Am J Physiol Endocrinol Metab. 2014 Jul 1;307(1):E34-46.

FFA-induced hepatic insulin resistance in vivo is mediated by PKCδ, NADPH oxidase, and oxidative stress.

Pereira S1, Park E1, Mori Y1, Haber CA1, Han P1, Uchida T1, Stavar L2, Oprescu AI3, Koulajian K1, Ivovic A1, Yu Z2, Li D2, Bowman TA4, Dewald J5, El-Benna J6, Brindley DN5, Gutierrez-Juarez R7, Lam TK1, Najjar SM4, McKay RA8, Bhanot S8, Fantus IG2, Giacca A9.
  • 1Department of Physiology, University of Toronto, Toronto, Ontario, Canada;
  • 2Department of Physiology, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, University of Toronto, Toronto, Ontario, Canada; and.
  • 3Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada;
  • 4Center for Diabetes and Endocrine Research and the Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, Ohio; and.
  • 5Metabolic and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, University of Alberta, Edmonton, Alberta, Canada;
  • 6Inserm, U1149, CNRS-ERL8252, Centre de Recherche sur l’Inflammation, Paris, France; Laboratoire d’Excellence Inflamex, Faculté de Médecine, Université Paris Diderot, Sorbonne Paris Cité, Site Xavier Bichat, Paris, France;
  • 7Department of Medicine, Diabetes Research Center, Albert Einstein College of Medicine, Bronx, New York;
  • 8ISIS Pharmaceuticals Inc., Carlsbad, California.
  • 9Department of Physiology, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, University of Toronto, Toronto, Ontario, Canada; and Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; adria.giacca@utoronto.ca.

 

Abstract

Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKβ- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKβ and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA → PKCδ → NADPH oxidase and oxidative stress → IKKβ/JNK → impaired hepatic insulin signaling.

KEYWORDS: antioxidant; antisense oligonucleotides; free fatty acids; hyperinsulinemic-euglycemic clamp; insulin resistance

PMID: 24824652

 

Supplements:

Obesity is a state of insulin resistance.   An increase in circulating levels of fatty acids is found in obesity due to release from expanded fat stores and fatty acids cause insulin resistance.  The objective of our paper was to determine how fatty acids cause insulin resistance and we focused on the liver, which is a primary site of insulin action.  We selectively increased levels of fatty acids in the circulation of normal rats by infusing i.v. a triglyceride emulsion and heparin, which favors the release of fatty acids from triglycerides.  Because oxidative stress had been implicated in insulin resistance in general, we started by asking whether an antioxidant, N-acetyl-L-cysteine (NAC), prevented insulin resistance in the liver caused by increased fatty acids in the circulation.  We found that NAC, which reduced oxidative stress in the liver, also prevented insulin resistance in the liver.  Next, we tested whether a kinase, protein kinase C (PKC)-δ, which we had shown to be activated by fatty acids, could be causing the increase in oxidative stress and insulin resistance.  Since reduced expression of PKC-δ in the liver improved insulin sensitivity, we concluded that PKC-δ was causing insulin resistance.  Our data also suggested that PKC-δ activates the enzyme NADPH oxidase, which produces reactive oxygen species causing oxidative stress.  In order to establish a relationship between oxidative stress and dampened insulin signaling, we studied kinases that were known to be activated by oxidative stress and to reduce the insulin signal.  We found that markers of activation of the inflammatory kinase IKKβ and the stress kinase JNK were increased by fatty acids, but this was prevented when NAC was administered simultaneously.  Our observations are summarized in Figure 1.  We conclude that this sequential pathway PKC-δ → NADPH oxidase → oxidative stress→ IKKβ/JNK is activated in the liver to cause impaired insulin signaling (i.e., insulin resistance) in response to increased circulating levels of fatty acids.  In addition, we showed that fatty acid- induced impairment in insulin signaling results in decreased removal of insulin from the circulation by the liver, and this is also prevented by NAC, although the mechanism requires further studies. In summary, we have identified a pathway whose interruption may have therapeutic implication for the improvement of insulin sensitivity in obesity, and thus, the prevention of type 2 diabetes.

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