 8-iso-PGF(2alpha) during the invasion period of the pregnancy, we hypothesized that 8-iso-PGF(2alpha) could reduce invasion of JAR cells, a choriocarcinoma cell line. We investigated JAR cell invasion with 2 types of Transwell assays and demonstrated that 8-iso-PGF(2alpha) (10 micromol/L) resulted in reduced cell invasion in both the colorimetric and radioactivity Transwell assays (P<0.01). Zymograms revealed reduced MMP-2 and MMP-9 activity in conditioned media from JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L) (P<0.02). 8-Iso-PGF(2alpha) (10 micromol/L) also reduced the collagenase type IV activity in the conditioned media of JAR cells (P=0.04). No effects on MMP-2 and MMP-9 mRNA levels were observed after incubation with 8-iso-PGF(2alpha) (10 micromol/L), whereas protein levels were significantly decreased (P<0.02), suggesting a posttranscriptional regulation. We hypothesize a potential role for 8-iso-PGF(2alpha) in the reduced trophoblast invasion in preeclampsia.

Elevated level of free 8-iso-prostaglandin F2alpha in the decidua basalis of women with preeclampsia.

OBJECTIVES: The prostaglandin-like compound 8-iso-prostaglandin F(2alpha) represents an index of oxidative stress and has the ability to induce endothelial derangement, platelet activation, and vasoconstriction. In women with preeclampsia the decidual spiral arteries contain lipid deposits (acute atherosis). Analogously to the elevated level of 8-iso-prostaglandin F(2alpha) demonstrated in atherosclerotic lesions, we hypothesized that 8-iso-prostaglandin F(2alpha) level would be elevated in preeclamptic decidua basalis tissues. STUDY DESIGN: Decidua basalis tissues were obtained by vacuum aspiration and placental tissues were obtained by excision at cesarean delivery from 16 preeclamptic and 15 normal pregnancies. Total and free 8-iso-prostaglandin F(2alpha) concentrations were quantified with an enzyme immunoassay technique after lipid extraction and separation. RESULTS: The content of free 8-iso-prostaglandin F(2alpha) in preeclamptic decidual tissues was found to be significantly elevated with respect to that in control tissues. The content of total 8-iso-prostaglandin F(2alpha) did not differ significantly between the groups in either placenta or decidua basalis. CONCLUSIONS: We propose that free 8-iso-prostaglandin F(2alpha) released from the decidua basalis in preeclampsia may mediate maternal vascular dysfunction and platelet activation.

Stimulation of serglycin and CD44 mRNA expression in endothelial cells exposed to TNF-alpha and IL-1alpha.

Serglycin is a widely distributed proteoglycan, previously assumed to be hematopoietic cell specific. However, the results presented show that serglycin mRNA is expressed outside the hematopoietic cell system. High levels of serglycin mRNA were detected in endothelial cells and smooth muscle cells, whereas low levels were detected in skin fibroblasts. To further analyze the importance of serglycin in endothelial cells, the expression of serglycin mRNA was measured following activation of an endothelial cell line derived from human umbilical cord vein (HUV-EC-C), by the proinflammatory cytokines TNF-alpha and IL-1alpha. The level of serglycin mRNA increased in a time- and dose-dependent way. TNF-alpha (7 ng/ml) was the most potent inducer, increasing the level of serglycin mRNA 2.5 times after 24 h of stimulation. Serglycin has been shown to be a ligand for CD44, a membrane protein expressed in endothelial cells. Following stimulation of the endothelial cells, the level of CD44 mRNA also increased. Again, TNF-alpha (7 ng/ml) turned out to be the most potent inducer, increasing the level of CD44 mRNA 5.5 times after 24 h of stimulation. Both TNF-alpha and IL-1alpha stimulation of the endothelial cells resulted in an increase in the total incorporation of [(35)S]sulfate into macromolecules, which probably indicates an increase in the total production of proteoglycans. A stimulation of endothelial cells by proinflammatory agents resulted in an increase in both serglycin and CD44 mRNA expression, indicating that serglycin, as well as CD44, may participate in the inflammatory process of leukocyte migration.

