TUB gene expression in hypothalamus and adipose tissue and its association with obesity in humans.

BACKGROUND/OBJECTIVES: Mutations in the Tubby gene (TUB) cause late-onset obesity and insulin resistance in mice and syndromic obesity in humans. Although TUB gene function has not yet been fully elucidated, studies in rodents indicate that TUB is involved in the hypothalamic pathways regulating food intake and adiposity. Aside from the function in central nervous system, TUB has also been implicated in energy metabolism in adipose tissue in rodents. We aimed to determine the expression and distribution patterns of TUB in man as well as its potential association with obesity. SUBJECTS/METHODS: In situ hybridization was used to localize the hypothalamic regions and cells expressing TUB mRNA. Using RT-PCR, we determined the mRNA expression level of the two TUB gene alternative splicing isoforms, the short and the long transcript variants, in the hypothalami of 12 obese and 12 normal-weight subjects, and in biopsies from visceral (VAT) and subcutaneous (SAT) adipose tissues from 53 severely obese and 24 non-obese control subjects, and correlated TUB expression with parameters of obesity and metabolic health. RESULTS: Expression of both TUB transcripts was detected in the hypothalamus, whereas only the short TUB isoform was found in both VAT and SAT. TUB mRNA was detected in several hypothalamic regions involved in body weight regulation, including the nucleus basalis of Meynert and the paraventricular, supraoptic and tuberomammillary nuclei. We found no difference in the hypothalamic TUB expression between obese and control groups, whereas the level of TUB mRNA was significantly lower in adipose tissue of obese subjects as compared to controls. Also, TUB expression was negatively correlated with indices of body weight and obesity in a fat-depot-specific manner. CONCLUSIONS: Our results indicate high expression of TUB in the hypothalamus, especially in areas involved in body weight regulation, and the correlation between TUB expression in adipose tissue and obesity. These findings suggest a role for TUB in human obesity.

Short communication: Supplementation of fructo-oligosaccharides does not improve insulin sensitivity in heavy veal calves fed different sources of carbohydrates.

Heavy veal calves (4-6 mo old) often develop problems with insulin sensitivity. This could lead to metabolic disorders and impaired animal growth performance. Studies in various animal species have shown that the supplementation of short-chain fructo-oligosaccharides (scFOS) can improve insulin sensitivity. We therefore studied the effects of scFOS supplementation on insulin sensitivity in heavy veal calves. Forty male Holstein-Friesian calves (BW = 190 ± 2.9 kg; age = 162 ± 1.4 d at the start of the trial) were fed either a control milk replacer (MR) diet or a diet in which one-third of the lactose was replaced by glucose, fructose, or glycerol for 10 wk prior to the start of the trial. At the start of the trial, calves were subjected to a frequently sampled intravenous glucose tolerance test to assess whole-body insulin sensitivity (muscle and hepatic insulin sensitivity). Calves within each dietary treatment group were ranked based on their insulin sensitivity value. Half of the calves received scFOS (12 mg/kg of BW) with the MR for 6 wk (supplementation was equally distributed over the insulin sensitivity range). Subsequently, a second frequently sampled intravenous glucose tolerance test was conducted to assess the effect of scFOS. In addition, fasting plasma levels of glucose, insulin, triglycerides, and cholesterol were determined to calculate the quantitative insulin sensitivity check index and triglyceride:high-density lipoprotein cholesterol ratio (fasting indicators of insulin sensitivity). Whole-body insulin sensitivity was low at the start of the trial and remained low in all groups [1.0 ± 0.1 and 0.8 ± 0.1 (mU/L)-1 · min-1 on average, respectively]. Supplementation of scFOS did not improve insulin sensitivity in any of the treatment groups. The quantitative insulin sensitivity check index and the triglyceride:high-density lipoprotein cholesterol ratio also did not differ between scFOS and non-scFOS calves and averaged 0.326 ± 0.003 and 0.088 ± 0.004, respectively, at the end of the trial. We conclude that scFOS supplementation does not improve insulin sensitivity in heavy veal calves regardless of the carbohydrate composition of the MR. This is in contrast to other animals (e.g., dogs and horses), where scFOS supplementation did improve insulin sensitivity. The absence of an effect of scFOS might be related to the dosage or to metabolic differences between ruminants and nonruminants. Increasing evidence indicates that dietary interventions in veal calves have little or no effect on insulin sensitivity, possibly because of low levels of insulin sensitivity.

