Human Milk Short-Chain Fatty Acid Composition is Associated with Adiposity Outcomes in Infants.

BACKGROUND: Presumed benefits of human milk (HM) in avoiding rapid infancy weight gain and later obesity could relate to its nutrient composition. However, data on breast milk composition and its relation with growth are sparse. OBJECTIVE: We investigated whether short-chain fatty acids (SCFAs), known to be present in HM and linked to energy metabolism, are associated with infancy anthropometrics. METHODS: In a prospective birth cohort, HM hindmilk samples were collected from 619 lactating mothers at 4-8 wk postnatally [median (IQR) age: 33.9 (31.3-36.5) y, body mass index (BMI) (kg/m2): 22.8 (20.9-25.2)]. Their offspring, born at 40.1 (39.1-41.0) wk gestation with weight 3.56 (3.22-3.87) kg and 51% male, were assessed with measurement of weight, length, and skinfold thickness at ages 3, 12, and 24 mo, and transformed to age- and sex-adjusted z scores. HM SCFAs were measured by 1H-nuclear magnetic resonance spectroscopy (NMR) and GC-MS. Multivariable linear regression models were conducted to analyze the relations between NMR HM SCFAs and infancy growth parameters with adjustment for potential confounders. RESULTS: NMR peaks for HM butyrate, acetate, and formic acid, but not propionate, were detected. Butyrate peaks were 17.8% higher in HM from exclusively breastfeeding mothers than mixed-feeding mothers (P = 0.003). HM butyrate peak values were negatively associated with changes in infant weight (standardized B  = -0.10, P = 0.019) and BMI (B = -0.10, P = 0.018) between 3 and 12 mo, and negatively associated with BMI (B = -0.10, P = 0.018) and mean skinfold thickness (B = -0.10, P = 0.049) at age 12 mo. HM formic acid peak values showed a consistent negative association with infant BMI at all time points (B < = -0.10, P < = 0.014), whereas HM acetate was negatively associated with skinfold thickness at 3 mo (B = -0.10, P = 0.028) and 24 mo (B = -0.10, P = 0.036). CONCLUSIONS: These results suggest that HM SCFAs play a beneficial role in weight gain and adiposity during infancy. Further knowledge of HM SCFA function may inform future strategies to support healthy growth.

Breast milk nutrient content and infancy growth.

AIM: Benefits of human breast milk (HM) in avoiding rapid infancy weight gain and later obesity could relate to its nutrient content. We tested the hypothesis that differential HM total calorie content (TCC) or macronutrient contents may be associated with infancy growth. METHODS: HM hindmilk samples were collected at ages 4-8 weeks from 614 mothers participating in a representative birth cohort, with repeated infancy anthropometry. HM triglyceride (fat), lipid analytes and lactose (carbohydrate) were measured by (1) H-NMR, and protein content by the Dumas method. TCC and %macronutrients were determined. RESULTS: In 614 HM samples, fat content was as follows: [median(IQR)]: 2.6 (1.7-3.6) g/100 mL, carbohydrate: 8.6 (8.2-8.8) g/100 mL, protein: 1.2 (1.1-1.2) g/100 mL; TCC: 61.8 (53.7-71.3) kcal/100 mL. HM of mothers exclusively breast feeding vs. mixed feeding was more calorific with higher %fat, lower %carbohydrate and lower %protein. Higher HM TCC was associated with lower 12-months body mass index (BMI)/adiposity, and lower 3-12 months gains in weight/BMI. HM %fat was inversely related to 3-12 months gains in weight, BMI and adiposity, whereas %carbohydrate was positively related to these measures. HM %protein was positively related to 12-months BMI. CONCLUSION: HM analysis showed wide variation in %macronutrients. Although data on milk intakes were unavailable, our findings suggest functional relevance of HM milk composition to infant growth.

Postnatal growth in preterm infants and later health outcomes: a systematic review.

UNLABELLED: In preterm infants, poor postnatal growth is associated with adverse neurocognitive outcomes; conversely, rapid postnatal growth is supposedly harmful for future development of metabolic diseases. CONCLUSION: In this systematic review, observational studies reported consistent positive associations between postnatal weight or head growth and neurocognitive outcomes; however, there was limited evidence from the few intervention studies. Evidence linking postnatal weight gain to later adiposity and other cardiovascular disease risk factors in preterm infants was also limited.

Correction: Long-chain polyunsaturated fatty acids and extensively hydrolyzed casein-induced browning in a Ucp-1 reporter mouse model of obesity.

