The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell

AbstractThe very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor

Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect beta-Oxidation

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial beta-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect beta-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies

Blood flow and metabolism in the corpus luteum of the rat : in vivo and in vitro studies on the ovarian luteal and follicular compartment of the rat

The ovary undergoes cyclic changes in follicular growth and luteogenesis due to the action of gonadotropins and steroids. The ovary and especially the corpus luteum has an exteremely high blood flow. There is a gap in our knowledge about the physiological role of the high blood flow of the corpus luteum. The production of lactate, progesterone and cyclic AMP of follicles and corpora lutea incubated in vitro was analyzed and related to the tissue content of ATP to elucidate possible connections between oxygen and substrate levels and energy consumption, steroid output and LH responsiveness in vitro. It was also considered of interest to investigate if the oxygen tensions needed for ATP and progesterone production of the follicle and the corpus luteum differed. A corpus luteum model using adult pseudopregnant rats was developed and characterized according to criteria for identification of corpora lutea as well as levels of plasma steroids and gonadotropins. In vitro progesterone production was compared to plasma progesterone levels. The absolute blood flow of corpora lutea of different ages and the response to injection of hCG, noradrenaline and antidiuretic hormone was investigated with the microsphere technique. Relative blood flow changes of follicles and corpora lutea during follicular growth and luteogenesis in vivo were studied by injecting radiolabelled microspheres to anaesthetized immature rats at different time periods after injection of an ovulatory dose of pregnant mare serum gonadotropin. This approach was chosen to investigate the possible relation between follicular/luteal blood flow, steroid output and morphology in relation to the endogenous gonadotropin surge, ovulation and luteogenesis. Hormonal stimulation by injection of hCG and noradrenaline increased total ovarian blood flow but no evidence was found for a parallelism between luteotropism and blood flow. The increasing effect of hCG on ovarian blood flow was partly due to a shunting of blood from the uterus towards the ovary. The antidiuretic hormone potently decreased ovarian and uterine blood flow by 80-90% while blood flow of some other organs (e.g. kidney and spleen) were hardly affected. The corpus luteum of pseudopregnancy was found to produce 15“ 20 times more progesterone in vitro as compared to the preovulatory follicle. The steroidogenesis and energy production of corpora lutea was found to be more sensitive to decreases in oxygen tension in terms of tissue ATP levels and LH responsiveness of progesterone production while the follicle could compensate by increasing glycolysis. A parallelism between follicular/luteal blood flow and progesterone production in vivo was found. It was shown that the formation, growth and progesterone production of the corpus luteum was accompanied by an increase in blood flow as well as vascularization as seen under the light microscope. The endogenous gonadotropin surge did not change follicular blood flow due to the development of a follicular oedema. We hypothesize that the corpus luteum function in vivo and in vitro is dependent on higher energy levels than the preovulatory follicle and that the transformation of the follicle to a corpus luteum is supported by a high nutritive blood flow possibly to support a high demand for energy-rich substrates.Diss. (sammanfattning) Umeå : Umeå universitet, 1987, härtill 7 uppsatser.digitalisering@um

Blood flow and metabolism in the corpus luteum of the rat : in vivo and in vitro studies on the ovarian luteal and follicular compartment of the rat

