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

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 IKKß- 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 IKKß 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 → IKKß/JNK → impaired hepatic insulin signaling.

Deficiency of a glycogen synthase-associated protein, Epm2aip1, causes decreased glycogen synthesis and hepatic insulin resistance.

Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.

Overexpression of glutathione peroxidase 4 prevents ß-cell dysfunction induced by prolonged elevation of lipids in vivo.

We have shown that oxidative stress is a mechanism of free fatty acid (FFA)-induced ß-cell dysfunction. Unsaturated fatty acids in membranes, including plasma and mitochondrial membranes, are substrates for lipid peroxidation, and lipid peroxidation products are known to cause impaired insulin secretion. Therefore, we hypothesized that mice overexpressing glutathione peroxidase-4 (GPx4), an enzyme that specifically reduces lipid peroxides, are protected from fat-induced ß-cell dysfunction. GPx4-overexpressing mice and their wild-type littermate controls were infused intravenously with saline or oleate for 48 h, after which reactive oxygen species (ROS) were imaged, using dihydrodichlorofluorescein diacetate in isolated islets, and ß-cell function was assessed ex vivo in isolated islets and in vivo during hyperglycemic clamps. Forty-eight-hour FFA elevation in wild-type mice increased ROS and the lipid peroxidation product malondialdehyde and impaired ß-cell function ex vivo in isolated islets and in vivo, as assessed by decreased disposition index. Also, islets of wild-type mice exposed to oleate for 48 h had increased ROS and lipid peroxides and decreased ß-cell function. In contrast, GPx4-overexpressing mice showed no FFA-induced increase in ROS and lipid peroxidation and were protected from the FFA-induced impairment of ß-cell function assessed in vitro, ex vivo and in vivo. These results implicate lipid peroxidation in FFA-induced ß-cell dysfunction.

In vitro and in vivo testing of glucose-responsive insulin-delivery microdevices in diabetic rats.

We have developed glucose-responsive implantable microdevices for closed-loop delivery of insulin and conducted in vivo testing of these devices in diabetic rats. The microdevices consist of an albumin-based bioinorganic membrane that utilizes glucose oxidase (GOx), catalase (CAT) and manganese dioxide (MnO(2)) nanoparticles to convert a change in the environmental glucose level to a pH stimulus, which regulates the volume of pH-sensitive hydrogel nanoparticles and thereby the permeability of the membrane. The membrane is integrated with microfabricated PDMS (polydimethylsiloxane) structures to form compact, stand-alone microdevices, which do not require tethering wires or tubes. During in vitro testing, the microdevices showed glucose-responsive insulin release over multiple cycles at clinically relevant glucose concentrations. In vivo, the microdevices were able to counter hyperglycemia in diabetic rats over a one-week period. The in vitro and in vivo testing results demonstrated the efficacy of closed-loop biosensing and rapid response of the 'smart' insulin delivery devices.