Research Description

Cellular and biochemical basis of insulin action and insulin resistance: focus on glucose transport

Glucose is the major energy substrate for most cells, and it is avidly stored as glycogen in the liver and muscle tissue, as well as processed into fat in adipose tissue. The liver provides the rest of the body with glucose between meals, especially the brain. However, during a meal, insulin derived from the pancreas vigorously promotes glucose uptake into muscle and fat cells and stops the liver from releasing glucose to the blood. The mechanism whereby insulin increases glucose uptake into muscle/fat has received much attention but is not completely understood. Insulin resistance, a key defect in type 2 diabetes, involves defective responses to insulin in muscle, adipose and hepatic tissues.

Klip’s laboratory has been studying the regulation of glucose uptake by insulin and muscle contraction, using an array of rat and mouse stable muscle cell lines that they have generated. The focus is the intracellular traffic of vesicles containing glucose transporters, primarily GLUT4. Current work focuses on how a series of signal transduction pathways activated by insulin within muscle cells impinge on intracellular stores of GLUT4, how the vesicles move to the cell surface, and how they are further turned-on into faster carriers. This recruitment of transporters and their subsequent activation requires participation of the actin cytoskeleton for faithful congregation of the pertinent signals and gathering of GLUT4 below the plasma membrane.

Her lab’s studies have revealed two important bifurcations in insulin action, one defined by different outcomes of  the insulin receptor substrate 1 (IRS-1) protein and insulin receptor substrate  2 (IRS-2) protein, the other defined by two signalling arms downstream of phosphatidylinositol 3-kinase (PI 3-kinase). The Akt/PKB arm, a serine/threonine protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration, leads to the inactivation of the RAb-GAP AS160 (Akt substrate of 160 kDa), which acts through distinct Rab molecules to mobilize and position GLUT4 vesicles. In muscle cells, their work identifies Rab8A, Rab13 and Rab14 as important mediators of these steps. On the other hand, the Rac activation arm functions to rapidly remodel actin filaments into a cortical mesh below the cell surface, as shown through a dynamic cycle of actin branching mediated by Arp2/3 and severing mediated by actin-binding proteins know as cofilin. Vesicles mobilized to the cell cortex interact with the actin mesh through actinin-4, possibly in preparation for optimal docking and fusion with the membrane. Klip’s team has also identified the SNARE molecules VAMP2, syntaxin4 and SNAP23 as mediators of this docking/fusion step. GLUT4 arrives at the cell surface in a state of low activity but are soon activated by a pathway that may involve the removal of ancillary inhibitors or the binding of activators of GLUT4. In particular, GAPDH binds to GLUT4 as Hexokinase II is displaced, possibly accounting for the activation of the transporter.

In a new series of studies, the Klip lab has also established cellular models to study insulin resistance:

1. Exposure of muscle cell cultures to high glucose and high insulin, emulating the environment in type 2 diabetes;
2. Exposure to high saturated fats or their derivative ceramides and reactive oxygen species, emulating the lipotoxic component of the metabolic syndrome; and
3. Exposure to conditioned media from palmitate-treated macrophages, emulating the inflammation component of insulin resistance.

In each case, GLUT4 translocation in response to insulin was dampened, but interestingly through distinct signalling defects, respectively: reduction in IRS-1 phosphorylation, in Rac activation and in Akt and AS160 phosphorylation. These findings suggest that insulin resistance in vivo must be analyzed at each signalling level and may require distinct and specific interventional therapies.

Publications

View all publications on PubMed

Calcium signaling in insulin action on striated muscle.
Contreras-Ferrat A, Lavandero S, Jaimovich E, Klip A.
Cell Calcium. 2014 Sep 6. pii: S0143-4160(14)00135-3.  Read

Nucleotides released from palmitate-challenged muscle cells through pannexin-3 attract monocytes.
Pillon NJ, Li YE, Fink LN, Brozinick JT, Nikolayev A, Kuo MS, Bilan PJ, Klip A.
Diabetes. 2014 Nov;63(11):3815-26.  Read

Mice lacking NOX2 are hyperphagic and store fat preferentially in the liver.
Costford SR, Castro-Alves J, Chan KL, Bailey LJ, Woo M, Belsham DD, Brumell JH, Klip A.
Am J Physiol Endocrinol Metab. 2014 Jun 15;306(12):E1341-53.  Read

Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide.
Foley KP, Klip A.
Biol Open. 2014 Apr 4;3(5):314-25.  Read