Here, we study the intricate relationship between gut microbiota and host cometabolic phenotypes associated with dietary-induced impaired glucose homeostasis and nonalcoholic fatty liver disease (NAFLD) in a mouse strain (129S6) known to be susceptible to these disease traits, using plasma and urine metabotyping, achieved by ¹ H NMR spectroscopy. Multivariate statistical modeling of the spectra shows that the genetic predisposition of the 129S6 mouse to impaired glucose homeostasis and NAFLD is associated with disruptions of choline metabolism, i.e., low circulating levels of plasma phosphatidylcholine and high urinary excretion of methylamines (dimethylamine, trimethylamine, and trimethylamine- N -oxide), coprocessed by symbiotic gut microbiota and mammalian enzyme systems. Conversion of choline into methylamines by microbiota in strain 129S6 on a high-fat diet reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing NAFLD. These data also indicate that gut microbiota may play an active role in the development of insulin resistance.

The nuclear receptor peroxisome proliferator-activated receptor δ [PPARδ/β (NR1C2)] has been implicated in colorectal carcinogenesis by various molecular genetic observations. These observations have recently been supported by studies of activation of PPARδ by pharmacological agents. Here we present the first report of the stimulation of breast and prostate cancer cell growth using PPARδ selective agonists. Activation of PPARδ with compound F stimulated proliferation in breast (T47D, MCF7) and prostate (LNCaP, PNT1A) cell lines, which are responsive to sex hormones. Conversely, we have found that several steroid-independent cell lines, including colon lines, were unresponsive to compound F. These findings were confirmed with an additional high-affinity PPARδ agonist, GW501516. Conditional expression of PPARδ in MCF7 Tet-On cells resulted in a doxycycline-enhanced response to GW501516, thus providing direct genetic evidence for the role of PPARδ in the proliferative response to this drug. Activation of PPARδ in T47D cells resulted in increased expression of the proliferation marker Cdk2 and also vascular endothelial growth factor α (VEGFα) and its receptor, FLT-1, thus, suggesting that PPARδ may initiate an autocrine loop for cellular proliferation and possibly angiogenesis. Consistent with this hypothesis, we demonstrated a pro-proliferative effect of GW501516 on human umbilical vein endothelial cell cultures and found that GW501516 also regulated the expression of VEGFα and FLT-1 in these cells. Our observations provide the first evidence that activation of PPARδ can result in increased growth in breast and prostate cancer cell lines and primary endothelial cells and supports the possibility that PPARδ antagonists may be of therapeutic value in the treatment of breast and prostate cancer.

Complex changes in gene expression are associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD) promoted by feeding a high-fat diet (HFD). We used functional genomic technologies to document molecular mechanisms associated with diet-induced NAFLD.

Insulin resistance plays a central role in type 2 diabetes and obesity, which develop as a consequence of genetic and environmental factors. Dietary changes including high fat diet (HFD) feeding promotes insulin resistance in rodent models which present useful systems for studying interactions between genetic background and environmental influences contributing to disease susceptibility and progression. We applied a combination of classical physiological, biochemical and hormonal studies and plasma ¹H NMR spectroscopy-based metabonomics to characterize the phenotypic and metabotypic consequences of HFD (40%) feeding in inbred mouse strains (C57BL/6, 129S6, BALB/c, DBA/2, C3H) frequently used in genetic studies. We showed the wide range of phenotypic and metabonomic adaptations to HFD across the five strains and the increased nutrigenomic predisposition of 129S6 and C57BL/6 to insulin resistance and obesity relative to the other strains. In contrast mice of the BALB/c and DBA/2 strains showed relative resistance to HFD-induced glucose intolerance and obesity. Hierarchical metabonomic clustering derived from ¹H NMR spectral data of the strains provided a phylometabonomic classification of strain-specific metabolic features and differential responses to HFD which closely match SNP-based phylogenetic relationships between strains. Our results support the concept of genomic clustering of functionally related genes and provide important information for defining biological markers predicting spontaneous susceptibility to insulin resistance and pathological adaptations to fat feeding.

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal‐cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial‐cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU 145 and an SV40 large T‐immortalized normal epithelial‐cell line, PNTIA, in double‐diffusion co‐culture chambers. PNTIA was stimulated by PNX (x 1.6) and more strongly by BH101 stromal cells (x 2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101‐conditioned medium (CM), obtained in serum‐free conditions, induced 90% inhibition of [³H]thymidine incorporation of the LNCaP androgen‐sensitive cell line. Two other androgen‐independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL‐Iβ, IL‐6 and IL‐8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL‐6. IL‐6 was demonstrated to be responsible for the strong inhibitory effect since an IL‐6‐neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL‐6. Recombinant IL‐6 growth inhibition was observed only on LNCaP prostate cancer androgen‐sensitive cells. © 1996 Wiley‐Liss, Inc.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) are used as cancer chemopreventative agents, their mechanism is unclear because NSAIDs have cyclooxygenase-independent actions. We investigated an alternative target for NSAIDs, peroxisome proliferator-activated receptor-γ (PPARγ), activation of which decreases cancer cell proliferation. NSAIDs have been shown to activate this receptor, but only at high concentrations. Here, we have examined binding of diclofenac to PPARγ using a cis-parinaric acid displacement assay and studied the effect of diclofenac effect on PPARγ trans-activation in a COS-1 cell reporter assay. Unexpectedly, diclofenac bound PPARγ at therapeutic concentrations (K_i = 700 nM) but induced only 2-fold activation of PPARγ at a concentration of 25 μM and antagonized PPARγ trans-activation by rosiglitazone. This antagonism was overcome with increasing rosiglitazone concentrations, indicating that diclofenac is a partial agonist. No effect of diclofenac was seen without exogenous receptor, confirming that it was working through a PPARγ-specific mechanism. This is the first description of an NSAID that can antagonize PPARγ. In addition, this is the first time that an NSAID has been shown to bind this receptor at clinically meaningful concentrations. The physiological relevance of these findings was tested using adipocyte differentiation and cancer cell proliferation assays. Diclofenac decreased PPARγ-mediated adipose cell differentiation by 60% and inhibited the action of rosiglitazone on the prostate cancer cell line, DU-145, allowing a 3-fold increase in proliferation. This work shows that standard doses of diclofenac may have pharmacodynamic interactions with rosiglitazone and this has therapeutic implications, both in the management of type 2 diabetes and during cancer treatment.