Timothy E. McGraw

The Akt kinases: isoform specificity in metabolism and cancer

The Akt (PKB) protein kinases are critical regulators of human physiology that control an impressive array of diverse cellular functions, including the modulation of growth, survival, proliferation and metabolism. The Akt kinase family is comprised of three highly homologous isoforms: Akt1 (PKBα), Akt2 (PKBβ) and Akt3 (PKBγ). Phenotypic analyses of Akt isoform knockout mice documented Akt isoform specific functions in the regulation of cellular growth, glucose homeostasis and neuronal development. Those studies establish that the functions of the different Akt kinases are not completely overlapping and that isoform-specific signaling contributes to the diversity of Akt activities. However, despite these important advances, a thorough understanding about the specific roles of Akt family members and the molecular mechanisms that determine Akt isoform functional specificity will be essential to elucidate the complexity of Akt regulated cellular processes and how Akt isoform-specific deregulation might contribute to disease states. Here, we summarize recent advances in understanding the roles of Akt isoforms in the regulation of metabolism and cancer, and possible mechanisms contributing to Akt isoform functional specificity.

Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects.

Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice. FAHFA isomers differ by the branched ester position on the hydroxy fatty acid (e.g., palmitic-acid-9-hydroxy-stearic-acid, 9-PAHSA). PAHSAs are synthesized in vivo and regulated by fasting and high-fat feeding. PAHSA levels correlate highly with insulin sensitivity and are reduced in adipose tissue and serum of insulin-resistant humans. PAHSA administration in mice lowers ambient glycemia and improves glucose tolerance while stimulating GLP-1 and insulin secretion. PAHSAs also reduce adipose tissue inflammation. In adipocytes, PAHSAs signal through GPR120 to enhance insulin-stimulated glucose uptake. Thus, FAHFAs are endogenous lipids with the potential to treat type 2 diabetes.

The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane

To perform vectorial secretory and transport functions that are critical for the survival of the organism, epithelial cells sort plasma membrane proteins into polarized apical and basolateral domains. Sorting occurs post-synthetically, in the trans Golgi network (TGN) or after internalization from the cell surface in recycling endosomes, and is mediated by apical and basolateral sorting signals embedded in the protein structure. Basolateral sorting signals include tyrosine motifs in the cytoplasmic domain that are structurally similar to signals involved in receptor internalization by clathrin-coated pits. Recently, an epithelial-specific adaptor protein complex, AP1B, was identified. AP-1B recognizes a subset of basolateral tyrosine motifs through its μ1B subunit. Here, we characterized the post-synthetic and post-endocytic sorting of the fast recycling low density lipoprotein receptor (LDLR) and transferrin receptor (TfR) in LLC-PK1 cells, which lack μ1B and mis-sort both receptors to the apical surface. Targeting and recycling assays in LLC-PK1 cells, before and after transfection with μ1B, and in MDCK cells, which express μ1B constitutively, suggest that AP1B sorts basolateral proteins post-endocytically.

Metabolic surgery: the role of the gastrointestinal tract in diabetes mellitus

Several conventional methods of bariatric surgery can induce long-term remission of type 2 diabetes mellitus (T2DM); novel gastrointestinal surgical procedures are reported to have similar effects. These procedures also dramatically improve other metabolic conditions, including hyperlipidemia and hypertension, in both obese and nonobese patients. Several studies have provided evidence that these metabolic effects are not simply the results of drastic weight loss and decreased caloric intake but might be attributable, in part, to endocrine changes resulting from surgical manipulation of the gastrointestinal tract. In this Review, we provide an overview of the clinical evidence that demonstrates the effects of such interventions—termed metabolic surgery—on T2DM and discuss the implications for future research. In light of the evidence presented here, we speculate that the gastrointestinal tract might have a role in the pathophysiology of T2DM and obesity.

Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling

The 3 Akt protein kinase isoforms have critical and distinct functions in the regulation of metabolism, cell growth, and apoptosis, yet the mechanisms by which their signaling specificity is achieved remain largely unclear. Here, we investigated potential mechanisms underlying Akt isoform functional specificity by using Akt2-specific regulation of glucose transport in insulin-stimulated adipocytes as a model system. We found that insulin activates both Akt1 and Akt2 in adipocytes, but differentially regulates the subcellular distribution of these Akt isoforms. The greater accumulation of Akt2 at the plasma membrane (PM) of insulin-stimulated adipocytes correlates with Akt2-specific regulation of the trafficking of the GLUT4 glucose transporter. Consistent with this pattern, Akt constructs that do not accumulate at the PM to the same degree as Akt2 fail to regulate GLUT4 translocation to the PM, whereas enhancement of Akt1 PM association through mutation in Akt1 PH domain is sufficient to overcome Akt-isoform specificity in GLUT4 regulation. Indeed, we found that this distinct insulin-induced PM accumulation of Akt kinases is translated into a differential regulation by the Akt isoforms of AS160, a RabGAP that regulates GLUT4 trafficking. Our data show that Akt2 specifically regulates AS160 phosphorylation and membrane association providing molecular basis for Akt2 specificity in the modulation of GLUT4 trafficking. Together, our findings reveal the stimulus-induced subcellular compartmentalization of Akt kinases as a mechanism contributing to specify Akt isoform functions.

CD2AP/CMS Regulates Endosome Morphology and Traffic to the Degradative Pathway Through its Interaction with Rab4 and c‐Cbl

The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4‐Q67L, the GTP‐bound form of Rab4. We cloned an ubiquitous 80‐kDa protein, identical to CD2‐associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4‐Q67L in the yeast two‐hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS‐positive vesicles. However, coexpression of Rab4‐Q67L with CD2AP/CMS induces a significant enlargement of EEA1‐positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c‐Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c‐Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c‐Cbl inhibits ligand‐induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c‐Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.

GLUT4 is internalized by a cholesterol‐dependent nystatin‐sensitive mechanism inhibited by insulin

Insulin slows GLUT4 internalization by an unknown mechanism. Here we show that in unstimulated adipocytes, GLUT4 is internalized by two mechanisms. Approximately 80% of GLUT4 is internalized by a mechanism that is sensitive to the cholesterol‐aggregating drug nystatin, and is independent of AP‐2 clathrin adaptor and two putative GLUT4 endocytic motifs. The remaining GLUT4 is internalized by an AP‐2‐dependent, nystatin‐resistant pathway that requires the FQQI GLUT4 motif. Insulin inhibits GLUT4 uptake by the nystatin‐sensitive pathway and, consequently, GLUT4 is internalized by the AP‐2‐dependent pathway in stimulated adipocytes. The phenylalanine‐based FQQI GLUT4 motif promotes AP‐2‐dependent internalization less rapidly than a tyrosine‐based motif, the classic form of aromatic‐based motifs. Thus, both a change in the predominant endocytosis pathway and the specific use of a suboptimal internalization motif contribute to the slowing of GLUT4 internalization in insulin‐stimulated adipocytes. Insulin also inhibits the uptake of cholera‐toxin B, indicating that insulin broadly regulates cholesterol‐dependent uptake mechanisms rather than specially targeting GLUT4. Our work thus identifies cholesterol‐dependent uptake as a novel target of insulin action in adipocytes.

GLUT4 Distribution between the Plasma Membrane and the Intracellular Compartments Is Maintained by an Insulin-modulated Bipartite Dynamic Mechanism

