Wayne Stevens

Dietary n‐3 polyunsaturated fatty acids decrease hepatic triglycerides in Fischer 344 rats

Dietary fatty acid composition modifies hepatic lipid metabolism. To determine the effects of fatty acids on hepatic triglyceride storage, rats were fed diets enriched in carbohydrates (control), fish oil, or lard. After 4 weeks, the animals were fasted overnight. In the morning, the animals were either sacrificed or fed 8 g of their respective diets before sacrifice. Animals ingested more food calories with diets containing fish oil than with other diets. However, fish oil–fed animals weighed less and had less body fat. In fish oil–fed animals, liver triglyceride was lower by 27% (P < .05) and 73% (P < .01) than in control‐ and lard‐fed animals, respectively. Fish oil altered the postprandial gene expression of hepatic regulators of fatty acid degradation and synthesis. Fish oil feeding blunted the normal postprandial decline in fatty acid degradation genes (PPARα, CPT1, and ACO) and blunted the normal postprandial rise in triglyceride synthesis genes (SREBP1‐c, FAS, SCD‐1). Therefore, the direct postprandial effect of fish oil ingestion decreases the propensity for hepatic triglyceride storage. In conclusion, n‐3 polyunsaturated fatty acids decrease total body weight, total body fat, and hepatic steatosis. (HEPATOLOGY 2004;39:608–616.)

Mechanisms of in vitro immunosuppression by hepatocyte-generated cyclophosphamide metabolites and 4-hydroperoxycyclophosphamide

Cyclophosphamide (CY) is metabolized to 4-hydroxy-CY which spontaneously breaks down to the reactive intermediates, phosphoramide mustard (PAM) and acrolein. The alkylating property of PAM is thought to mediate the anti-proliferative and cytotoxic actions of CY. Acrolein is known to bind sulfhydryl groups of cellular molecules and may contribute to the action of CY. However, the role of acrolein in the CY-induced immunosuppression remains unclear. The results of studies in which a hepatocyte co-culture system was used suggest that acrolein may play an important role in the cytotoxic action of CY, whereas those investigations using activated derivatives of CY indicate that acrolein is not an important factor. Thus, both approaches of CY exposure were utilized in the present study. Splenocytes of B6C3F1 mice were incubated with syngeneic isolated hepatocytes and CY or with 4-hydroperoxycyclophosphamide (4-HC) (which spontaneously decomposes to 4-hydroxy-CY). The in vitro antibody forming cell (AFC) response was found to be suppressed with both methods of exposure to CY metabolites. The addition of DNA to bind extracellular PAM was ineffective in preventing the suppression produced by hepatocyte-activated CY. However, it was also observed that DNA was able to attenuate the PAM-induced suppression. The sulfhydryl compounds 2-mercaptoethanesulfonate (MESNA) (15 microM) or reduced glutathione (GSH) (1 mM) inhibited the suppression of the AFC response of splenocytes incubated with CY and mouse hepatocytes. The suppression produced by 4-HC, however, was not affected by MESNA and only slightly inhibited by GSH. Similarly, the PAM-induced suppression was not affected by MESNA and slightly attenuated with GSH. In contrast, both MESNA and GSH were very effective in abrogating the acrolein-induced suppression, whereas DNA was ineffective. The findings of this study suggest that in the hepatocyte co-culture system, the immunosuppressive actions of CY are mediated by acrolein generated outside of the splenocyte, whereas the 4-HC induced suppression is not mediated by extracellular acrolein. Thus, this difference may explain the contradictory findings of previous studies that used different means of exposing cells to activated CY.

Immunosuppression in adult female B6C3F1 mice by chronic exposure to ethanol in a liquid diet.

The overall objective of these studies was to characterize the effects of ethanol on the immunocompetence of adult female B6C3F1 mice. To obtain a significant suppression in the antibody response to SRBC, splenocytes from untreated mice had to be directly exposed to concentrations of ethanol from 0.3% to 3.0%, or to acetaldehyde at concentrations greater than 0.03%. We do not believe that these results are consistent with a role by a direct effect by either ethanol or its primary metabolite because these concentrations are higher than what could be obtained as reasonable blood levels. For in vivo exposure, we employed a pair-feeding regimen which was based on a liquid diet containing 5% ethanol (v/v) that provided 36% of the caloric intake as ethanol. Our results indicated that there was a definite temporal relationship to the consequent suppression of the antibody response to SRBC in that no effect was observed after 14 days exposure, and that the magnitude of the suppression increased from 18% after 21 days to 70% after 42 days. We also monitored the liver for histopathology and observed that the ethanol-induced liver damage was restricted to steatosis (fatty liver), which was also manifested with time and which was most pronounced after 42 days exposure. In contrast to our results with the in vivo antibody response, we saw no effect on mitogen-induced proliferation by splenocytes from ethanol-treated mice. These results prompted us to measure in vitro antibody responses by splenocytes from ethanol-treated mice. We saw no suppression of the in vitro antibody responses to SRBC, DNP-Ficoll or LPS after any length of exposure to ethanol, and speculated that the basis for the suppression of the in vivo antibody response was an indirect consequence of exposure. We subsequently determined that when normal splenocytes were cultured in 5% serum from ethanol-exposed mice (42-day group), there was a > 80% suppression relative to the serum from the pair-fed controls. As important controls for these studies, we have demonstrated that there was no difference between the responses of normal lymphocytes cultured in 5% normal mouse serum and in 5% serum taken from the pair-fed restricted controls. A determination of the ethanol content in the serum from ethanol-exposed mice (42-day group) indicated that the amount of ethanol present in these cultures was < 0.003%.(ABSTRACT TRUNCATED AT 400 WORDS)

