High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

BACKGROUND:High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS:To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-(13)C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 microg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 microg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8+/-0.4% vs. 8.1+/-0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized. CONCLUSIONS/SIGNIFICANCE:High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice

Stable isotope dilution analysis of N-acetylaspartic acid in CSF, blood, urine and amniotic fluid: Accurate postnatal diagnosis and the potential for prenatal diagnosis of canavan disease

A sensitive and selective analytical technique is described for the determination of N-acetylaspartic acid in body fluids using stable isotope dilution in combination with positive chemical ionization mass spectrometry with selected ion monitoring. Control mean and ranges have been established: in urine 19.5 and 6.6-35.4 μmol/mmol creat.; in plasma 0.44 and 0.17-0.81 μmol/L; in cerebrospinal fluid 1.51 and 0.25-2.83 μmol/L; and in amniotic fluid 1.27 and 0.30-2.55 μmol/L. In a patient with Canavan disease, N-acetylaspartic acid concentration was elevated 80-fold in urine and 20-fold in plasma compared to the control means. A subsequent pregnancy of the mother was monitored and the N-acetylaspartic acid concentration in the amniotic fluid was within the control range and a healthy child was born

Plasma bile acids are not associated with energy metabolism in humans

Bile acids (BA) have recently been shown to increase energy expenditure in mice, but this concept has not been tested in humans. Therefore, we investigated the relationship between plasma BA levels and energy expenditure in humans. Type 2 diabetic (T2DM) patients (n = 12) and gender, age and BMI-matched healthy controls (n = 12) were studied before and after 8 weeks of treatment with a BA sequestrant. In addition, patients with liver cirrhosis (n = 46) were investigated, since these display elevated plasma BA together with increased energy expenditure. This group was compared to gender-, age- and BMI-matched healthy controls (n = 20). Fasting plasma levels of total BA and individual BA species as well as resting energy expenditure were determined. In response to treatment with the BA sequestrant, plasma deoxycholic acid (DCA) levels decreased in controls (-60%, p &lt;0.05) and T2DM (-32%, p &lt;0.05), while chenodeoxycholic acid (CDCA) decreased in controls only (-33%, p &lt;0.05). Energy expenditure did not differ between T2DM and controls at baseline and, in contrast to plasma BA levels, was unaffected by treatment with the BA sequestrant. Total BA as well as individual BA species did not correlate with energy expenditure at any time throughout the study. Patients with cirrhosis displayed on average an increase in energy expenditure of 18% compared to values predicted by the Harris-Benedict equation, and plasma levels of total BA (up to 12-fold) and individual BA (up to 20-fold) were increased over a wide range. However, neither total nor individual plasma BA levels correlated with energy expenditure. In addition, energy expenditure was identical in patients with a cholestatic versus a non-cholestatic origin of liver disease while plasma total BA levels differed four-fold between the groups. In conclusion, in the various (patho) physiological conditions studied, plasma BA levels were not associated with changes in energy expenditure. Therefore, our data do not support an important role of circulating BA in the control of human energy metabolism.</p

Decreased expression of breast cancer resistance protein in the duodenum in patients with obstructive cholestasis

Background/Aims: The expression of transporters involved in bile acid homeostasis is differentially regulated during obstructive cholestasis. Since the drug efflux transporter breast cancer resistance protein (BCRP) is known to transport bile acids, we investigated whether duodenal BCRP expression could be altered during cholestasis. Methods: Using real-time RT-PCR analysis we determined mRNA expression levels in duodenal tissue of 19 cholestatic patients. Expression levels were compared to 14 healthy subjects. BCRP protein staining was determined in biopsies of 6 cholestatic and 6 healthy subjects by immunohistochemistry. Results: We found that in patients with obstructive cholestasis mean duodenal BCRP mRNA levels were significantly reduced to 53% and mean protein staining was reduced to 57%. Conclusions: BCRP, a transporter for bile acids and numerous drugs, appears to be down-regulated in the human duodenum during cholestasis. The clinical impact of these results has to be investigated in further studies. Copyright (c) 2006 S. Karger AG, Basel

Gall-bladder dysmotility - A risk factor for gall-stone formation in hypertriglyceridaemia and reversal on triglyceride-lowering therapy with bezafibrate and fish oil