The ovary undergoes cyclic changes in follicular growth and luteogenesis due to the action of gonadotropins and steroids. The ovary and especially the corpus luteum has an exteremely high blood flow. There is a gap in our knowledge about the physiological role of the high blood flow of the corpus luteum. The production of lactate, progesterone and cyclic AMP of follicles and corpora lutea incubated in vitro was analyzed and related to the tissue content of ATP to elucidate possible connections between oxygen and substrate levels and energy consumption, steroid output and LH responsiveness in vitro. It was also considered of interest to investigate if the oxygen tensions needed for ATP and progesterone production of the follicle and the corpus luteum differed. A corpus luteum model using adult pseudopregnant rats was developed and characterized according to criteria for identification of corpora lutea as well as levels of plasma steroids and gonadotropins. In vitro progesterone production was compared to plasma progesterone levels. The absolute blood flow of corpora lutea of different ages and the response to injection of hCG, noradrenaline and antidiuretic hormone was investigated with the microsphere technique. Relative blood flow changes of follicles and corpora lutea during follicular growth and luteogenesis in vivo were studied by injecting radiolabelled microspheres to anaesthetized immature rats at different time periods after injection of an ovulatory dose of pregnant mare serum gonadotropin. This approach was chosen to investigate the possible relation between follicular/luteal blood flow, steroid output and morphology in relation to the endogenous gonadotropin surge, ovulation and luteogenesis. Hormonal stimulation by injection of hCG and noradrenaline increased total ovarian blood flow but no evidence was found for a parallelism between luteotropism and blood flow. The increasing effect of hCG on ovarian blood flow was partly due to a shunting of blood from the uterus towards the ovary. The antidiuretic hormone potently decreased ovarian and uterine blood flow by 80-90% while blood flow of some other organs (e.g. kidney and spleen) were hardly affected. The corpus luteum of pseudopregnancy was found to produce 15“ 20 times more progesterone in vitro as compared to the preovulatory follicle. The steroidogenesis and energy production of corpora lutea was found to be more sensitive to decreases in oxygen tension in terms of tissue ATP levels and LH responsiveness of progesterone production while the follicle could compensate by increasing glycolysis. A parallelism between follicular/luteal blood flow and progesterone production in vivo was found. It was shown that the formation, growth and progesterone production of the corpus luteum was accompanied by an increase in blood flow as well as vascularization as seen under the light microscope. The endogenous gonadotropin surge did not change follicular blood flow due to the development of a follicular oedema. We hypothesize that the corpus luteum function in vivo and in vitro is dependent on higher energy levels than the preovulatory follicle and that the transformation of the follicle to a corpus luteum is supported by a high nutritive blood flow possibly to support a high demand for energy-rich substrates.Diss. (sammanfattning) Umeå : Umeå universitet, 1987, härtill 7 uppsatser.digitalisering@um

A fast semi-quantitative LC-MS method for measurement of intact apolipoprotein A-I reveals novel proteoforms in serum.

Surrogate markers for reverse cholesterol transport (RCT) efficiency such as HDL cholesterol and immune methods for apolipoprotein A-I (ApoA-I) may not fully reflect the actual efficiency of the RCT pathway. Several genetic variants and different posttranslational proteoforms of ApoA-I may unevenly affect the functionality of the HDL particle to efflux cholesterol. A method employing top-down immunoaffinity LC-MS of ApoA-I in order to characterize the most prevalent ApoA-I proteoforms in human plasma is described

Falsely elevated thyroid-stimulating hormone results dueto Interference by M-component of IgG-lambda type

Heterophilic antibodies but also M-components can interfere with laboratory tests causing erroneous results. We report the case of a 75-year-old man with myeloma and a monoclonal immunoglobulin component (M-component) that caused elevated thyroid-stimulating hormone (TSH) results. The M-component was of the IgG-lambda type. Thyroid markers were analyzed repeatedly, and there was a clear association between IgG concentrations and TSH values (R 2 = 0.724). The highest TSH value was 75 mIU/L. Polyethylene glycol (PEG) precipitation of intact immunoglobulins was used to investigate if there was an antibody-related interference problem. The PEG treatment normalized the TSH value, showing that the cause of the elevated TSH result was due to interference caused by the M-component. In conclusion, it is important to remember that both heterophilic antibodies and M-components may cause erroneous results

Increased whole body energy expenditure and protection against diet-induced obesity in Cyp8b1-deficient mice is accompanied by altered adipose tissue features

The aim of this study was to elucidate mechanisms whereby bile acids exert beneficial metabolic effects, using theCyp8b1(-/-)mouse as model. These mice are unable to synthesize cholic acid, resulting in increased synthesis of chenodeoxycholic acid and enlarged bile acid pool.Cyp8b1(-/-)mice were found to be protected against high-fat diet induced obesity. Bomb calorimetry measurements showed increased faecal energy output inCyp8b1(-/)mice. Indirect calorimetry measurements demonstrated increased energy expenditure inCyp8b1(-/-)mice. Meal tolerance tests revealed no differences in glucose disposal, but the insulin response was lower inCyp8b1(-/-)mice. Intravenous glucose tolerance tests, as well as static incubations of isolated islets, showed no difference between the groups, whereas insulin tolerance tests demonstrated improved insulin sensitivity inCyp8b1(-/-)mice. The genes encoding mitochondrial transcription factor A (TFAM) and type 2-iodothyronine deiodinase were upregulated in brown adipose tissue ofCyp8b1(/-)mice and Western blot analyses showed increased abundance of TFAM, and a trend towards increased abundance of UCP1. The upregulation of TFAM and UCP1 was accompanied by increased mitochondrial density, as shown by transmission electron microscopy. White adipocytes ofCyp8b1(-/-)mice exhibited increased responsiveness to both catecholamines and insulin in lipolysis experiments and increased insulin-stimulated lipogenesis. In conclusion, increased energy expenditure, mitochondrial density of brown adipocytes and faecal energy output may all contribute to the protection against diet-induced obesity ofCyp8b1(-/-)mice. Enhanced insulin sensitivity ofCyp8b1(-/-)mice is accompanied by increased hormonal responsiveness of white adipocytes