Correction for 'Long-chain polyunsaturated fatty acids and extensively hydrolyzed casein-induced browning in a Ucp-1 reporter mouse model of obesity' by Liufeng Mao et al., Food Funct., 2018, 9, 2362-2373, https://doi.org/10.1039/C7FO01835E.

Prebiotic Galacto-Oligosaccharides Impact Stool Frequency and Fecal Microbiota in Self-Reported Constipated Adults: A Randomized Clinical Trial.

Constipation is a major issue for 10-20% of the global population. In a double-blind randomized placebo-controlled clinical trial, we aimed to determine a dose-response effect of galacto-oligosaccharides (GOS) on stool characteristics and fecal microbiota in 132 adults with self-reported constipation according to Rome IV criteria (including less than three bowel movements per week). Subjects (94% females, aged: 18-59 years) received either 11 g or 5.5 g of BiotisTM GOS, or a control product, once daily for three weeks. Validated questionnaires were conducted weekly to study primarily stool frequency and secondary stool consistency. At base- and endline, stool samples were taken to study fecal microbiota. A trend towards an increased stool frequency was observed after the intervention with 11 g of GOS compared to control. While during screening everybody was considered constipated, not all subjects (n = 78) had less than three bowel movements per week at baseline. In total, 11 g of GOS increased stool frequency compared to control in subjects with a low stool frequency at baseline (≤3 bowel movements per week) and in self-reported constipated adults 35 years of age or older. A clear dose-response of GOS was seen on fecal Bifidobacterium, and 11 g of GOS significantly increased Anaerostipes hadrus. In conclusion, GOS seems to be a solution to benefit adults with a low stool frequency and middle-aged adults with self-reported constipation.

Sex-Specific Differences in Fat Storage, Development of Non-Alcoholic Fatty Liver Disease and Brain Structure in Juvenile HFD-Induced Obese Ldlr-/-.Leiden Mice.

BACKGROUND: Sex-specific differences play a role in metabolism, fat storage in adipose tissue, and brain structure. At juvenile age, brain function is susceptible to the effects of obesity; little is known about sex-specific differences in juvenile obesity. Therefore, this study examined sex-specific differences in adipose tissue and liver of high-fat diet (HFD)-induced obese mice, and putative alterations between male and female mice in brain structure in relation to behavioral changes during the development of juvenile obesity. METHODS: In six-week-old male and female Ldlr-/-.Leiden mice (n = 48), the impact of 18 weeks of HFD-feeding was examined. Fat distribution, liver pathology and brain structure and function were analyzed imunohisto- and biochemically, in cognitive tasks and with MRI. RESULTS: HFD-fed female mice were characterized by an increased perigonadal fat mass, pronounced macrovesicular hepatic steatosis and liver inflammation. Male mice on HFD displayed an increased mesenteric fat mass, pronounced adipose tissue inflammation and microvesicular hepatic steatosis. Only male HFD-fed mice showed decreased cerebral blood flow and reduced white matter integrity. CONCLUSIONS: At young age, male mice are more susceptible to the detrimental effects of HFD than female mice. This study emphasizes the importance of sex-specific differences in obesity, liver pathology, and brain function.

Long-chain polyunsaturated fatty acids and extensively hydrolyzed casein-induced browning in a Ucp-1 reporter mouse model of obesity.

Browning in adipose tissues, which can be affected by diet, may mitigate the detrimental effects of adiposity and improve longer-term metabolic health. Here, browning-inducing effects of long-chain polyunsaturated fatty acids, e.g., arachidonic acid (ARA)/docosahexaenoic acid (DHA) and extensively hydrolyzed casein (eHC) were investigated in uncoupling protein 1 (Ucp-1) reporter mice. To address the overall functionality, their potential role in supporting a healthy metabolic profile under obesogenic dietary challenges later in life was evaluated. At weaning Ucp1+/LUC reporter mice were fed a control low fat diet (LFD) with or without ARA + DHA, eHC or eHC + ARA + DHA for 8 weeks until week 12 after which interventions continued for another 12 weeks under a high-fat diet (HFD) challenge. Serology (metabolic responses and inflammation) and in vivo and ex vivo luciferase activity were determined; in the meantime browning-related proteins UCP-1 and the genes peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), PR domain containing 16 (PRDM16) and Ucp-1 were examined. ARA + DHA, eHC or their combination reduced body weight gain and adipose tissue weight compared to the HFD mice. The interventions induced Ucp-1 expression in adipose tissues prior to and during the HFD exposure. Ucp-1 induction was accompanied by higher PGC1a and PRDM16 expression. Glucose tolerance and insulin sensitivity were improved coinciding with lower serum cholesterol, triglycerides, free fatty acids, insulin, leptin, resistin, fibroblast growth factor 21, alanine aminotransferase, aspartate aminotransferase and higher adiponectin than the HFD group. HFD-associated increased systemic (IL-1ß and TNF-α) and adipose tissue inflammation (F4/80, IL-1ß, TNF-α, IL-6) was reduced. Studies in a Ucp-1 reporter mouse model revealed that early intervention with ARA/DHA and eHC improves metabolic flexibility and attenuates obesity during HFD challenge later in life. Increased browning is suggested as, at least, part of the underlying mechanism.