The GLUT4 glucose transporter is predominantly retained inside basal fat and muscle cells, and it is rapidly recruited to the plasma membrane with insulin stimulation. There is controversy regarding the mechanism of basal GLUT4 retention. One model is that GLUT4 retention is dynamic, based on slow exocytosis and rapid internalization of the entire pool of GLUT4 (Karylowski, O., Zeigerer, A., Cohen, A., and McGraw, T. E. (2004) Mol. Biol. Cell 15, 870–882). In this model, insulin increases GLUT4 in the plasma membrane by modulating GLUT4 exocytosis and endocytosis. The second model is that GLUT4 retention is static, with ∼90% of GLUT4 stored in compartments that are not in equilibrium with the cell surface in basal conditions (Govers, R., Coster, A. C., and James, D. E. (2004) Mol. Cell Biol. 24, 6456–6466). In this model, insulin increases GLUT4 in the plasma membrane by releasing it from the static storage compartment. Here we show that under all experimental conditions examined, basal GLUT4 retention is by a bipartite dynamic mechanism involving slow efflux and rapid internalization. To establish that the dynamic model developed in studies of the extreme conditions of >100 nm insulin and no insulin also describes GLUT4 behavior at more physiological insulin concentrations, we characterized GLUT4 trafficking in 0.5 nm insulin. This submaximal insulin concentration promotes an intermediate effect on both GLUT4 exocytosis and endocytosis, resulting in an intermediate degree of redistribution to the plasma membrane. These data establish that changes in the steady-state surface/total distributions of GLUT4 are the result of gradated, insulin-induced changes in GLUT4 exocytosis and endocytosis rates.

Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor

Insulin resistance is a component of the metabolic syndrome and Type 2 diabetes. It has been recently shown that in liver insulin resistance is not complete. This so-called selective insulin resistance is characterized by defective insulin inhibition of hepatic glucose output while insulin-induced lipogenesis is maintained. How this occurs and whether uncoupled insulin action develops in other tissues is unknown. Here we show in a model of chronic hyperinsulinemia that adipocytes develop selective insulin resistance in which translocation of the GLUT4 glucose transporter to the cell surface is blunted yet nuclear exclusion of the FoxO1 transcription factor is preserved, rendering uncoupled insulin-controlled carbohydrate and lipid metabolisms. We found that in adipocytes FoxO1 nuclear exclusion has a lower half-maximal insulin dose than GLUT4 translocation, and it is because of this inherent greater sensitivity that control of FoxO1 by physiological insulin concentrations is maintained in adipocytes with compromised insulin signaling. Pharmacological and genetic interventions revealed that insulin regulates GLUT4 and FoxO1 through the PI3-kinase isoform p110α, although FoxO1 showed higher sensitivity to p110α activity than GLUT4. Transient down-regulation and overexpression of Akt isoforms in adipocytes demonstrated that insulin-activated PI3-kinase signals to GLUT4 primarily through Akt2 kinase, whereas Akt1 and Akt2 signal to FoxO1. We propose that the lower threshold of insulin activity for FoxO1’s nuclear exclusion is in part due to its regulation by both Akt isoforms. Identification of uncoupled insulin action in adipocytes suggests this condition might be a general phenomenon of insulin target tissues contributing to insulin resistance’s pathophysiology.

Molecular Mechanisms Controlling GLUT4 Intracellular Retention

In basal adipocytes, glucose transporter 4 (GLUT4) is sequestered intracellularly by an insulin-reversible retention mechanism. Here, we analyze the roles of three GLUT4 trafficking motifs (FQQI, TELEY, and LL), providing molecular links between insulin signaling, cellular trafficking machinery, and the motifs in the specialized trafficking of GLUT4. Our results support a GLUT4 retention model that involves two linked intracellular cycles: one between endosomes and a retention compartment, and the other between endosomes and specialized GLUT4 transport vesicles. Targeting of GLUT4 to the former is dependent on the FQQI motif and its targeting to the latter is dependent on the TELEY motif. These two motifs act independently in retention, with the TELEY-dependent step being under the control of signaling downstream of the AS160 rab GTPase activating protein. Segregation of GLUT4 from endosomes, although positively correlated with the degree of basal retention, does not completely account for GLUT4 retention or insulin-responsiveness. Mutation of the LL motif slows return to basal intracellular retention after insulin withdrawal. Knockdown of clathrin adaptin protein complex-1 (AP-1) causes a delay in the return to intracellular retention after insulin withdrawal. The effects of mutating the LL motif and knockdown of AP-1 were not additive, establishing that AP-1 regulation of GLUT4 trafficking requires the LL motif.