Substantial replacement of lactose with fat in a high-lactose milk replacer diet increases liver fat accumulation but does not affect insulin sensitivity in veal calves.

In veal calves, the major portion of digestible energy intake originates from milk replacer (MR), with lactose and fat contributing approximately 45 and 35%, respectively. In veal calves older than 4 mo, prolonged high intakes of MR may lead to problems with glucose homeostasis and insulin sensitivity, ultimately resulting in sustained insulin resistance, hepatic steatosis, and impaired animal performance. The contribution of each of the dietary energy sources (lactose and fat) to deteriorated glucose homeostasis and insulin resistance is currently unknown. Therefore, an experiment was designed to compare the effects of a high-lactose and a high-fat MR on glucose homeostasis and insulin sensitivity in veal calves. Sixteen male Holstein-Friesian calves (120±2.8kg of BW) were assigned to either a high-lactose (HL) or a high-fat (HF) MR for 13 consecutive weeks. After at least 7 wk of adaptation, whole-body insulin sensitivity and insulin secretion were assessed by euglycemic-hyperinsulinemic and hyperglycemic clamps, respectively. Postprandial blood samples were collected to assess glucose, insulin, and triglyceride responses to feeding, and 24-h urine was collected to quantify urinary glucose excretion. At the end of the trial, liver and muscle biopsies were taken to assess triglyceride contents in these tissues. Long-term exposure of calves to HF or HL MR did not affect whole-body insulin sensitivity (averaging 4.2±0.5×10-2 [(mg/kg∙min)/(µU/mL)]) and insulin secretion. Responses to feeding were greater for plasma glucose and tended to be greater for plasma insulin in HL calves than in HF calves. Urinary glucose excretion was substantially higher in HL calves (75±13g/d) than in HF calves (21±6g/d). Muscle triglyceride content was not affected by treatment and averaged 4.5±0.6g/kg, but liver triglyceride content was higher in HF calves (16.4±0.9g/kg) than in HL calves (11.2±0.7g/kg), indicating increased hepatic fat accumulation. We conclude that increasing the contribution of fat to the digestible energy intake from the MR from 20 to 50%, at the expense of lactose does not affect whole-body insulin sensitivity and insulin secretion in calves. However, a high-lactose MR increases postprandial glucose and insulin responses, whereas a high-fat MR increases fat accumulation in liver but not muscle.

Insulin sensitivity in calves decreases substantially during the first 3 months of life and is unaffected by weaning or fructo-oligosaccharide supplementation.

Veal calves at the age of 4 to 6 mo often experience problems with glucose homeostasis, as indicated by postprandial hyperglycemia, hyperinsulinemia, and insulin resistance. It is not clear to what extent the ontogenetic development of calves or the feeding strategy [e.g., prolonged milk replacer (MR) feeding] contribute to this pathology. The objective of this study was therefore to analyze effects of MR feeding, weaning, and supplementation of short-chain fructo-oligosaccharides (FOS) on the development of glucose homeostasis and insulin sensitivity in calves during the first 3 mo of life. Thirty male Holstein-Friesian calves (18±0.7 d of age) were assigned to 1 of 3 dietary treatments: the control (CON) group received MR only, the FOS group received MR with the addition of short-chain FOS, and the solid feed (SF) group was progressively weaned to SF. The CON and FOS calves received an amount of MR, which gradually increased (from 400 to 1,400 g/d) during the 71-d trial period. For the SF calves, the amount of MR increased from 400 to 850 g/d at d 30, and then gradually decreased, until completely weaned to only SF at d 63. The change in whole body insulin sensitivity was assessed by intravenous glucose tolerance tests. Milk tolerance tests were performed twice to assess changes in postprandial blood glucose, insulin, and nonesterified fatty acid responses. Whole-body insulin sensitivity was high at the start (16.7±1.6×10(-4) [µU/mL](-1)), but decreased with age to 4.2±0.6×10(-4) [µU/mL](-1) at the end of the trial. The decrease in insulin sensitivity was most pronounced (~70%) between d 8 and 29 of the trial. Dietary treatments did not affect the decrease in insulin sensitivity. For CON and FOS calves, the postprandial insulin response was 3-fold higher at the end of the trial than at the start, whereas the glucose response remained similar. The SF calves, however, showed pronounced hyperglycemia and hyperinsulinemia at the end of the trial, although weaning did not affect insulin sensitivity. We conclude that whole body insulin sensitivity decreases by 75% in calves during the first 3 mo of life. Weaning or supplementation of short-chain FOS does not affect this age-related decline in insulin sensitivity. Glucose homeostasis is not affected by supplementation of short-chain FOS in young calves, whereas postprandial responses of glucose and insulin to a MR meal strongly increase after weaning.