LXR Driven Induction of HDL-Cholesterol is Independent of Intestinal Cholesterol Absorption and ABCA1 Protein Expression

We investigated whether: (1) liver X receptor (LXR)-driven induction of high-density lipoprotein cholesterol (HDL-C) and other LXR-mediated effects on cholesterol metabolism depend on intestinal cholesterol absorption; and (2) combined treatment with the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe results in synergistic effects on cholesterol metabolism that could be beneficial for treatment of atherosclerosis. Mice were fed 0.2 % cholesterol and treated with GW3965+ezetimibe, GW3965 or ezetimibe. GW3965+ezetimibe treatment elevated serum HDL-C and Apolipoprotein (Apo) AI, effectively reduced the intestinal cholesterol absorption and increased the excretion of faecal neutral sterols. No changes in intestinal ATP-binding cassette (ABC) A1 or ABCG5 protein expression were observed, despite increased mRNA expression, while hepatic ABCA1 was slightly reduced. The combined treatment caused a pronounced down-regulation of intestinal Niemann-Pick C1-like 1 (NPC1L1) and reduced hepatic and intestinal cholesterol levels. GW3965 did not affect the intestinal cholesterol absorption, but increased serum HDL-C and ApoAI levels. GW3965 also increased Apoa1 mRNA levels in primary mouse hepatocytes and HEPA1-6 cells. Ezetimibe reduced the intestinal cholesterol absorption, ABCA1 and ABCG5, but did not affect the serum HDL-C or ApoAI levels. Thus, the LXR-driven induction of HDL-C and ApoAI was independent of the intestinal cholesterol absorption and increased expression of intestinal or hepatic ABCA1 was not required. Inhibited influx of cholesterol via NPC1L1 and/or low levels of intracellular cholesterol prevented post-transcriptional expression of intestinal ABCA1 and ABCG5, despite increased mRNA levels. Combined LXR activation and blocked intestinal cholesterol absorption induced effective faecal elimination of cholesterol

Hepatic gene expression and lipid content.

<p>A) Hepatic cholesteryl esters in 7 and 12 days old TK2^+/+ and TK2^−/− mice determined with GC-MS. B) Relative hepatic mRNA expression of <i>3-hydroxy-3-methyl-glutaryl-Coenzyme A reductase</i> (<i>Hmgcr</i>) and <i>3-hydroxy-3-methyl-glutaryl-Coenzyme A synthase</i> (<i>Hmgcs</i>) in 7 and 12 days old TK2^+/+ and TK2^−/− mice determined with real-time PCR. C) Hepatic triglycerides in 7, 10 and 12 days old TK2^+/+ and TK2^−/− mice determined with an enzymatic assay. D) Relative hepatic mRNA expression of <i>fatty acid synthase</i> (<i>Fas</i>) and <i>sterol regulatory element binding protein 1c</i> (<i>Srebp1c</i>) in 7 and 12 days old TK2^+/+ and TK2^−/− mice determined with real-time PCR. Data presented as mean ± SEM. Statistically significant difference (two-tailed unpaired Student’s t-test) compared to TK2^+/+ of the same age, *p≤0.05.</p

Hepatic expression of adipophilin, voltage dependent anion channel (VDAC) and cytochrome oxidase subunit II (COXII).

<p>Protein expression in livers of A) 7 days old and B) 12 days old TK2^+/+ and TK2^−/− mice detected with Western blot. Protein samples are from three individuals of each genotype and age. Bio-RAD Quantity one software was used to determine the intensity of the bands and statistical comparisons (two-tailed unpaired Student’s t-test) between TK2^+/+ and TK2^−/− were done. VDAC was used to normalize the data. Only 12 days old TK2^−/− mice exhibit 150% higher expression of adipophilin compared to TK2^+/+ mice (p≤0.05), no other difference was detected.</p