Macrophage-specific MyD88 deletion and pharmacological inhibition prevents liver damage in non-alcoholic fatty liver disease via reducing inflammatory response

Activation of the innate immune system through toll-like receptors (TLRs) has been repeatedly demonstrated in non-alcoholic fatty liver disease (NAFLD) and several TLRs have been shown to contribute. Myeloid differentiation primary response 88 (MyD88) is as an adapter protein for the activation of TLRs and bridges TLRs to NF-κB-mediated inflammation in macrophages. However, whether myeloid cell MyD88 contributes to NAFLD are largely unknown. To test this approach, we generated macrophage-specific MyD88 knockout mice and show that these mice are protected against high-fat diet (HFD)-induced hepatic injury, lipid accumulation, and fibrosis. These protective effects were associated with reduced macrophage numbers in liver tissues and surpassed inflammatory responses. In cultured macrophages, saturated fatty acid palmitate utilizes MyD88 to activate NF-κB and induce inflammatory and fibrogenic factors. In hepatocytes, these factors may cause lipid accumulation and a further elaboration of inflammatory cytokines. In hepatic stellate cells, macrophage-derived factors, especially TGF-β, cause activation and hepatic fibrosis. We further show that pharmacological inhibition of MyD88 is also able to reduce NAFLD injury in HFD-fed mice. Therefore, our study has provided empirical evidence that macrophage MyD88 participates in HFD-induced NAFLD and could be targeted to prevent the development and progression of NAFLD/NASH.     

Intragastric administration with recombinant Lactococcus lactis producing heme oxygenase-1 prevents lipopolysaccharide-induced endotoxemia in rats

Gut injury is a pivotal initiating event in the dysfunctional inflammatory response that causes postinjury multiple organ failure. Heme oxygenase-1 (HO-1) is an important enzyme that provides cellular protection against oxidative stress in different in vitro and in vivo systems. In this study, we evaluated the protective effects of intragastrically administered live Lactococcus lactis secreting bioactive HO-1 to treat intestinal mucosal injury induced by lipopolysaccharide in rats. Intragastric administration with this recombinant L. lactis strain led to active delivery of HO-1 at the mucosa and significantly decreased morbidity and mortality of lipopolysaccharide -induced endotoxemia as confirmed by blinded macroscopic and microscopic inflammatory scores (Chiu's grade), myeloperoxidase activity, mortality, and tumor necrosis factor-alpha and IL-10 cytokine stimulation. This protective effect could be abolished by an HO-1 inhibitor, the zinc protoporphyrin-IX. Our results suggest that a food-grade bacterium genetically modified to deliver bioactive HO-1 in situ exerts a protective effect against intestinal mucosal injury in rats with endotoxemia via modulation of the immune system. This novel approach may be beneficial for the maintenance of the intestinal barrier and anti-inflammatory response of the lower intestine.     

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.     

Dextromethorphan prevents circulatory failure in rats with endotoxemia

Dextromethorphan (DM), an antitussive agent, has been claimed to have anti-inflammatory and immunomodulatory effects in vitro. Thus, the aim of this study was to evaluate the effects of DM on sepsis induced by intravenous (i.v.) administration of lipopolysaccharide (LPS) in anesthetized Wistar rats and by intraperitoneal administration in conscious ICR mice. Results demonstrated that pretreatment with DM (1,5 and 10 mg/kg, i.v.) significantly attenuated the deleterious hemodynamic changes (e.g., hypotension and tachycardia) in rats treated with LPS. Meanwhile, DM (5 mg/kg) significantly inhibited the elevation of plasma tumor necrosis factor- and interleukin-10 levels, as well as values of GOT and GPT (as an index of liver function), and BUN and creatinine (as an index of renal function) caused by LPS. The induction of inducible NO synthase and the overproduction of NO and superoxide anions by LPS were also reduced by DM. Moreover, infiltration of neutrophils into the lungs and liver of rats 6 h after treatment with LPS was also reduced by DM. In conclusion, the beneficial effects of DM on LPS-induced sepsis result from its anti-inflammatory and antioxidant effects. Thus, DM can possibly be used as a prophylactic agent for sepsis in the future.     