Lactose in milk replacer can partly be replaced by glucose, fructose, or glycerol without affecting insulin sensitivity in veal calves.

Calf milk replacer (MR) contains 40 to 50% lactose. Lactose strongly fluctuates in price and alternatives are desired. Also, problems with glucose homeostasis and insulin sensitivity (i.e., high incidence of hyperglycemia and hyperinsulinemia) have been described for heavy veal calves (body weight >100 kg). Replacement of lactose by other dietary substrates can be economically attractive, and may also positively (or negatively) affect the risk of developing problems with glucose metabolism. An experiment was designed to study the effects of replacing one third of the dietary lactose by glucose, fructose, or glycerol on glucose homeostasis and insulin sensitivity in veal calves. Forty male Holstein-Friesian (body weight=114 ± 2.4 kg; age=97 ± 1.4 d) calves were fed an MR containing 462 g of lactose/kg (CON), or an MR in which 150 g of lactose/kg of MR was replaced by glucose (GLU), fructose (FRU), or glycerol (GLY). During the first 10d of the trial, all calves received CON. The CON group remained on this diet and the other groups received their experimental diets for a period of 8 wk. Measurements were conducted during the first (baseline) and last week of the trial. A frequently sampled intravenous glucose tolerance test was performed to assess insulin sensitivity and 24 h of urine was collected to measure glucose excretion. During the last week of the trial, a bolus of 1.5 g of [U-(13)C] substrates was added to their respective meals and plasma glucose, insulin, and (13)C-glucose responses were measured. Insulin sensitivity was low at the start of the trial and remained low [1.2 ± 0.1 and 1.0 ± 0.1 (mU/L)(-1) × min(-1)], and no treatment effect was noted. Glucose excretion was low at the start of the trial (3.4 ± 1.0 g/d), but increased in CON and GLU calves (26.9 ± 3.9 and 43.0 ± 10.6g/d) but not in FRU and GLY calves. Postprandial glucose was higher in GLU, lower in FRU, and similar in GLY compared with CON calves. Postprandial insulin was lower in FRU and GLY and similar in GLU compared with CON calves. Postprandial (13)C-glucose increased substantially in FRU and GLY calves, indicating that calves are able to partially convert these substrates to glucose. We concluded that replacing one third of lactose in MR by glucose, fructose, or glycerol in MR differentially influences postprandial glucose homeostasis but does not affect insulin sensitivity in veal calves.

Anabolic competence: Assessment and integration of the multimodality interventional approach in disease-related malnutrition.

