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PUFA Receptors vs. Proteolysis pt. 4 - Supporting Documents

After establishing an hypothesis, it ought to be put it to the test. Outside of novel experimentation, currently preferred to justify salary and materials budgets, you can apply your ideas to previous experiments and see how they fit. While reading on mechanisms for PUFA inhibition of SREBP-1, I came across a few papers that serve as testing opportunities. “Polyunsaturated Fatty Acids Suppress Sterol Regulatory Element Binding Protein 1c Promoter Activity by Inhibition of Liver X Receptor (LXR) Binding to LXR Response Elements” is a 2002 manuscript from a Japanese research group that attempted to show PUFA interaction with LXR interferes with its ability to bind to response elements in the SREBP1c transcriptional domain and upregulate transcription (1). Within the proteolytic inhibition hypothetical framework the explanation would be that PUFA prevent the cleavage of SREBP-1 from the cytoplasmic matrix, thus blocking both its cellular actions and auto-upregulation of nuclear transcription and translation.

In the introduction, the SREBP1c auto-loop is acknowledged but promptly forgotten and never subsequently considered as acting in the experiments to follow. That is to say that all increases in SREBP1c activation as measured by plasmid transfected luciferase assay are credited to LXR response elements, one half of their introduced mechanism for SREBP1c upregulation. They do not directly measure LXR binding to response elements and increasing transcription; an inhibition of the SREBP1 auto-loop from cytoplasmic sequestering could explain all of their findings.

They begin with the idea that PUFA acts through binding with PPARs. This was quickly abandoned because SREBP1c inhibition was not achieved by addition of synthetic PPAR agonists, a strong piece of evidence against the PUFA receptor theory and something fairly well ignored by the hundreds of scientists still using that model in their experimental interpretations. This paper also contributes some handsome figures showing, more or less in order of increasing chain length and degree of unsaturation, the inhibition of SREBP1c via reduced luciferase activity.

EtOH = ethanol, SA = stearic acid, OA = oleic acid, LA = linoleic acid, DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, AA = arachidonic acid

This is precisely what one would predict using the PUFA proteolytic inhibition hypothesis.

Coincidentally, while researching ammonia metabolism, a couple of papers by Rowland et al. in Australia provide more cross-disciplinary evidence for the PUFA proteolytic inhibition hypothesis. In 2007 (2) and 2008 (3) experiments were performed on the ability of albumin to enhance drug clearance via glucuronidation. The mechanism in both papers was binding of “inhibitory fatty acids,” their term for the PUFA that inhibit the glucuronosyltransferases. The measured inhibition came in the form of an increased Km, or the concentration of substrate necessary to achieve half of the maximal reaction velocity (Vmax), without changing the Vmax. Bovine serum albumin and fatty acid free human serum albumin, up to 2% concentration, decreased reaction Km while Vmax stayed unchanged. Crude human serum albumin, containing approximately ten times the concentration of fatty acids as the other serum albumin reagents, decreased reaction Km at 0.1% concentration but increased Km at higher concentrations, a switch from potentiation to inhibition.

This is characteristic of competitive inhibition, where the enzyme is capable of performing at the same rate given enough substrate in excess of its competitor; the enzyme itself is not damaged. This same inhibition was achieved by addition of unsaturated fatty acids alone.

Having not extended this investigation into other enzyme types, I nonetheless would be surprised if many enzymes are not generally inhibited by PUFA. The largest source of PUFA production is within plant seeds grown in moderate and cold climates, where they are most likely used to inhibit the enzymatic sprouting processes until conditions are ideal. After sprouting, the sprout rapidly loses its PUFA content relative to the seed, never to return in any tissue save newly formed seeds. Warm blooded animals, mammals and birds, only produce saturated and monounsaturated fatty acids. This would be passing strange if PUFA were “essential” or beneficial to cellular processes in animals.

1.      Yoshikawa T, Shimano H, Yahagi N, Ide T, Amemiya-Kudo M, Matsuzaka T, et al. Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements. J Biol Chem. 2002;277(3):1705–11.

2.      Rowland A, Gaganis P, Elliot DJ, Mackenzie PI, Knights KM, Miners JO. Binding of inhibitory fatty acids is responsible for the enhancement of UDP-glucuronosyltransferase 2B7 activity by albumin: implications for in vitro-in vivo extrapolation. J Pharmacol Exp Ther. 2007;321(1):137–47.

3.      Rowland A, Knights KM, Mackenzie PI, Miners JO. The “albumin effect” and drug glucuronidation: Bovine serum albumin and fatty acid-free human serum albumin enhance the glucuronidation of UDP-glucuronosyltransferase (UGT) 1A9 substrates but not UGT1A1 and UGT1A6 activities. Drug Metab Dispos. 2008;36(6):1056–62.