Total RNA was p38 inhibitors clinical trials prepared and analyzed by real-time polymerase chain reaction (PCR) as previously described using primer pairs detailed in Supporting Table 1. Data are presented as mean ± standard error (SE) unless otherwise noted. Differences between means were evaluated using an unpaired two-sided Student t test (P < 0.05 considered significant; Microsoft Excel). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by analysis of variance (ANOVA) with Dunnett's test for post-hoc analysis. Surveying messenger RNA (mRNA) expression of Fabp family members in quiescent (day 1) and activated (day 7) primary mouse HSCs revealed L-Fabp
to be the most abundantly expressed member and, unlike other Fabp family members (Fig. 1A), decreased by >90% upon HSC activation, with a gradual decline in L-Fabp mRNA (Fig. 1B) and protein (Fig. 1C) abundance from 3 to 7 days of culture. Freshly isolated HSCs from wild-type (WT) mice manifest abundant (oil-red-O staining) LDs, as expected (Fig. 1D). By contrast, intracellular LDs were less abundant in freshly isolated HSCs from L-FABP−/− mice (day 1) and almost undetectable this website by day 3. We also examined mRNA abundance of α-SMA and α(I)I collagen (αI(I)Col), as representative markers of HSC activation.8, 21 These
mRNAs were barely detectable in freshly isolated HSCs from WT mice, increasing ∼5 fold after culture (Fig. 1E, left panel), as expected.21 By contrast, expression of α-SMA and αI(I)Col mRNAs were readily detected in HSCs
from L-FABP−/− mice after 1 day of culture (Fig. 1E, right panel), with continued up-regulation after 7 days. Taken together, these findings suggest that L-Fabp may play a role in LD accumulation selleck compound and activation of HSCs in vitro. Based on the coupled observations of a decline in L-Fabp expression with decreased lipid accumulation and increased activation of HSCs, we asked whether forced expression of L-Fabp would modulate lipid content and the patterns of FA utilization. Ad-L-Fabp transduction of cultured HSCs (Fig. 2A) increased both cellular FA and TG content (Fig. 2B,C) and revealed enrichment with palmitic acid (C16:0) as the major FA species (Fig. 2D). These findings suggest that rescuing L-Fabp expression in cultured HSCs reverses lipid depletion and leads to enrichment in 16:0 FA. In line with these findings, the FA profile in freshly isolated HSCs from L-FABP−/− mice revealed depletion of 16:0 with a shift to 18:0, 18:1, and 18:2 species in the free FA pool (Fig. 2E). HSC triglyceride species, however, were comparable between the genotypes (Fig. 2F). These findings demonstrate corresponding gain- and loss-of-function effects of L-Fabp on the FA profile in passaged HSCs transduced with Ad-L-Fabp and in freshly isolated HSCs from L-FABP−/− mice, respectively, each approach revealing a role for L-Fabp in modulating palmitate abundance.