Disease-related malnutrition (DRM) is a frequent clinical problem, characterized by loss of lean body mass and decreased function, including muscle function and immunocompetence. In DRM, nutritional intervention is necessary, but it has not consistently been shown to be sufficient. Other factors, for example, physical activity and hormonal or metabolic influencers of the internal milieu, are also important in the treatment of DRM. A prerequisite for successful treatment of DRM is the positive balance between anabolism and catabolism. The aim of this review was to approach DRM using this paradigm of anabolic competence, for conceptual and practical reasons. Anabolic competence is defined as "that state which optimally supports protein synthesis and lean body mass, global aspects of muscle and organ function, and immune response." Anabolic competence and interdisciplinary, multimodality interventions create a practical foundation to approach DRM in a proactive comprehensive way. Here, we describe the paradigm of anabolic competence, and its operationalization by measuring factors related to anabolic competence and suited for clinical management of patients with DRM.

The role of colonic metabolism in lactose intolerance.

Lactose maldigestion and intolerance affect a large part of the world population. The underlying factors of lactose intolerance are not fully understood. In this review, the role of colonic metabolism is discussed, i.e. fermentation of lactose by the colonic microbiota, colonic processing of the fermentation metabolites and how these processes would play a role in the pathophysiology of lactose intolerance. We suggest that the balance between the removal and production rate of osmotic-active components (lactose, and intermediate metabolites, e.g. lactate, succinate, etc.) in the colon is a key factor in the development of symptoms. The involvement of the colon may provide the basis for designing new targeted strategies for dietary and clinical management of lactose intolerance.

A curve fitting approach to estimate the extent of fermentation of indigestible carbohydrates.

BACKGROUND: Information about the extent of carbohydrate digestion and fermentation is critical to our ability to explore the metabolic effects of carbohydrate fermentation in vivo. We used cooked (13)C-labelled barley kernels, which are rich in indigestible carbohydrates, to develop a method which makes it possible to distinguish between and to assess carbohydrate digestion and fermentation. MATERIALS AND METHODS: Seventeen volunteers ingested 86 g (dry weight) of cooked naturally (13)C enriched barley kernels after an overnight fast. (13)CO(2) and H(2) in breath samples were measured every half hour for 12 h. The data of (13)CO(2) in breath before the start of the fermentation were used to fit the curve representing the digestion phase. The difference between the area under curve (AUC) of the fitted digestion curve and the AUC of the observed curve was regarded to represent the fermentation part. Different approaches were applied to determine the proportion of the (13)C-dose available for digestion and fermentation. RESULTS: Four hours after intake of barley, H(2)-excretion in breath started to rise. Within 12 h, 24-48% of the (13)C-dose was recovered as (13)CO(2), of which 18-19% was derived from colonic fermentation and the rest from digestion. By extrapolating the curve to baseline, it was estimated that eventually 24-25% of the total available (13)C in barley would be derived from colon fermentation. CONCLUSION: Curve fitting, using (13)CO(2)- and H(2)-breath data, is a feasible and non-invasive method to assess carbohydrate digestion and fermentation after consumption of (13)C enriched starchy food.

Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactose-intolerant subjects.

AIMS: Colonic metabolism of lactose may play a role in lactose intolerance. We investigated whether a 2-week supplementation of Bifidobacterium longum (in capsules) and a yogurt enriched with Bifidobacterium animalis could modify the composition and metabolic activities of the colonic microbiota in 11 Chinese lactose-intolerant subjects. METHODS AND RESULTS: The numbers of total cells, total bacteria and the Eubacterium rectale/Clostridium coccoides group in faeces as measured with fluorescent in situ hybridization and the faecal beta-galactosidase activity increased significantly during supplementation. The number of Bifidobacterium showed a tendency to increase during and after supplementation. With PCR-denaturing gradient gel electrophoresis, in subjects in which B. animalis and B. longum were not detected before supplementation, both strains were present in faeces during supplementation, but disappeared after supplementation. The degree of lactose digestion in the small intestine and the oro-caecal transit time were not different before and after supplementation, whereas symptom scores after lactose challenge decreased after supplementation. CONCLUSIONS: The results suggest that supplementation modifies the amount and metabolic activities of the colonic microbiota and alleviates symptoms in lactose-intolerant subjects. The changes in the colonic microbiota might be among the factors modified by the supplementation which lead to the alleviation of lactose intolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence for the possibility of managing lactose intolerance with dietary lactose (yogurt) and probiotics via modulating the colonic microbiota.

