Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents
Stephen C. Benoit, … , William L. Holland, Deborah J. Clegg
Stephen C. Benoit, … , William L. Holland, Deborah J. Clegg
Published August 10, 2009
Citation Information: J Clin Invest. 2009;119(9):2577-2589. https://doi.org/10.1172/JCI36714.
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Research Article

Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

Abstract

Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity.

Authors

Stephen C. Benoit, Christopher J. Kemp, Carol F. Elias, William Abplanalp, James P. Herman, Stephanie Migrenne, Anne-Laure Lefevre, Céline Cruciani-Guglielmacci, Christophe Magnan, Fang Yu, Kevin Niswender, Boman G. Irani, William L. Holland, Deborah J. Clegg

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Figure 5

Saturated fatty acids increased PKC-θ translocation to the membrane.

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Saturated fatty acids increased PKC-θ translocation to the membrane. To ...
To confirm our protocol to isolate membrane from cytosolic fractions, medial basal hypothalamic cell lysate was ultracentrifuged. (A) The membrane fraction was stained with membrane-associated proteins, IR, SNAP 25, and Histone 3, a nuclear protein. Rats (n = 8–10/group), gavaged 3 times per day for 3 days with palmitic acid, oleic acid, or vehicle had no change in body weight (data not shown). Western blots were performed in 3 replications. There was a significant (P < 0.05) increase in medial basal hypothalamic membrane translocation of PKC-θ in the palmitic acid–gavaged animals but not the oleic acid– or vehicle-gavaged animals. Cyt, cytosolic; NUC, nuclear; TOT, total. (B) The GAPDH control was run on the same blot as AKT. Quantification (mean ± SEM) of 3 representative blots (n = 3/group) in C (vehicle and palmitic acid lanes were run on the same gel but were noncontiguous; oleic acid was run on a separate gel but under the same conditions; GAPDH lanes were run on separate gels). There was a significant decrease in medial basal hypothalamic cytosolic location of PKC-θ. (D) Quantified data (mean ± SEM) from 2 separate Western blots (n = 4/group), of which E is a representative blot. Cell membrane content of medial basal hypothalamic PKC-θ was also increased by i.c.v. osmotic minipump infusion of palmitic acid relative to oleic acid or vehicle. (F) Quantification (mean ± SEM) from 2 separate Western blots (n = 4–5/group), with a representative of the blot shown in G (vehicle, palmitic acid, and oleic acid lanes were run on the same gel but were noncontiguous). No significant change in cytosolic PKC-θ was found. (H) Quantification (mean ± SEM) from 2 separate Western blots (n = 4–5/group), representative of the blot shown in I (vehicle, palmitic acid, and oleic acid lanes were run on the same gel but were noncontiguous). (J) Represents increased serine phosphorylation of IRS-1, which is associated with reductions in insulin signaling in the animals i.c.v. infused the palmitic acid but not the oleic acid or vehicle infusion. (*P < 0.05 compared with vehicle treatment; quantification [mean ± SEM] from 3 separate Western blots [n = 5–6/group]).