Glucose Directly Links to Lipid Metabolism through High Affinity Interaction with Peroxisome Proliferator-activated Receptor α

Abstract

The pathophysiology of diabetes is characterized not only by elevated glucose but also elevated long chain fatty acid levels. We show for the first time that the peroxisome proliferatoractivated receptor-␣ (PPAR␣) binds glucose and glucose metabolites with high affinity, resulting in significantly altered PPAR␣ secondary structure. Glucose decreased PPAR␣ interaction with fatty acid metabolites and steroid receptor coactivator-1 while increasing PPAR␣ interaction with DNA. Concomitantly, glucose increased PPAR␣ interaction with steroid receptor coactivator-1, DNA binding, and transactivation of ␤-oxidation pathways in the presence of activating ligands. Heterodimerization of PPAR␣ to the retinoid X receptor-␣ resulted in even larger increases in transactivation with the addition of glucose. These data suggest that PPAR␣ is responsible for maintaining energy homeostasis through a concentration-dependent regulation of both lipids and sugars and that hyperglycemic injury mediated by PPAR␣ occurs not only indirectly through elevated long chain fatty acid levels but also through direct action of glucose on PPAR␣.Energy homeostasis is a highly complex and strictly regulated process. Free fatty acids compete with glucose for oxidation, and increased free fatty acid concentrations are associated with reduced muscle glycogen synthesis. Dysregulation at any step may elicit severe pathophysiological complications, as seen in diabetes. Maintained low levels of blood glucose are critical for preventing or delaying the clinical complications of diabetes, such as insulin resistance and cardiovascular disease (1). Although the liver plays essential roles in the control of blood glucose levels by modulating gluconeogenesis and glycogen synthesis, the specific mechanism(s) of this regulation is unclear. Several studies suggest that peroxisome proliferatoractivated receptor ␣ (PPAR␣), 2 a ligand-regulated transcription factor belonging to the nuclear hormone receptor superfamily, contributes to this regulation. PPAR␣ is highly expressed in liver and is the target of potent hypolipidemic drugs, such as fibrates, used to treat cardiovascular disease (2). Although a variety of compounds bind and activate PPAR␣, long chain fatty acids (LCFA) and their metabolites (i.e. long chain fatty acyl-CoAs, LCFA-CoA) function as high affinity, endogenous ligands (3-5), which could play an important role because diabetes is characterized not only by elevated glucose levels but also elevated LCFA and LCFA-CoA levels (6). Ligand binding initiates PPAR␣ transcription of multiple genes in fatty acid and glucose metabolism while concomitantly down-regulating genes in insulin signaling (7-9). Furthermore, expression of PPAR␣ is elevated in humans with type 2 diabetes (10), and PPAR␣-null mice are protected from high fat diet-induced insulin resistance (11). EXPERIMENTAL PROCEDURESProtein Expression and Purification-The bacterial expression vector containing murine PPAR␣ (pET-PPAR␣⌬AB) was expressed in the BL21(DE3)pLysS strain of Escherichia coli as de...

Glucose regulates fatty acid binding protein interaction with lipids and peroxisome proliferator-activated receptor α

“…L-FABP is thought to function as a shuttle that delivers these metabolites to the nucleus for interaction with nuclear receptors, such as PPAR ␣ ( 15,19,20 ). Since glucose potentiates PPAR ␣ activation by LCFA ( 13,14 ), a potential effect of glucose on L-FABP and/or the L-FABP-PPAR ␣ interaction could play an important role in the maintenance of energy homeostasis. To begin to resolve the role L-FABP may play in such regulation, the effect of glucose directly on L-FABP and the PPAR ␣ -L-FABP complex was examined.…”

“…To determine glucose effects on protein secondary structure, circular dichroic (CD) spectra of 0.8 M PPAR ␣ ⌬ AB, 2.4 M L-FABP, and equal amino acid molar concentrations of the two proteins [0.4 M PPAR ␣ ⌬ AB + 1.2 M L-FABP] were taken in the absence and presence of glucose, maltose, and glucose metabolites with a J-815 spectropolarimeter (JASCO, Easton, MD) as previously described ( 13,20 ). Ten scans per replicate were averaged for percent composition of secondary structures by three different methods (SELCON3, CDSSTR, and CONTIN/LL) with the software package CDPro ( 23 ) as previously described ( 13,20 ).…”

“…Next, protein mixtures were incubated with anti bodylinked resin (rabbit polyclonal anti-PPAR ␣ [Affi nity BioReagents, Golden, CO] or goat polyclonal anti-L-FABP [Santa Cruz Biotechnology, Santa Cruz, CA]) for 1 1/2 h at room temperature according to the manufacturers' instructions (ProFound TM mammalian co-immunoprecipitation kit, Pierce Biotechnology, Rockford, IL). Eluted proteins were examined by SDS-PAGE analysis as previously described ( 13 ). Values were normalized to the amount of protein detected from the control (e.g., the protein used for co-immunoprecipitation [CoIP]), and samples in absence of glucose were arbitrarily set to 100 percent.…”

“…Ten scans per replicate were averaged for percent composition of secondary structures by three different methods (SELCON3, CDSSTR, and CONTIN/LL) with the software package CDPro ( 23 ) as previously described ( 13,20 ). The CD spectrum of the mixed proteins was compared with a theoretical spectrum of combined but noninteracting proteins in the absence and presence of sugar.…”

“…The parinaric lipids were excited at 317 nm, and emission measured from 380 nm to 450 nm. Data were corrected for background (buffer, fl uorescent ligands, and solvent effects), and maximal intensities were used to calculate the percent change in LCFA or LCFA-CoA binding as previously described ( 13 ).…”