Increased contents of phospholipids, cholesterol, and lipid peroxides in decidua basalis in women with preeclampsia.

OBJECTIVES: Accelerated recovery from preeclampsia has been reported after postpartum curettage. Lipid deposition in decidual spiral arteries (acute atherosis) is a histologic feature of preeclampsia. Increased tissue content of lipids is associated with enhanced formation of lipid peroxides, which are compounds that may induce endothelial dysfunction. We hypothesized that the content of lipids and lipid peroxides is elevated in decidua basalis tissues of women with preeclampsia compared with those of women with uneventful pregnancies. STUDY DESIGN: Decidua basalis tissues were obtained with a vacuum aspiration technique during cesarean delivery from 30 preeclamptic and 34 uneventful pregnancies. Total cholesterol, phospholipids, triglycerides, free fatty acids, and lipid peroxides were quantified. RESULTS: Significantly elevated contents of phospholipids, total cholesterol, and lipid peroxides were found in preeclamptic decidua basalis tissues, whereas the contents of triglycerides and free fatty acids did not differ significantly from those of the control group. CONCLUSIONS: Decidua basalis tissues, with their elevated lipid content, may be a source of lipid compounds that can cause maternal endothelial dysfunction in preeclampsia.

Effect of a coffee lipid (cafestol) on cholesterol metabolism in human skin fibroblasts.

Consumption of boiled coffee promotes an elevation of plasma cholesterol concentration in humans. The active compounds found in the lipid fraction of the coffee have been identified as the diterpenes cafestol and kahweol. We have studied the effects of pure cafestol on cholesterol metabolism in human skin fibroblasts (HSF). The uptake of [125I]-labeled tyramine cellobiose-labeled low density lipoprotein ([125I]TC-LDL) was decreased by about 50% (P< 0.05) after 18 h preincubation time with cafestol (20 microg/ml), as compared to the control cells. The specific binding of radiolabeled LDL was reduced by 54% (P < 0.05) after preincubation for 18 h with cafestol. A reduced amount of LDL receptors was demonstrated by a protein-normalized Scatchard plot analysis (20% decrease in Bmax) as well as by immunoblotting (25%) after cafestol incubation. No significant effect was observed on the level of mRNA for the LDL receptor after 11 and 23 h incubation with cafestol. Furthermore, we transfected HSF cells with a promoter region for the LDL receptor gene linked to a reporter gene, chloramphenicol acetyl transferase (CAT). No change was seen in the CAT activity after incubation with cafestol (20 microg/ml). Moreover, cafestol caused a 2.3-fold (P < 0.05) higher incorporation of radiolabeled [14C]oleic acid into cholesteryl esters after 24 h incubation, as compared to control cells, suggesting an increased acyl-CoA:cholesterol acyl transferase (ACAT) activity. Incorporation of [14C]acetate into cholesterol was reduced by approximately 40% (P < 0.05) with cafestol (20 microg/ml), as compared to control after 24 h preincubation, indicating a decreased 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity. Our results suggest that intake of cafestol may cause increased concentration of plasma cholesterol via the down-regulation of low density lipoprotein receptors by post-transcriptional mechanisms.

Interaction between BTBR and C57BL/6J genomes produces an insulin resistance syndrome in (BTBR x C57BL/6J) F1 mice.