A casein hydrolysate based formulation attenuates obesity and associated non-alcoholic fatty liver disease and atherosclerosis in LDLr-/-.Leiden mice.

BACKGROUND: Obesity frequently associates with the development of non-alcoholic fatty liver disease (NAFLD) and atherosclerosis. Chronic inflammation in white adipose tissue (WAT) seems to be an important driver of these manifestations. OBJECTIVE: This study investigated a combination of an extensively hydrolyzed casein (eHC), docosahexaenoic acid (DHA), arachidonic acid (ARA), and Lactobacillus Rhamnosus GG (LGG) (together referred to as nutritional ingredients, NI) on the development of obesity, metabolic risk factors, WAT inflammation, NAFLD and atherosclerosis in high-fat diet-fed LDLr-/-.Leiden mice, a model that mimics disease development in humans. METHODS: LDLr-/-.Leiden male mice (n = 15/group) received a high-fat diet (HFD, 45 Kcal%) for 21 weeks with or without the NI (23.7% eHC, 0.083% DHA, 0.166% ARA; all w/w and 1x109 CFU LGG gavage 3 times/week). HFD and HFD+NI diets were isocaloric. A low fat diet (LFD, 10 Kcal%) was used for reference. Body weight, food intake and metabolic risk factors were assessed over time. At week 21, tissues were analyzed for WAT inflammation (crown-like structures), NAFLD and atherosclerosis. Effects of the individual NI components were explored in a follow-up experiment (n = 7/group). RESULTS: When compared to HFD control, treatment with the NI strongly reduced body weight to levels of the LFD group, and significantly lowered (P<0.01) plasma insulin, cholesterol, triglycerides, leptin and serum amyloid A (P<0.01). NI also reduced WAT mass and inflammation. Strikingly, NI treatment significantly reduced macrovesicular steatosis, lobular inflammation and liver collagen (P<0.05), and attenuated atherosclerosis development (P<0.01). Of the individual components, the effects of eHC were most pronounced but could not explain the entire effects of the NI formulation. CONCLUSIONS: A combination of eHC, ARA, DHA and LGG attenuates obesity and associated cardiometabolic diseases (NAFLD, atherosclerosis) in LDLr-/-.Leiden mice. The observed reduction of inflammation in adipose tissue and in the liver provides a rationale for these comprehensive health effects.

Early intake of long-chain polyunsaturated fatty acids preserves brain structure and function in diet-induced obesity.

Worldwide, the incidence of obesity is increasing at an alarming rate, and the number of children with obesity is especially worrisome. These developments raise concerns about the physical, psychosocial and cognitive consequences of obesity. It was shown that early dietary intake of arachidonic acid (ARA) and docosahexaenoic acid (DHA) can reduce the detrimental effects of later obesogenic feeding on lipid metabolism and adipogenesis in an animal model of mild obesity. In the present study, the effects of early dietary ARA and DHA on cognition and brain structure were examined in mildly obesogenic ApoE*3Leiden mouse model. We used cognitive tests and neuroimaging during early and later life. During their early development after weaning (4-13weeks of age), mice were fed a chow diet or ARA and DHA diet for 8 weeks and then switched to a high-fat and high-carbohydrate (HFHC) diet for 12weeks (14-26weeks of age). An HFHC-diet led to increased energy storage in white adipose tissue, increased cholesterol levels, decreased triglycerides levels, increased cerebral blood flow and decreased functional connectivity between brain regions as well as cerebrovascular and gray matter integrity. ARA and DHA intake reduced the HFHC-diet-induced increase in body weight, attenuated plasma triglycerides levels and improved cerebrovasculature, gray matter integrity and functional connectivity in later life. In conclusion, an HFHC diet causes adverse structural brain and metabolic adaptations, most of which can be averted by dietary ARA and DHA intake early in life supporting metabolic flexibility and cerebral integrity later in life.

Nutritional ingredients modulate adipokine secretion and inflammation in human primary adipocytes.