Whole grain foods for the prevention of type 2 diabetes mellitus.

BACKGROUND: Diet as one aspect of lifestyle is thought to be one of the modifiable risk factors for the development of type 2 diabetes mellitus (T2DM). Information is needed as to which components of the diet could be protective for this disease. OBJECTIVES: To asses the effects of whole-grain foods for the prevention of T2DM. SEARCH STRATEGY: We searched CENTRAL, MEDLINE, EMBASE, CINAHL and AMED. SELECTION CRITERIA: We selected cohort studies with a minimum duration of five years that assessed the association between intake of whole-grain foods or cereal fibre and incidence of T2DM. Randomised controlled trials lasting at least six weeks were selected that assessed the effect of a diet rich in whole-grain foods compared to a diet rich in refined grain foods on T2DM and its major risk factors. DATA COLLECTION AND ANALYSIS: Two authors independently selected the studies, assessed study quality and extracted data. Data of studies were not pooled because of methodological diversity. MAIN RESULTS: One randomised controlled trial and eleven prospective cohort studies were identified. The randomised controlled trial, which was of low methodological quality, reported the change in insulin sensitivity in 12 obese hyperinsulinemic participants after six-week long interventions. Intake of whole grain foods resulted in a slight improvement of insulin sensitivity and no adverse effects. Patient satisfaction, health related quality of life, total mortality and morbidity was not reported. Four of the eleven cohort studies measured cereal fibre intake, three studies whole grain intake and two studies both. Two studies measured the change in whole grain food intake and one of them also change in cereal fibre intake. The incidence of T2DM was assessed in nine studies and changes in weight gain in two studies. The prospective studies consistently showed a reduced risk for high intake of whole grain foods (27% to 30%) or cereal fibre (28% to 37%) on the development of T2DM. AUTHORS' CONCLUSIONS: The evidence from only prospective cohort trials is considered to be too weak to be able to draw a definite conclusion about the preventive effect of whole grain foods on the development of T2DM. Properly designed long-term randomised controlled trials are needed. To facilitate this, further mechanistic research should focus on finding a set of relevant intermediate endpoints for T2DM and on identifying genetic subgroups of the population at risk that are most susceptible to dietary intervention.

Adenosine triphosphate-dependent taurocholate transport in human liver plasma membranes.

Transport systems involved in uptake and biliary secretion of bile salts have been extensively studied in rat liver; however, little is known about these systems in the human liver. In this study, we investigated taurocholate (TC) transport in canalicular and basolateral plasma membrane vesicles isolated from 15 human livers (donor age 6-64 yr). ATP stimulated the uptake of TC into both canalicular and basolateral human liver plasma membrane vesicles (cLPM and blLPM, respectively). Considerable interindividual variations in the transport velocity were observed in the different membrane preparations used: 9.0 +/- 1.3 (mean +/- SEM, n = 17; range 1.6-18.0) and 9.3 +/- 2.0 (range 1.1-29.8) pmol TC.mg protein-1.min-1 at 1.0 microM TC for cLPM and blLPM, respectively. TC transport was temperature sensitive and showed saturation kinetics with a high affinity for TC (Km 4.2 +/- 0.7 microM and 3.7 +/- 0.5 microM for cLPM and blLPM, respectively). Transport was dependent on the ATP concentration and saturable (Km 0.25 +/- 0.03 mM, n = 3). Neither nitrate, which reduces membrane potential, nor the protonophore FCCP strongly inhibited ATP-dependent TC transport, indicating that membrane potential and proton gradient are not involved in this process. TC transport was significantly inhibited by the classical anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (250 microM) and the glutathione conjugate S-(2,4-dinitrophenyl)glutathione (100 microM). In conclusion, high affinity ATP-dependent TC transport is present in human liver at both the canalicular and the basolateral sides of the hepatocyte.

Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT.

Dietary fish oil-induced changes in intrahepatic cholesterol transport and bile acid synthesis in rats.