Tetradecylthioacetic acid prevents the inflammatory response in two-kidney, one-clip hypertension

ANG II promotes inflammation through nuclear factor-kappaB (NF-kappaB)-mediated induction of cytokines and reactive oxygen species (ROS). The aim of the present study was to examine the effect of tetradecylthioacetic acid (TTA), a modified fatty acid, on NF-kappaB, proinflammatory markers, ROS, and nitric oxide (NO) production in two-kidney, one-clip (2K1C) hypertension. The 2K1C TTA-treated group had lower blood pressure (128 +/- 3 mmHg) compared with 2K1C nontreated (178 +/- 5 mmHg, P < 0.001). The p50 and p65 subunits of NF-kappaB were higher in the clipped kidney (0.44 +/- 0.01 and 0.22 +/- 0.01, respectively) compared with controls (0.25 +/- 0.03 and 0.12 +/- 0.02, respectively, P < 0.001). In the 2K1C TTA-treated group, these values were similar to control levels. The same pattern of response was seen in the nonclipped kidney. In 2K1C hypertension, cytokines plasma were higher than in control: TNF-alpha was 13.5 +/- 2 pg/ml (P < 0.03), IL-1beta was 58.8 +/- 10 pg/ml (P = 0.003), IL-6 was 210 +/- 33 pg/ml (P < 0.001), and monocyte chemoattractant protein-1 was 429 +/- 21 pg/ml (P = 0.04). In the 2K1C TTA-treated group, these values were similar to controls, and the same pattern was seen in the clipped kidney. Clipping increased 8-iso-PGF-2alpha (P < 0.01) and decreased NO production (P < 0.01 vs. control) in the urine. TTA treatment normalized these values. NO production was also lower in clipped and nonclipped kidney (P < 0.001). After TTA treatment, these values were similar to controls. The results indicate that TTA has a potent anti-inflammatory effect in 2K1C by inhibition of p50/p65 NF-kappaB subunit activation, reduction of cytokines production and ROS, and enhanced NO production.     

Exercise training prevents the inflammatory response to hypoxia in cremaster venules.

Systemic hypoxia produces microvascular inflammation in several tissues, including skeletal muscle. Exercise training (ET) has been shown to reduce the inflammatory component of several diseases. Alternatively, ET could influence hypoxia-induced inflammation by improving tissue oxygenation or increasing mechanical antiadhesive forces at the leukocyte-endothelial interface. The effect of 5 wk of treadmill ET on hypoxia-induced microvascular inflammation was studied in the cremaster microcirculation of rats using intravital microscopy. In untrained rats, hypoxia (arterial Po(2) = 32.3 +/- 2.1 Torr) increased leukocyte-endothelial adherence from 2.3 +/- 0.4 to 10.2 +/- 0.3 leukocytes per 100 microm of venule (P < 0.05) and was accompanied by extravasation of FITC-labeled albumin after 4 h of hypoxia (extra-/intravascular fluorescence intensity ratio = 0.50 +/- 0.07). These responses were attenuated in ET (leukocyte adherence was 1.5 +/- 0.4 during normoxia and 1.8 +/- 0.7 leukocytes per 100 mum venule after 10 min of hypoxia; extra-/intravascular fluorescence intensity ratio = 0.11 +/- 0.02; P < 0.05 vs. untrained) despite similar reductions of arterial (32.4 +/- 1.8 Torr) and microvascular Po(2) (measured with an oxyphor-quenching method) in both groups. Shear rate decreased during hypoxia to similar extents in ET and untrained rats. In addition, circulating blood leukocyte count was similar in ET and untrained rats. The effects of ET on hypoxia-induced leukocyte-endothelial adherence remained up to 4 wk after discontinuing training. Thus ET attenuated hypoxia-induced inflammation despite similar effects of hypoxia on tissue Po(2), venular shear rate, and circulating leukocyte count.     