Insulin resistance is a common syndrome that often precedes the development of noninsulin-dependent diabetes mellitus (NIDDM). Both diet and genetic factors are associated with insulin resistance. BTBR and C57BL/6J (B6) mice have normal insulin responsiveness and normal fasting plasma insulin levels. However, a cross between these two strains yielded male offspring with severe insulin resistance. Surprisingly, on a basal diet (6.5% fat), the insulin resistance was not associated with fasting hyperinsulinemia. However, a 15% fat diet produced significant hyperinsulinemia in the male mice (twofold at 10 weeks; P < .05). At 10 weeks of age, visceral fat contributed approximately 4.3% of the total body weight in the males versus 1.8% in females. In the males, levels of plasma triacylglycerol and total cholesterol increased 40% and 30%, respectively, compared to females. Plasma free fatty acid concentrations were unchanged. Oral glucose tolerance tests revealed significant levels of hyperglycemia and hyperinsulinemia 15 to 90 minutes after oral glucose administration in the male mice. This was particularly dramatic in males on a 15% fat diet. Glucose transport was examined in skeletal muscles in (BTBR x B6)F1 mice. In the nonhyperinsulinemic animals (females), insulin stimulated 2-deoxyglucose transport 3.5-fold in the soleus and 2.8-fold in the extensor digitorum longus muscles. By contrast, glucose transport was not stimulated in the hyperinsulinemic male mice. Hypoxia stimulates glucose transport through an insulin-independent mechanism. This is known to involve the translocation of GLUT4 from an intracellular pool to the plasma membrane. In the insulin-resistant male mice, hypoxia induced glucose transport as effectively as it did in the insulin-responsive mice. Thus, defective glucose transport in the (BTBR x B6)F1 mice is specific for insulin-stimulated glucose transport. This is similar to what has been observed in muscles taken from obese NIDDM patients. These animals represent an excellent genetic model for studying insulin resistance and investigating the transition from insulin resistance in the absence of hyperinsulinemia to insulin resistance with hyperinsulinemia.

Effect of coffee lipids (cafestol and kahweol) on regulation of cholesterol metabolism in HepG2 cells.

We studied the effect of the coffee diterpene alcohols, cafestol and kahweol, on cholesterol metabolism in HepG2 cells. Uptake of 125I-tyramine cellobiose-labeled LDL was decreased by 15% to 20% (P < .05) after 18 hours of preincubation with cafestol (20 micrograms/mL), whereas 25-hydroxycholesterol reduced uptake by 55% to 65% (P < .05). Degradation of LDL in the presence of cafestol was decreased by 20% to 30% (P < .05) under the same conditions. The effect of cafestol (20 micrograms/mL) on uptake and degradation of LDL was greatest (35% to 40%, P < .05) after 6 and 10 hours of preincubation, respectively. Furthermore, the effect of cafestol was also dependent on its concentration, and a significant decrease in the LDL uptake (19%) was observed at 10 micrograms/mL (P < .05). Specific binding of LDL was reduced by 17% (P < .05) and 60% (P < .05) after preincubation with cafestol (20 micrograms/mL) and 25-hydroxycholesterol (5 micrograms/mL) for 6 hours, respectively, compared with control cells. Analysis of LDL binding showed that cafestol reduced the number of binding sites for LDL on the cell surface (capacity) by 35% (P < .05). In contrast, no significant effect on the level of mRNA for the LDL receptor was observed after incubation with cafestol, whereas 25-hydroxycholesterol reduced the mRNA level for the LDL receptor by 40% to 50% (P < .05). A fusion gene construct consisting of a synthetic sterol regulatory element-1 (SRE-1) promoter for the human LDL receptor coupled to the reporter gene for chloramphenicol acetyltransferase (CAT) was transfected into HepG2 cells. No change was observed in CAT activity in SRE-1-transfected cells after incubation with cafestol, whereas 25-hydroxycholesterol reduced CAT activity by 30% to 40% (P < .05). Incorporation of [14C]acetate into unesterified cholesterol and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were unaffected in cells incubated with cafestol as well as the cafestol-kahweol mixture compared with control cells. Moreover, cafestol and the cafestol-kahweol mixture did not promote increased incorporation of radiolabeled [14C]oleic acid into cholesteryl esters after short-term incubation compared with control cells. On the other hand, 25-hydroxycholesterol caused a 70% to 90% reduction of cholesterol synthesis (P < .05) and HMG-CoA reductase activity (P < .05), decreased HMG-CoA reductase mRNA level by 70% to 80% (P < .05), and promoted a twofold increase in cholesterol esterification (P < .05). Finally, no effect of the coffee diterpenes on bile acid formation was observed. These results suggest that cafestol (and kahweol) may reduce the activity of hepatic LDL receptors and thereby cause extracellular accumulation of LDL.