Total Parenteral Nutrition Increases Serum Leptin Concentration in Hospitalized, Undernourished Patients

BACKGROUND The hormone leptin has putative roles in both body weight homeostasis (chronic) and satiety (acute). To determine if this dual regulation is observed in hospitalized, undernourished patients, serum leptin concentration was measured before and during total parenteral nutrition (TPN) infusion. METHODS Six consecutive patients were considered undernourished, as assessed by an independent multidisciplinary nutrition team, and TPN was prescribed at an initial rate of between 5023.2 and 7333.2 kJ in the first 24 hours. Serum leptin, insulin, and glucose were measured before the infusion and at 3 and 22 hours after initiation of TPN. RESULTS Baseline serum leptin concentrations correlated well with the patients body mass index (BMI; r2 = .85, p<.05). Three hours of TPN infusion produced only modest changes in circulating leptin. However, after 22 hours, leptin concentrations increased by 1.8+/-0.5-fold (p<.05), and this increase was independent of any change in body weight. CONCLUSIONS Basal leptin concentrations correlate well with BMI. TPN induces a rise in leptin concentration independent of body weight. Leptin secretion is dually regulated in hospitalized, undernourished patients.

Serum modulation of the effects of TCDD on the in vitro antibody response and on enzyme induction in primary hepatocytes

We have recently reported that the effects of TCDD on the in vitro antibody response can vary considerably depending on the serum conditions used in the culture media. To further investigate this phenomenon, studies were performed to compare the effects of TCDD on both splenocyte antibody responses and P450 enzyme induction (EROD) in primary hepatocytes (HPTC) derived from B6C3F1 and DBA/2 mice when evaluated in the presence of either fetal bovine serum (FBS), newborn calf serum (NBCS) or normal mouse serum (NMS). The latter studies with NMS also included crossovers where splenocytes and HPTC from B6C3F1 mice were cultured in the presence of DBA/2 serum and vice versa. Results with NBCS showed comparable suppression of antibody responses by TCDD in splenocytes from B6C3F1 and DBA/2 mice where we detected IC50 values of 3.0 and 2.8 nM, respectively. In contrast, responses in the presence of NMS showed an Ah-dependency that was characterized by a dose-related suppression of antibody responses by B6C3F1 splenocytes, but a lack of suppression in the responses by DBA/2 splenocytes. Distribution studies with radiolabelled TCDD indicated that the observed profile of activity could not be attributed to a differential uptake of the chemical into splenocytes from B6C3F1 or DBA/2 under the various serum conditions. Serum was also found to modulate the TCDD-induced EROD activity in primary HPTC and the profile of activity was identical to the effects of TCDD on in vitro antibody responses. We observed an enhanced induction of EROD in the presence of NBCS (immunosuppressive conditions) and a lower induction in the presence of FBS (non-immunosuppressive conditions), each giving the same relative magnitude of induction regardless of the mouse strain used as the source of HPTC. In contrast, induction in the presence of NMS showed an Ah-dependency and resulted in a dose-related enhancement in EROD activity in B6C3F1 HPTC but decreased activity in the DBA/2 HPTC. Cross-over studies further showed that the pattern of effects on both splenocytes and HPTC was not altered by changing the strain of mouse used as the source of serum, where each gave equivalent results. These findings demonstrate that the Ah-dependency for the effects of TCDD on both the in vitro antibody response and P450 enzyme induction are modulated by the serum environment in which the cells are exposed. The studies with NMS indicate that it is the genotype of the lymphocyte (i.e., or the HPTC), and not the strain-specific hormone environment, which confers sensitivity to TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Bile Acid Composition and Manipulation of Enterohepatic Circulation on Leptin Gene Regulation