Axin1 prevents Salmonella invasiveness and inflammatory response in intestinal epithelial cells

Background

Axin1 and its homolog Axin2 are scaffold proteins essential for regulating Wnt signaling. Axin-dependent regulation of Wnt is important for various developmental processes and human diseases. However, the involvement of Axin1 and Axin2 in host defense and inflammation remains to be determined.

Methods/Principal Findings

Here, we report that Axin1, but not Axin2, plays an essential role in host-pathogen interaction mediated by the Wnt pathway. Pathogenic Salmonella colonization greatly reduces the level of Axin1 in intestinal epithelial cells. This reduction is regulated at the posttranslational level in early onset of the bacterial infection. Further analysis reveals that the DIX domain and Ser614 of Axin1 are necessary for the Salmonella-mediated modulation through ubiquitination and SUMOylation.

Conclusion/Significance

Axin1 apparently has a preventive effect on bacterial invasiveness and inflammatory response during the early stages of infection. The results suggest a distinct biological function of Axin1 and Axin2 in infectious disease and intestinal inflammation while they are functionally equivalent in developmental settings.     

Circulating levels of glucagon-like peptide-2 in human subjects with inflammatory bowel disease

Glucagon-like peptide-2 (GLP-2) is a recently characterized intestine-derived peptide that exerts trophic activity in the small and large intestine. Whether circulating levels of GLP-2 are perturbed in the setting of human inflammatory bowel disease (IBD) remains unknown. The circulating levels of bioactive GLP-2-(1-33) compared with its degradation product GLP-2-(3-33) were assessed using a combination of RIA and HPLC in normal and immunocompromised control human subjects and patients hospitalized for IBD. The activity of the enzyme dipeptidyl peptidase IV (DP IV), a key determinant of GLP-2-(1-33) degradation was also assessed in the plasma of normal controls and subjects with IBD. The circulating levels of bioactive GLP-2-(1-33) were increased in patients with either ulcerative colitis (UC) or Crohn's Disease (CD; to 229 +/- 65 and 317 +/- 89%, P < 0.05, of normal, respectively). Furthermore, the proportion of total immunoreactivity represented by intact GLP-2-(1-33), compared with GLP-2-(3-33), was increased from 43 +/- 3% in normal healthy controls to 61 +/- 6% (P < 0.01) and 59 +/- 2% (P < 0.01) in patients with UC and CD, respectively. The relative activity of plasma DP IV was significantly reduced in subjects with IBD compared with normal subjects (1.4 +/- 0.3 vs. 5.0 +/- 1.1 mU/ml, respectively; P < 0.05). These results suggest that patients with active IBD may undergo an adaptive response to intestinal injury by increasing the circulating levels of bioactive GLP-2-(1-33), facilitating enhanced repair of the intestinal mucosal epithelium in vivo.     

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1

Intestinal ischemia-reperfusion has been implicated in the systemic inflammatory response and organ injury in hemorrhagic shock, but the exact role of the intestine has never been directly demonstrated. Preconditioning (PC) with brief periods of intermittent ischemia is a known potent anti-ischemic intervention and thus can be used as a tool to assess the role of local intestinal ischemia-reperfusion injury in systemic inflammatory response. Thus rats were first subjected to sham surgery or intestinal preconditioning with four cycles of 1-min ischemia and 10 min of reperfusion 24 h before hemorrhagic shock followed by resuscitation. PC reduced fluid requirements, lung edema, and lactate and tumor necrosis factor-alpha production. These effects were abolished by the heme-oxygenase-1 (HO-1) inhibitor tin protoporphyrin (Sn-PP). PC induced more than fivefold in intestinal HO-1 expression. These results suggest that intestinal ischemia-reperfusion is a major trigger for inflammatory response and organ injury in nonseptic shock. HO-1 appears to play an important role in the protective effect of intestinal preconditioning.     

