Differential regulation of alcohol consumption and reward by the transcriptional cofactor LMO4.

Repeated alcohol exposure leads to changes in gene expression that are thought to underlie the transition from moderate to excessive drinking. However, the mechanisms by which these changes are integrated into a maladaptive response that leads to alcohol dependence are not well understood. One mechanism could involve the recruitment of transcriptional co-regulators that bind and modulate the activity of transcription factors. Our results indicate that the transcriptional regulator LMO4 is one such candidate regulator. Lmo4-deficient mice (Lmo4gt/+) consumed significantly more and showed enhanced preference for alcohol in a 24 h intermittent access drinking procedure. shRNA-mediated knockdown of Lmo4 in the nucleus accumbens enhanced alcohol consumption, whereas knockdown in the basolateral amygdala (BLA) decreased alcohol consumption and reduced conditioned place preference for alcohol. To ascertain the molecular mechanisms that underlie these contrasting phenotypes, we carried out unbiased transcriptome profiling of these two brain regions in wild type and Lmo4gt/+ mice. Our results revealed that the transcriptional targets of LMO4 are vastly different between the two brain regions, which may explain the divergent phenotypes observed upon Lmo4 knockdown. Bioinformatic analyses revealed that Oprk1 and genes related to the extracellular matrix (ECM) are important transcriptional targets of LMO4 in the BLA. Chromatin immunoprecipitation revealed that LMO4 bound Oprk1 promoter elements. Consistent with these results, disruption of the ECM or infusion of norbinaltorphimine, a selective kappa opioid receptor antagonist, in the BLA reduced alcohol consumption. Hence our results indicate that an LMO4-regulated transcriptional network regulates alcohol consumption in the BLA.

A novel murine model of multi-day moderate ethanol exposure reveals increased intestinal dysfunction and liver inflammation with age.

BACKGROUND: There are currently > 600 million people over the age of 65 globally and this number is expected to double by the year 2050. Alcohol use among this population is on the rise, which is concerning as aging is associated with increased risk for a number of chronic illnesses. As most studies investigating the effects of alcohol have focused on young/middle-aged populations, there is a dearth of information regarding the consequences of alcohol use in older consumers. In addition, most murine ethanol models have concentrated on exposure to very high levels of ethanol, while the vast majority of elderly drinkers do not consume alcohol in excess; instead, they drink on average 2 alcoholic beverages a day, 3-4 days a week. METHODS: We designed a murine model of aging and moderate ethanol consumption to determine if the deleterious effects of alcohol on the gut-liver axis are exacerbated in aged, relative to younger, animals. Aged and young mice were exposed to a multi-day moderate exposure ethanol regimen for 4 weeks and changes in gut permeability along with intestinal tight junction protein and antimicrobial peptide gene expression were measured. In addition, hepatic inflammation was assessed by histological analysis, inflammatory gene expression and flow cytometric analysis of inflammatory infiltrate. RESULTS: Our results reveal that in aged, but not young mice, moderate ethanol exposure yielded significantly worsened intestinal permeability, including increased bacterial translocation from the gut, elevated serum iFABP and leakage of FITC-dextran from the gut. Interestingly, moderate ethanol exposure in young animals led to gut protective transcriptional changes in the ileum while this protective response was blunted in aged mice. Finally, moderate ethanol exposure in aged mice also resulted in marked inflammatory changes in the liver. CONCLUSIONS: These results demonstrate that aged mice are more susceptible to ethanol-induced gut barrier dysfunction and liver inflammation, even at moderate doses of ethanol. This increased vulnerability to ethanol's gastrointestinal effects has important implications for alcohol use in the aging population. Future studies will explore whether improving intestinal barrier function can reverse these age-related changes.

Advanced age exacerbates intestinal epithelial permeability after burn injury in mice.

BACKGROUND: Advanced age is an independent risk factor for morbidity and mortality after burn injury. Following burn, the intestines can become permeable leading to the leakage of bacteria and their products from the lumen of the ileum to the portal and systemic circulation. Here, we sought to determine the effects of advanced age on intestinal permeability post burn injury and assess intestinal inflammatory biomarkers. METHODS: Young (4-5 months) and aged (18-22 months) female BALB/cBy mice were subjected to a 12-15% total body surface area (TBSA) sham or burn injury. 24 h after injury, mice were euthanized, and organs collected. Colony-forming units (CFU) were counted from plated mesenteric lymph nodes (MLN). Gene expression of ileal tight junctional proteins, occludin and zonula occludens 1 (ZO-1), in addition to ileal damage associated molecular pattern (DAMP) proteins, S100A8 and S100A9, as well as ileal inflammatory markers IL-6 and TNF-α were measured by qPCR. Intestinal cell death was measured by ELISA. Intestinal permeability was determined by FITC fluorescence in serum; 4kD FITC-dextran was given by oral gavage 3 h before euthanasia. RESULTS: Aged mice subjected to burn injury had increased intestinal permeability as evidenced by a 5.8-fold higher level of FITC-dextran in their serum when compared to all other groups (p < 0.05). In addition, aged burn-injured mice exhibited heightened bacterial accumulation in the MLN with a 15.5-fold increase over all other groups (p < 0.05). Histology of ileum failed to show differences in villus length among all groups. Analysis of ileal tight junctional proteins and inflammatory marker gene expression revealed no difference in Ocln, Tjp1, Il6, or Tnf expression among all groups, but 2.3 and 2.9-fold upregulation of S100a8 and S100a9, respectively, in aged burn-injured mice relative to both young groups and aged sham-injured mice (p < 0.05). Lastly, cell death in the ileum was elevated more than two-fold in aged burn-injured mice relative to young animals regardless of injury (p < 0.05). CONCLUSIONS: These data demonstrate that advanced age exacerbates intestinal epithelial permeability after burn injury. Heightened apoptosis may be responsible for the elevated intestinal leakiness and accumulation of bacteria in mesenteric lymph nodes. In addition, S100a8/9 may serve as a biomarker of elevated inflammation within the intestine.