In the rat, the ob gene product, leptin, putatively regulates energy balance via appetite control and energy expenditure. Bile acids in the intestinal lumen are necessary for efficient absorption of dietary lipids and may trigger the release of regulatory peptides. To investigate whether bile acids play a role in leptin gene expression, we altered the bile acid pool and then measured leptin mRNA levels in adipose tissue. Rats fed cholic acid (1% of chow wt/wt) for 2 weeks did not gain weight as rapidly as pair-fed control animals. Despite the lower weight, normalized leptin mRNA levels were 24% greater in cholic acid-fed rats compared with controls. Conversely, cholestyramine, a bile acid sequestrant, in chow (5% wt/wt) resulted in a 26% decline in leptin mRNA. Ligation of the common bile duct or chronic biliary diversion, experimental manipulations that decreased the intestinal concentration of bile salts, decreased leptin gene expression by 30% and 50%, respectively. A fluid and electrolyte (F/E) solution with and without taurocholate (36 micromol/h x 100 g rat[-1]) was then infused for 12 hours into the duodenum in animals with chronic biliary diversion. Taurocholate infusion resulted in a fourfold increase in steady-state adipocyte leptin mRNA levels compared with F/E infusion. Intravenous infusion of taurocholate or incubation of cultured adipocytes with taurocholate had no effect on leptin mRNA levels. We conclude that bile acids upregulate leptin gene expression indirectly, probably via effects on the absorption of dietary lipids or the release of neurohumoral mediators.

The role of metabolism in carbon tetrachloride-mediated immunosuppression. In vitro studies.

In vitro studies were performed to determine the role of metabolic bioactivation in mediating immunosuppression by CCl4. Direct addition of CCl4 to naive spleen cell cultures sensitized with either sheep erythrocytes, DNP-Ficoll or lipopolysaccharide (LPS) resulted in a marked inhibition of the antibody forming cell (AFC) response to all three of the selected antigens at 3.0 mM concentration in culture. However, this inhibition was primarily due to the direct cytotoxic effects of CCl4 on spleen cells following 3-5 days of culture in the presence of the chemical as evidenced by a decrease in cell number and viability and by the absence of selective effects on T-cell dependent humoral responses which is contradictory to the effects observed in vivo. Co-incubation of splenocytes for 1 h with primary hepatocytes, but not with subcellular metabolic activation systems, such as S9 or microsomes, enhanced the immunotoxic effects of CCl4 in vitro. Interestingly, a 3-h co-incubation of spleen cells with metabolically active hepatocytes in primary culture resulted in an even greater potentiation of the immunotoxic effects of CCl4 as determined by the T-cell dependent IgM AFC response. Conversely, under identical conditions, CCl4 did not suppress humoral responses to the polyclonal B-cell activator LPS which is in agreement with the effects produced by in vivo exposure to CCl4. It is important to emphasize that for the metabolic activation studies (i.e. co-incubation with either S9, microsomes or hepatocytes), spleen cells were washed free of CCl4 immediately following the co-incubation period. Control splenocyte cultures (i.e. no metabolic activation system) incubated in the presence of CCl4 alone at 3.0 mM over a 3-h time-period, had no effect on spleen cell function, number or viability. In agreement with our previous findings which indicate that pretreatment of mice with inducers and inhibitors of the mixed function oxygenase system prior to CCl4 administration modulated the immunotoxic effects of CCl4 in vivo, these results lead us to conclude that immunotoxicity by CCl4 requires metabolic activation.

Role of hydrocortisone in dimethylnitrosamine-induced suppression of antibody response in the mixed culture of murine hepatocytes and splenocytes.

We have previously reported that the hormone-supplemented culture condition for primary hepatocytes is required in dimethylnitrosamine (DMN)-induced suppression of antibody response to sheep erythrocytes in the mixed cultures of murine hepatocytes and splenocytes. In the present investigation, the components of the hormone supplement were screened to identify the component(s) responsible for the increased ability of hepatocytes to activate DMN to its immunosuppressive form. The presence of hydrocortisone in the hepatocyte culture media had the primary role in DMN activation in the co-culture system. Other components of the hormone supplement showed slight or no effects. The effects of hydrocortisone were clearly confirmed through the dose-response study of both DMN and hydrocortisone. To characterize whether the effect of hydrocortisone is glucocorticoid-dependent we tested another potent glucocorticoid, dexamethasone (DEX), and determined if the activity by hydrocortisone could be reversed by RU 486. It was found that hepatocytes cultured in DEX-containing media could also activate DMN to its immunosuppressive form. However, the activity by hydrocortisone to increase DMN-induced immunosuppression was not reversed by RU 486. Furthermore, a possible direct interaction between DMN and hydrocortisone was ruled out. Finally, we transferred DMN-pre-treated culture supernatant from hepatocytes to spleen cell cultures, and found that the metabolite of DMN was very unstable, and that DMN-induced suppression of T-dependent antibody response was hepatocyte-dependent. The present results suggest that glucocorticoids, including hydrocortisone and DEX, in hepatocyte culture media can affect DMN-induced immunosuppression in the hepatocyte/splenocyte co-culture system via a pathway which does not appear to be related to the glucocorticoid receptor.