Hepatic cholesterol metabolism was studied in rats fed purified diets supplemented (9% wt/wt) with either fish oil (FO) (n-3 fatty acids) or corn oil (CO) (n-6 fatty acids) for 4 wk. Rats were equipped with permanent catheters in heart, bile duct, and duodenum to allow studies under normal feeding conditions. [3H]-cholesteryl oleate-labeled small unilamellar liposomes, which are rapidly endocytosed by hepatocytes, were intravenously injected to label intrahepatic cholesterol pools, and plasma and bile were collected. FO as compared to CO induced a lowering of plasma cholesterol levels by 38% and of triglyceride levels by 69%. This reduction in plasma lipids in FO rats was accompanied by: (a) an increased bile acid pool size (28%); (b) a fourfold increase in the ratio cholic acid/chenodeoxycholic acid in bile; (c) increased biliary excretion of cholesterol (51%); (d) accelerated excretion of endocytosed free cholesterol into bile; (e) accelerated incorporation of endocytosed cholesterol in bile acids; (f) a significant increase in the bile acid-independent fraction of bile flow; and (g) a threefold increase in hepatic alkaline phosphatase activity. The results show that FO induces changes in transport and metabolic pathways of cholesterol in the rat liver, which result in a more rapid disposition of plasma-derived cholesterol into the bile.

Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy.

Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.

Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E- deficient mouse hepatocytes.

To explore mechanisms underlying triglyceride (TG) accumulation in livers of chow-fed apo E-deficient mice (Kuipers, F., J.M. van Ree, M.H. Hofker, H. Wolters, G. In't Veld, R.J. Vonk, H.M.G. Princen, and L.M. Havekes. 1996. Hepatology. 24:241-247), we investigated the effects of apo E deficiency on secretion of VLDL-associated TG (a) in vivo in mice, (b) in isolated perfused mouse livers, and (c) in cultured mouse hepatocytes. (a) Hepatic VLDL-TG production rate in vivo, determined after Triton WR1339 injection, was reduced by 46% in apo E-deficient mice compared with controls. To eliminate the possibility that impaired VLDL secretion is caused by aspecific changes in hepatic function due to hypercholesterolemia, VLDL-TG production rates were also measured in apo E-deficient mice after transplantation of wild-type mouse bone marrow. Bone marrow- transplanted apo E-deficient mice, which do not express apo E in hepatocytes, showed normalized plasma cholesterol levels, but VLDL-TG production was reduced by 59%. (b) VLDL-TG production by isolated perfused livers from apo E-deficient mice was 50% lower than production by livers from control mice. Lipid composition of nascent VLDL particles isolated from the perfusate was similar for both groups. (c) Mass VLDL-TG secretion by cultured apo E-deficient hepatocytes was reduced by 23% compared with control values in serum-free medium, and by 61% in the presence of oleate in medium (0. 75 mM) to stimulate lipogenesis. Electron microscopic evaluation revealed a smaller average size for VLDL particles produced by apo E-deficient cells compared with control cells in the presence of oleate (38 and 49 nm, respectively). In short-term labeling studies, apo E-deficient and control cells showed a similar time-dependent accumulation of [3H]TG formed from [3H]glycerol, yet secretion of newly synthesized VLDL-associated [3H]TG by apo E-deficient cells was reduced by 60 and 73% in the absence and presence of oleate, respectively. We conclude that apo E, in addition to its role in lipoprotein clearance, has a physiological function in the VLDL assembly-secretion cascade.

Changes in rectal epithelial cell proliferation and intestinal bile acids after subtotal colectomy in familial adenomatous polyposis.