Doel: Onderzoeken van de pathofysiologische mechanismen die de kans op galstenen verhogen bij hypertriglyceridemie (HTG) en het vergelijken van de effecten van triglycerideverlagende therapie met bezafibraat en visolie op determinanten van cholelithiasis (biliaire lipidesamenstelling en galblaasmotoriek) bij HTG-patiënten.Opzet: Gekruiste opzet met ‘random’-volgorde.Patiënten en methoden: De galblaasmotoriek werd postprandiaal en tijdens cholecystokinine(CCK)-infusie echografisch onderzocht. De determinanten van cholelithiasis en de serumlipiden werden vergeleken tussen 9 HTG-patiënten en 10 normolipidemische controlepersonen van hetzelfde geslacht, dezelfde leeftijd en ‘body mass’-index. Bij de HTG-patiënten werden de effecten van bezafibraat en gezuiverde omega-3-olie (‘visolie’) bepaald.Resultaten: De serumtriglyceride(TG)-spiegel van de HTG-patiënten was 14-voudig verhoogd, vergeleken met de controlepersonen. De lipidesamenstelling van de gal, de nuchtere galblaasvolumen en de serum-CCK-spiegels verschilden niet tussen HTG-patiënten en controlepersonen. De galblaaslediging was verminderd bij HTG-patiënten versus controlepersonen tijdens CCK-infusie (–22) en ook na een maaltijd (–37; beide p &lt; 0,001). De postprandiale serum-CCK-spiegels waren significant hoger bij HTG-patiënten. Zowel bezafibraat als visolie verlaagde de serum-TG-spiegel (–68 en –51 ten opzichte van de uitgangswaarde; beide: p &lt; 0,01). Nuchtere CCK-spiegels verschilden niet, terwijl de CCK-geïnduceerde galblaaslediging onder bezafibraat toenam met 29 (p &lt; 0,001) en met visolie met 13 (p = 0,07). De postprandiale galblaasmotoriek verbeterde tijdens zowel bezafibraat- (+47) als visoliebehandeling (+25; beide: p &lt; 0,02), waarschijnlijk gedeeltelijk door een toegenomen gevoeligheid van de galblaas voor CCK (voor beide: p &lt; 0,05 vergeleken met de uitgangsfase). Bezafibraat, in tegenstelling tot visolie, verhoogde de molaire cholesterol-galzuurratio (+40; p ≤ 0,05), terwijl beide behandelingen geen effect hadden op de cholesterolsaturatie-index.Conclusies: De verminderde galblaasmotoriek bij HTG-patiënten lijkt het gevolg te zijn van verminderde gevoeligheid voor CCK, wat kan bijdragen aan het verhoogde risico op galsteenvorming. Bij HTG-patiënten verbetert triglycerideverlagende therapie met visolie of bezafibraat de verminderde galblaasmotoriek zonder nadelig effect op de biliaire cholesterolverzadiging.Objective. To unravel the mechanisms responsible for the increased risk of gall-stone disease in hypertriglyceridaemia (HTG) and to compare the effects of triglyceride-lowering therapy with bezafibrate and fish oil on determinants of cholelithiasis (biliary-lipid composition and gall-bladder motility) in HTG patients. Design. Randomised cross over. Patients and methods. Gall-bladder motility (ultrasonography) was studied postprandially and during infusion of cholecystokinin (CCK). Determinants of cholelithiasis and serum lipids were compared between 9 HTG patients and 10 age, sex and body-mass index matched normolipidaemic controls. The effects of bezafibrate and purified omega-3-oil ('fish oil') in HTG patients were studied. Results. HTG patients showed 14-fold higher serum-triglyceride (TG) levels than controls. Biliary-lipid composition, fasting gall-bladder volumes, and CCK levels did not differ between HTG patients and controls. Gall-bladder emptying was reduced in HTG patients compared with controls during CCK infusion (-22%) as well as in response to a meal (-37%; both p &lt; 0.001). Postprandial CCK levels were significantly higher in HTG patients. Both bezafibrate and fish oil reduced serum TG levels (-68 and -51% versus baseline, respectively; both p &lt; 0.01). Fasting CCK levels were not affected whereas CCK-induced gall-bladder emptying increased during bezafibrate (+29%; p &lt; 0.001) and tended to increase upon fish-oil therapy (+13%; p = 0.07). Postprandial gall-bladder motility improved at least partly with bezafibrate and fish oil (+47 and +25% versus baseline, respectively; both p &lt; 0.02) due to increased gallbladder sensitivity to CCK (both p &lt; 0.05 versus baseline). Bezafibrate but not fish oil increased the molar ratio of cholesterol to bile acids (+40%; p ≤ 0.05), but no effects on the cholesterol-saturation index were seen with either treatment. Conclusions. We suggest that impaired gall-bladder motility occurs in HTG patients due to decreased sensitivity to CCK, which may add to the enhanced risk of gall-stone disease in HTG patients. Triglyceride-lowering therapy by both fish oil and bezafibrate improves gall-bladder dysmotility without adversely affecting biliary-cholesterol saturation.</p