Nutritional factors such as casein hydrolysates and long chain polyunsaturated fatty acids have been proposed to exert beneficial metabolic effects. We aimed to investigate how a casein hydrolysate (eCH) and long chain polyunsaturated fatty acids could affect human primary adipocyte function in vitro. Incubation conditions with the different nutritional factors were validated by assessing cell vitality with lactate dehydrogenase (LDH) release and neutral red incorporation. Intracellular triglyceride content was assessed with Oil Red O staining. The effect of eCH, a non-peptidic amino acid mixture (AA), and long-chain polyunsaturated fatty acids (LC-PUFAs) on adiponectin and leptin secretion was determined by enzyme-linked immunosorbent assay (ELISA). Intracellular adiponectin expression and nuclear factor-κB (NF-κB) activation were analyzed by Western blot, while monocyte chemoattractant protein-1 (MCP-1) release was explored by ELISA. The eCH concentration dependently increased adiponectin secretion in human primary adipocytes through its intrinsic peptide bioactivity, since the non-peptidic mixture, AA, could not mimic eCH's effects on adiponectin secretion. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and DHA combined with arachidonic acid (ARA) upregulated adiponectin secretion. However, only DHA and DHA/ARA exerted a potentanti-inflammatory effect reflected by prevention of tumor necrosis factor-α (TNF-α) induced NF-κB activation and MCP-1 secretion in human adipocytes. eCH and DHA alone or in combination with ARA, may hold the key for nutritional programming through their anti-inflammatory action to prevent diseases with low-grade chronic inflammation such as obesity or diabetes.

Arachidonic acid/docosahexaenoic acid-supplemented diet in early life reduces body weight gain, plasma lipids, and adiposity in later life in ApoE*3Leiden mice.

SCOPE: This study addresses whether early life arachidonic acid (ARA)/docosahexaenoic acid (DHA) supplementation or eicosapentaenoic acid (EPA)/DHA (Omacor) supplementation affects body weight gain, lipid metabolism, and adipose tissue quantity and quality in later life in ApoE*3Leiden-transgenic mice, a humanized model for hyperlipidemia and mild obesity. METHODS AND RESULTS: Four-week-old male ApoE*3Leiden mice were fed chow diet with or without a mixture of ARA (0.129 wt%) and DHA (0.088 wt%) or Omacor (0.30 wt% EPA, 0.25 wt% DHA). At age 12 weeks, mice were fed high-fat/high-carbohydrate (HFHC) diet without above supplements until age 20 weeks. Control mice received chow diet throughout the study. Mice receiving ARA/DHA gained less body weight compared to control and this effect was sustained when fed HFHC. Omacor had no significant effect on body weight gain. Plasma cholesterol and triglycerides were significantly lowered by both supplementations. At 20 weeks, epididymal fat mass was less in ARA/DHA-supplemented mice, while Omacor had no significant effect on fat mass. Both ARA/DHA and Omacor reduced inguinal adipocyte cell size; only ARA/DHA significantly reduced epididymal macrophage infiltration. CONCLUSION: This study shows that early life ARA/DHA, but not Omacor supplementation improves body weight gain later in life. ARA/DHA and to a lesser extent Omacor both improved adipose tissue quality.

PDGF-receptor beta-targeted adenovirus redirects gene transfer from hepatocytes to activated stellate cells.

Chronic liver damage may lead to liver fibrosis. In this process, hepatic activated stellate cells are the key players. Thus, activated stellate cells are attractive targets for antifibrotic gene therapy. Recombinant adenovirus is a promising vehicle for delivering therapeutic genes to liver cells. However, this vector has considerable tropism for hepatocytes and Kupffer cells. The aim of this study is therefore to retarget the adenovirus to the activated stellate cells while reducing its affinity for hepatocytes. We constructed a fusion protein with affinity for both the adenovirus and the platelet derived growth factor-receptor beta (PDGF-Rbeta). In contrast to other cells, the PDFG-Rbeta is highly expressed on activated stellate cells. The targeting moiety, the PDGF peptide CSRNLIDC, was cloned in front of the single-chain antibody fragment (S11) directed against the adenoviral knob. This fusion protein enhanced adenoviral gene transfer in both 3T3 fibroblasts and primary isolated activated rat stellate cells by 10-60-fold. A fusion protein with a scrambled PDGF peptide (CIDNLSRC) did not accomplish this effect. Importantly, the PDGF-Rbeta-retargeted adenovirus showed a 25-fold reduced tropism for primary rat hepatocytes. Our novel approach demonstrates that therapeutic genes can be selectively directed to stellate cells. This opens new possibilities for the treatment of liver fibrosis.

Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases.

BACKGROUND/AIMS: In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the mechanisms are not completely elucidated. To elucidate the mechanisms of hepatocyte cell death induced by the ROS superoxide anions and hydrogen peroxide, primary cultures of hepatocytes were exposed to the superoxide anion donor menadione (10-50 micromol/L) or H2O2 (1-5 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors, superoxide dismutase (PEG-SOD) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9, -6, -3 activation and cleaved PARP, and necrotic cell death by Sytox Green staining. RESULTS: (1) Menadione (50 micromol/L) induces JNK phosphorylation, caspase-9, -6, -3 activation, PARP cleavage and apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50 micromol/L) induces the phosphorylation of ERK1/2 and this attenuates cell death. (2) H2O2 increases necrotic cell death at high concentration or when H2O2 detoxification is impaired. H2O2 does not activate MAPKs signalling. (3) PEG-SOD prevents ERK1/2-, JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases menadione-induced apoptosis. (4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death. CONCLUSIONS: In normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on JNK activity and totally abolished by superoxide scavengers.

Defying death: the hepatocyte's survival kit.

Acute liver injury can develop as a consequence of viral hepatitis, drug- or toxin-induced toxicity or rejection after liver transplantation, whereas chronic liver injury can be due to long-term exposure to alcohol, chemicals, chronic viral hepatitis, metabolic or cholestatic disorders. During liver injury, liver cells are exposed to increased levels of cytokines, bile acids and oxidative stress. This results in death of hepatocytes. In contrast, stellate cells become active and are resistant against cell death. Eventually, acute and chronic liver injury is followed by loss of liver function for which no effective therapies are available. Hepatocytes are well equipped with protective mechanisms to prevent cell death. As long as these protective mechanisms can be activated, the balance will be in favour of cell survival. However, the balance between cell survival and cell death is delicate and can be easily tipped towards cell death during liver injury. Therefore understanding the cellular mechanisms controlling death of liver cells is of clinical and scientific importance and can lead to the identification of novel intervention targets. This review describes some of the mechanisms that determine the balance between cell death and cell survival during liver diseases. The strict regulation of apoptotic cell death allows therapeutic intervention strategies. In this light, receptor-mediated apoptosis and mitochondria-mediated cell death are discussed and strategies are provided to selectively interfere with these processes.

Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis.

BACKGROUND/AIMS: In acute liver failure, hepatocytes are exposed to various cytokines that activate both cell survival and apoptotic pathways. NF-kappaB is a central transcription factor in these responses. Recent studies indicate that blocking NF-kappaB causes apoptosis, indicating the existence of NF-kappaB-regulated anti-apoptotic genes. In the present study the relationship between NF-kappaB activation and apoptosis has been investigated in hepatocytes. METHODS: Primary rat hepatocytes were exposed to a cytokine mixture of tumor necrosis factor alpha, interleukin-1beta, interferon-gamma and lipopolysaccharide. Modulation of signalling pathways was performed by using dominant negative adenoviral constructs. Apoptosis and NF-kappaB activation were determined by caspase-3 activity, Hoechst staining and electrophoretic mobility shift assay, respectively. Furthermore, expression and regulation of apoptosis-related genes were investigated. RESULTS: (1) Inhibition of NF-kappaB activation results in apoptosis. (2) Inhibitor of apoptosis protein (IAP) family members, inhibitor of apoptosis protein1 (cIAP1), and X-chromosome-linked IAP, are expressed in rat hepatocytes. cIAP2 is induced by cytokines in an NF-kappaB-dependent manner and overexpression of cIAP2 inhibits apoptosis. (3) The anti-apoptotic Bcl-2 family member A1/Bfl-1 and the pro-apoptotic members Bak and Bid are induced by cytokines and NF-kappaB-dependent. (4) Nitric oxide inhibits caspase-3 activity in hepatocytes. CONCLUSIONS: In inflammatory conditions, hepatocyte survival is dependent on NF-kappaB activation and cIAP2 contributes significantly to this protection.

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine-conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco-conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol-3 kinase pathway (PI3K) and nuclear factor-kappaB were inhibited using LY 294002 and adenoviral overexpression of dominant-negative IkappaB, respectively. The role of p38 and extracellular signal-regulated protein kinase mitogen-activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin-D. Apoptosis was determined by measuring caspase-3, -9, and -8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase-8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA-induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF-kappaB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA-induced mitochondria-controlled apoptosis by activating survival pathways.

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.

BACKGROUND/AIMS: To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS: Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS: Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS: GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.