Subtotal colectomy and ileorectal anastomosis in familial adenomatous polyposis patients can induce temporary regression of adenomas in the rectum. The mechanism for this phenomenon is unclear. We evaluated the effect of colectomy on rectal mucosal proliferation, in relation to changes in bile acid metabolism. Four familial adenomatous polyposis patients were studied before and 3-6 months after surgery, and eight others 7-22 years postoperatively. Within 6 months after surgery, the size of the proliferative zone of the colonic crypts was found to be reduced (P less than 0.05). The proliferative activity of total colonic crypts was not affected within this period. More than 7 years postoperatively, increased cell proliferation of total crypts (P less than 0.02), as well as mid (P less than 0.05) and basal (P less than 0.05) crypt compartments, were observed compared to shortly after colectomy. In duodenal bile, deoxycholic acid was absent shortly after operation, whereas several years after operation only a small fraction (2%) was present. Fecal secondary bile acid excretion diminished after colectomy and did not change several years postoperatively. In postoperative stools only, small proportions of ursocholic and ursodeoxycholic acids (about 5% each) were consistently found. As subtotal colectomy causes a temporary decrease in the length of the proliferative zone of rectal crypts toward a normal pattern, this may explain regression of rectal polyps. This temporary effect may be mediated, at least in part, by decreased amounts of cytotoxic secondary bile acids in the rectal lumen.

Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans.

Dietary calcium may reduce the risk of colon cancer, probably by precipitating cytotoxic surfactants, such as secondary bile acids, in the colonic lumen. We previously showed that milk mineral, an important source of calcium, decreases metabolic risk factors and colonic proliferation in rats. We now report the effects of the habitual intake of milk calcium on metabolic risk factors in healthy subjects. A double-blind, cross-over metabolic study was performed in 13 healthy males. Placebo milk products (calcium, 3 mM) were compared with regular milk products (calcium, 30 mm). In each 1-week period, the habitual diet was recorded, and urine and feces were collected for 1 and 3 days, respectively. Milk calcium significantly increased fecal pH and fecal excretion of phosphate (132%), total fat (139%), free fatty acids (195%), and bile acids (141%), indicating intestinal complexation. In fecal water, the concentrations of long-chain fatty acids, secondary bile acids (deoxycholic and lithocholic acid), neutral sterols, and phospholipids were about halved (P <0.05). Consistent with these changes in soluble hydrophobic surfactants, calcium decreased the cytotoxicity of fecal water from 68 +/- 9 to 28 +/- 12% (P < 0.005). Calcium in milk products precipitates luminal cytotoxic surfactants and thus inhibits colonic cytotoxicity. Therefore, habitual dietary calcium may contribute to a nutritional modulation of colon cancer risk.

Isolation and characterization of canalicular and basolateral plasma membrane fractions from human liver.

A method is described for the isolation of subfractions from human liver plasma membranes, enriched in canalicular domains (cLPM) and basolateral domains (blLPM), respectively, and the results are compared to those obtained with rat liver. The studies were performed in 18 human livers. The cLPM (isolated at densities 1.103-1.127 for human and 1.036-1.127 for rat cLPM) from human as well as rat liver showed a lower density than the blLPM (1.141-1.161 for human and 1.151-1.172 for rat blLPM). Human and rat blLPM were characterized by increased levels of (Na+/K+)-ATPase (relative enrichment 33 and 21, respectively). Both human and rat cLPM showed high specific activities of leucine aminopeptidase; relative enrichment factors were 42 and 31, respectively. Mg(2+)-ATPase and alkaline phosphatase, specific canalicular enzymes in rat liver, were only slightly enriched in the cLPM of human liver, which indicates that these enzymes are not suitable as marker enzymes for human liver cLPM. Both cLPM and blLPM of human and rat origin were only slightly contaminated with mitochondria, lysosomes, Golgi membranes and endoplasmic reticulum. Total recoveries of cLPM and blLPM were 0.02 mg protein/g liver each for the human membrane preparations, compared to 0.07 and 0.16 mg protein/g liver for the membranes prepared from rat liver. Analysis of membrane fluidity revealed that the human liver cLPM were more rigid than blLPM (mean difference in fluorescence polarization PDPH 0.024). They contained more cholesterol (0.43 vs. 0.30 mumol/mg protein) and phospholipids (0.54 vs. 0.39 mumol/mg protein, respectively), which was compatible to rat liver plasma membrane fractions. This study shows that besides similarities, there are several differences between human and rat liver plasma membrane fractions.

Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid.

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.