Gall-bladder dysmotility - A risk factor for gall-stone formation in hypertriglyceridaemia and reversal on triglyceride-lowering therapy with bezafibrate and fish oil

Doel: Onderzoeken van de pathofysiologische mechanismen die de kans op galstenen verhogen bij hypertriglyceridemie (HTG) en het vergelijken van de effecten van triglycerideverlagende therapie met bezafibraat en visolie op determinanten van cholelithiasis (biliaire lipidesamenstelling en galblaasmotoriek) bij HTG-patiënten.Opzet: Gekruiste opzet met ‘random’-volgorde.Patiënten en methoden: De galblaasmotoriek werd postprandiaal en tijdens cholecystokinine(CCK)-infusie echografisch onderzocht. De determinanten van cholelithiasis en de serumlipiden werden vergeleken tussen 9 HTG-patiënten en 10 normolipidemische controlepersonen van hetzelfde geslacht, dezelfde leeftijd en ‘body mass’-index. Bij de HTG-patiënten werden de effecten van bezafibraat en gezuiverde omega-3-olie (‘visolie’) bepaald.Resultaten: De serumtriglyceride(TG)-spiegel van de HTG-patiënten was 14-voudig verhoogd, vergeleken met de controlepersonen. De lipidesamenstelling van de gal, de nuchtere galblaasvolumen en de serum-CCK-spiegels verschilden niet tussen HTG-patiënten en controlepersonen. De galblaaslediging was verminderd bij HTG-patiënten versus controlepersonen tijdens CCK-infusie (–22) en ook na een maaltijd (–37; beide p &lt; 0,001). De postprandiale serum-CCK-spiegels waren significant hoger bij HTG-patiënten. Zowel bezafibraat als visolie verlaagde de serum-TG-spiegel (–68 en –51 ten opzichte van de uitgangswaarde; beide: p &lt; 0,01). Nuchtere CCK-spiegels verschilden niet, terwijl de CCK-geïnduceerde galblaaslediging onder bezafibraat toenam met 29 (p &lt; 0,001) en met visolie met 13 (p = 0,07). De postprandiale galblaasmotoriek verbeterde tijdens zowel bezafibraat- (+47) als visoliebehandeling (+25; beide: p &lt; 0,02), waarschijnlijk gedeeltelijk door een toegenomen gevoeligheid van de galblaas voor CCK (voor beide: p &lt; 0,05 vergeleken met de uitgangsfase). Bezafibraat, in tegenstelling tot visolie, verhoogde de molaire cholesterol-galzuurratio (+40; p ≤ 0,05), terwijl beide behandelingen geen effect hadden op de cholesterolsaturatie-index.Conclusies: De verminderde galblaasmotoriek bij HTG-patiënten lijkt het gevolg te zijn van verminderde gevoeligheid voor CCK, wat kan bijdragen aan het verhoogde risico op galsteenvorming. Bij HTG-patiënten verbetert triglycerideverlagende therapie met visolie of bezafibraat de verminderde galblaasmotoriek zonder nadelig effect op de biliaire cholesterolverzadiging.Objective. To unravel the mechanisms responsible for the increased risk of gall-stone disease in hypertriglyceridaemia (HTG) and to compare the effects of triglyceride-lowering therapy with bezafibrate and fish oil on determinants of cholelithiasis (biliary-lipid composition and gall-bladder motility) in HTG patients. Design. Randomised cross over. Patients and methods. Gall-bladder motility (ultrasonography) was studied postprandially and during infusion of cholecystokinin (CCK). Determinants of cholelithiasis and serum lipids were compared between 9 HTG patients and 10 age, sex and body-mass index matched normolipidaemic controls. The effects of bezafibrate and purified omega-3-oil ('fish oil') in HTG patients were studied. Results. HTG patients showed 14-fold higher serum-triglyceride (TG) levels than controls. Biliary-lipid composition, fasting gall-bladder volumes, and CCK levels did not differ between HTG patients and controls. Gall-bladder emptying was reduced in HTG patients compared with controls during CCK infusion (-22%) as well as in response to a meal (-37%; both p &lt; 0.001). Postprandial CCK levels were significantly higher in HTG patients. Both bezafibrate and fish oil reduced serum TG levels (-68 and -51% versus baseline, respectively; both p &lt; 0.01). Fasting CCK levels were not affected whereas CCK-induced gall-bladder emptying increased during bezafibrate (+29%; p &lt; 0.001) and tended to increase upon fish-oil therapy (+13%; p = 0.07). Postprandial gall-bladder motility improved at least partly with bezafibrate and fish oil (+47 and +25% versus baseline, respectively; both p &lt; 0.02) due to increased gallbladder sensitivity to CCK (both p &lt; 0.05 versus baseline). Bezafibrate but not fish oil increased the molar ratio of cholesterol to bile acids (+40%; p ≤ 0.05), but no effects on the cholesterol-saturation index were seen with either treatment. Conclusions. We suggest that impaired gall-bladder motility occurs in HTG patients due to decreased sensitivity to CCK, which may add to the enhanced risk of gall-stone disease in HTG patients. Triglyceride-lowering therapy by both fish oil and bezafibrate improves gall-bladder dysmotility without adversely affecting biliary-cholesterol saturation.</p