Sauchinone prevents IL‐1β‐induced inflammatory response in human chondrocytes

Sauchinone is one of the active lignan isolated from Saururus chinensis, which has been considered to possess various pharmacological activities, such as antitumor, hepatoprotective, antioxidant, and anti‐inflammatory effects. However, the functional roles of sauchinone in interleukin‐1 beta (IL‐1β)‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Thus, in this study, we investigated the anti‐inflammatory effects of sauchinone in IL‐1β‐stimulated chondrocytes. Our results demonstrated that sauchinone significantly attenuated NO and PGE2 production, as well as inhibited iNOS and COX‐2 expression in IL‐1β‐stimulated OA chondrocytes. In addition, sauchinone efficiently inhibited IL‐1β‐induced MMP‐3 and MMP‐13 release in human OA chondrocytes. Furthermore, sauchinone significantly attenuated the activation of NF‐κB in human OA chondrocytes. In conclusion, we showed for the first time that sauchinone inhibited inflammatory response in IL‐1β‐stimulated human chondrocytes probably through inhibiting the activation of NF‐κB signaling pathway. These data suggest that sauchinone may be a potential agent in the treatment of OA.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Lipoic acid prevents liver metabolic changes induced by administration of a fructose-rich diet

Background

To evaluate whether co-administration of R/S-α-lipoic acid can prevent the development of oxidative stress and metabolic changes induced by a fructose-rich diet (F).

Methods

We assessed glycemia in the fasting state and during an oral glucose tolerance test, triglyceridemia and insulinemia in rats fed with standard diet (control) and fructose without or with R/S-α-lipoic acid. Insulin resistance and hepatic insulin sensitivity were also calculated. In liver, we measured reduced glutathione, protein carbonyl groups, antioxidant capacity by ABTS assay, antioxidant enzymes (catalase and superoxide dismutase 1 and 2), uncoupling protein 2, PPARδ and PPARγ protein expressions, SREBP-1c, fatty acid synthase and glycerol-3-phosphate acyltransferase-1 gene expression, and glucokinase activity.

Results

R/S-α-lipoic acid co-administration to F-fed rats a) prevented hyperinsulinemia, hypertriglyceridemia and insulin resistance, b) improved hepatic insulin sensitivity and glucose tolerance, c) decreased liver oxidative stress and increased antioxidant capacity and antioxidant enzymes expression, d) decreased uncoupling protein 2 and PPARδ protein expression and increased PPARγ levels, e) restored the basal gene expression of PPARδ, SREBP-1c and the lipogenic genes fatty acid synthase and glycerol-3-phosphate acyltransferase, and f) decreased the fructose-mediated enhancement of glucokinase activity.

Conclusions

Our results suggest that fructose-induced oxidative stress is an early phenomenon associated with compensatory hepatic metabolic mechanisms, and that treatment with an antioxidant prevented the development of such changes.

General significance

This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced oxidative stress and to develop effective strategies to prevent and treat, at early stages, obesity and type 2 diabetes mellitus.     

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.     

Intradermal administration of thymic stromal lymphopoietin induces a T cell- and eosinophil-dependent systemic Th2 inflammatory response

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is sufficient to induce asthma or atopic dermatitis-like phenotypes when selectively overexpressed in transgenic mice, or when driven by topical application of vitamin D3 or low-calcemic analogues. Although T and B cells have been reported to be dispensable for the TSLP-induced inflammation in these models, little is known about the downstream pathways or additional cell types involved in the inflammatory response driven by TSLP. To characterize the downstream effects of TSLP in vivo, we examined the effects of exogenous administration of TSLP protein to wild-type and genetically deficient mice. TSLP induced a systemic Th2 inflammatory response characterized by increased circulating IgE and IgG1 as well as increased draining lymph node size and cellularity, Th2 cytokine production in draining lymph node cultures, inflammatory cell infiltrates, epithelial hyperplasia, subcuticular fibrosis, and up-regulated Th2 cytokine and chemokine messages in the skin. Responses to TSLP in various genetically deficient mice demonstrated T cells and eosinophils were required, whereas mast cells and TNF-alpha were dispensable. TSLP-induced responses were significantly, but not completely reduced in IL-4- and IL-13-deficient mice. These results shed light on the pathways and cell types involved in TSLP-induced inflammation.     