Gall-bladder dysmotility - A risk factor for gall-stone formation in hypertriglyceridaemia and reversal on triglyceride-lowering therapy with bezafibrate and fish oil

Doel: Onderzoeken van de pathofysiologische mechanismen die de kans op galstenen verhogen bij hypertriglyceridemie (HTG) en het vergelijken van de effecten van triglycerideverlagende therapie met bezafibraat en visolie op determinanten van cholelithiasis (biliaire lipidesamenstelling en galblaasmotoriek) bij HTG-patiënten.Opzet: Gekruiste opzet met ‘random’-volgorde.Patiënten en methoden: De galblaasmotoriek werd postprandiaal en tijdens cholecystokinine(CCK)-infusie echografisch onderzocht. De determinanten van cholelithiasis en de serumlipiden werden vergeleken tussen 9 HTG-patiënten en 10 normolipidemische controlepersonen van hetzelfde geslacht, dezelfde leeftijd en ‘body mass’-index. Bij de HTG-patiënten werden de effecten van bezafibraat en gezuiverde omega-3-olie (‘visolie’) bepaald.Resultaten: De serumtriglyceride(TG)-spiegel van de HTG-patiënten was 14-voudig verhoogd, vergeleken met de controlepersonen. De lipidesamenstelling van de gal, de nuchtere galblaasvolumen en de serum-CCK-spiegels verschilden niet tussen HTG-patiënten en controlepersonen. De galblaaslediging was verminderd bij HTG-patiënten versus controlepersonen tijdens CCK-infusie (–22) en ook na een maaltijd (–37; beide p &lt; 0,001). De postprandiale serum-CCK-spiegels waren significant hoger bij HTG-patiënten. Zowel bezafibraat als visolie verlaagde de serum-TG-spiegel (–68 en –51 ten opzichte van de uitgangswaarde; beide: p &lt; 0,01). Nuchtere CCK-spiegels verschilden niet, terwijl de CCK-geïnduceerde galblaaslediging onder bezafibraat toenam met 29 (p &lt; 0,001) en met visolie met 13 (p = 0,07). De postprandiale galblaasmotoriek verbeterde tijdens zowel bezafibraat- (+47) als visoliebehandeling (+25; beide: p &lt; 0,02), waarschijnlijk gedeeltelijk door een toegenomen gevoeligheid van de galblaas voor CCK (voor beide: p &lt; 0,05 vergeleken met de uitgangsfase). Bezafibraat, in tegenstelling tot visolie, verhoogde de molaire cholesterol-galzuurratio (+40; p ≤ 0,05), terwijl beide behandelingen geen effect hadden op de cholesterolsaturatie-index.Conclusies: De verminderde galblaasmotoriek bij HTG-patiënten lijkt het gevolg te zijn van verminderde gevoeligheid voor CCK, wat kan bijdragen aan het verhoogde risico op galsteenvorming. Bij HTG-patiënten verbetert triglycerideverlagende therapie met visolie of bezafibraat de verminderde galblaasmotoriek zonder nadelig effect op de biliaire cholesterolverzadiging.Objective. To unravel the mechanisms responsible for the increased risk of gall-stone disease in hypertriglyceridaemia (HTG) and to compare the effects of triglyceride-lowering therapy with bezafibrate and fish oil on determinants of cholelithiasis (biliary-lipid composition and gall-bladder motility) in HTG patients. Design. Randomised cross over. Patients and methods. Gall-bladder motility (ultrasonography) was studied postprandially and during infusion of cholecystokinin (CCK). Determinants of cholelithiasis and serum lipids were compared between 9 HTG patients and 10 age, sex and body-mass index matched normolipidaemic controls. The effects of bezafibrate and purified omega-3-oil ('fish oil') in HTG patients were studied. Results. HTG patients showed 14-fold higher serum-triglyceride (TG) levels than controls. Biliary-lipid composition, fasting gall-bladder volumes, and CCK levels did not differ between HTG patients and controls. Gall-bladder emptying was reduced in HTG patients compared with controls during CCK infusion (-22%) as well as in response to a meal (-37%; both p &lt; 0.001). Postprandial CCK levels were significantly higher in HTG patients. Both bezafibrate and fish oil reduced serum TG levels (-68 and -51% versus baseline, respectively; both p &lt; 0.01). Fasting CCK levels were not affected whereas CCK-induced gall-bladder emptying increased during bezafibrate (+29%; p &lt; 0.001) and tended to increase upon fish-oil therapy (+13%; p = 0.07). Postprandial gall-bladder motility improved at least partly with bezafibrate and fish oil (+47 and +25% versus baseline, respectively; both p &lt; 0.02) due to increased gallbladder sensitivity to CCK (both p &lt; 0.05 versus baseline). Bezafibrate but not fish oil increased the molar ratio of cholesterol to bile acids (+40%; p ≤ 0.05), but no effects on the cholesterol-saturation index were seen with either treatment. Conclusions. We suggest that impaired gall-bladder motility occurs in HTG patients due to decreased sensitivity to CCK, which may add to the enhanced risk of gall-stone disease in HTG patients. Triglyceride-lowering therapy by both fish oil and bezafibrate improves gall-bladder dysmotility without adversely affecting biliary-cholesterol saturation.</p