Down-regulation of prostaglandin F2 alpha receptors in rat liver during chronic endotoxemia.

Prostaglandin (PG) F2 alpha binding parameters were measured in purified plasma membrane preparations isolated from livers of chronically endotoxin-(ET) treated rats and corresponding controls. Two classes of binding sites were detected in both groups: high affinity, low capacity, with a KD of 44.4 +/- 8.8 nM for saline- and 28.6 +/- 11.3 nM for ET-treated rats (n = 5 for both, p greater than 0.05) and low affinity, high capacity with a KD of 1.12 +/- 0.49 microM for saline- and 1.24 +/- 0.43 microM for ET-treated rats (p greater than 0.05). Bmax values for high affinity sites were 1.01 +/- 0.18 fmol.mg-1 protein for saline- and 1.02 +/- 0.54 (same units) for ET-treated rats (p greater than 0.05). There was a significant difference (p less than 0.01) between the Bmax values for low affinity sites in saline- (675 +/- 332 fmol.mg-1 protein) and ET-treated rats (12 +/- 1, same units). This decrease in the amount of PGF2 alpha low affinity high capacity binding sites may underlie the depression of the PGF2 alpha stimulatory effect on hepatic gluconeogenesis induced by non-lethal, chronic ET treatment of rats, recently described by us (9).     

Inorganic polyphosphate from the immunobiotic Lactobacillus rhamnosus CRL1505 prevents inflammatory response in the respiratory tract

Lactobacillus (L.) rhamnosus CRL1505 accumulates inorganic polyphosphate (polyP) in its cytoplasm in response to environmental stress. The aim of this study was to evaluate the potential effects of polyP from the immunobiotic CRL1505 on an acute respiratory inflammation murine animal model induced by lipopolysaccharide (LPS).First, the presence of polyP granules in the cytoplasm of CRL1505 strain was evidenced by specific staining. Then, it was demonstrated in the intracellular extracts (ICE) of CRL1505 that polyP chain length is greater than 45 phosphate residues. In addition, the functionality of the genes involved in the polyP metabolism (ppk, ppx1 and ppx2) was corroborated by RT-PCR. Finally, the possible effect of the ICE of CRL1505 strain containing polyP and a synthetic polyP was evaluated in vivo using a murine model of acute lung inflammation. It was observed that the level of cytokines pro-inflammatory (IL-17, IL-6, IL-2, IL-4, INF-γ) in serum was normalized in mice treated with ICE, which would indicate that polyP prevents the local inflammatory response in the respiratory tract. The potential application of ICE from L. rhamnosus CRL1505 as a novel bioproduct for the treatment of respiratory diseases is one of the projections of this work.     

High-fat diet prevents eating response and attenuates liver ATP decline in rats given 2,5-anhydro-D-mannitol

Administration of the fructose analog 2,5-anhydro-D-mannitol (2,5-AM) stimulates eating in rats fed a low-fat diet but not in those fed a high-fat diet that enhances fatty acid oxidation. The eating response to 2,5-AM treatment is apparently triggered by a decrease in liver ATP content. To assess whether feeding a high-fat diet prevents the eating response to 2,5-AM by attenuating the decrease in liver ATP, we examined the effects of the analog on food intake, liver ATP content, and hepatic phosphate metabolism [using in vivo 31P-NMR spectroscopy (NMRS)]. Injection (intraperitoneal) of 300 mg/kg 2,5-AM increased food intake in rats fed a high-carbohydrate/low-fat diet, but not in those fed high-fat/low-carbohydrate (HF/LC) food. Liver ATP content decreased in all rats given 2,5-AM compared with saline, but it decreased about half as much in rats fed the HF/LC diet. NMRS on livers of anesthetized rats indicated that feeding the HF/LC diet attenuates the effects of 2,5-AM on liver ATP by reducing phosphate trapping. These results suggest that rats consuming a high-fat diet do not increase food intake after injection of 2,5-AM, because the analog is not sufficiently phosphorylated and therefore fails to decrease liver energy status below a level that generates a signal to eat.