Restoration of impaired intestinal barrier function by the hydrolysed casein diet contributes to the prevention of type 1 diabetes in the diabetes-prone BioBreeding rat

Aims/hypothesis Impaired intestinal barrier function is observed in type I diabetes patients and animal models of the disease. Exposure to diabetogenic antigens from the intestinal milieu due to a compromised intestinal barrier is considered essential for induction of the autoimmune process leading to type I diabetes. Since a hydrolysed casein (HC) diet prevents autoimmune diabetes onset in diabetes-prone (DP)-BioBreeding (BB) rats, we studied the role of the HC diet on intestinal barrier function and, therefore, prevention of autoimmune diabetes onset in this animal model. Methods DP-BB rats were fed the HC diet from weaning onwards and monitored for autoimmune diabetes development. Intestinal permeability was assessed in vivo by lactulose mannitol test and ex vivo by measuring trans-epithelial electrical resistance (TEER). Levels of serum zonulin, a physiological tight junction modulator, were measured by ELISA. heal mRNA expression of Myo9b, Cldn1, Cldn2 and Ocln (which encode the tight junction-related proteins myosin IXb, claudin-1, claudin-2 and occludin) and Il-10, Tgf-beta (also known as Il10 and Tgfb, respectively, which encode regulatory cytokines) was analysed by quantitative PCR. Results The HC diet reduced autoimmune diabetes by 50% in DP-BB rats. In DP-BB rats, prediabetic gut permeability negatively correlated with the moment of autoimmune diabetes onset. The improved intestinal barrier function that was induced by HC diet in DP-BB rats was visualised by decreasing lactulose:mannitol ratio, decreasing serum zonulin levels and increasing ileal TEER. The HC diet modified ileal mRNA expression of Myo9b, and Cldn1 and Cldn2, but left Ocln expression unaltered. Conclusions/interpretation Improved intestinal barrier function might be an important intermediate in the prevention of autoimmune diabetes by the HC diet in DP-BB rats. Effects on tight junctions, ileal cytokines and zonulin production might be important mechanisms for this effect

Use of dual isotope tracers in biomedical research

Biomedical stable isotope studies involve administration of tracer and measurement of isotope enrichment in blood, urine, feces or breath. The aim of the studies is to gather quantitative information about a specific metabolic function. However, the measured isotope enrichment may be affected by other metabolic events than only this function. In this case, a correction is necessary. The best approach is to add a second tracer simultaneously which is known to be metabolised by all interfering metabolic events but not by the function of interest. This dual isotope approach also enables simultaneous measurement of two interrelated functions. A summary of selected applications involving dual isotope tracer studies is presented. The applications deal with energy expenditure ( doubly labelled water technique), cholesterol absorption, starch and lactose digestion, fat digestion, bile acid metabolism and the combination of stable and radioactive carbon isotopes in breath testing