In the sciences, experiments are performed within a specific system where, hopefully, all variables can be controlled - thus allowing manipulation of a single unknown factor. An ideal system will be consistent, and therefore predictable, which facilitates interpretation of purposeful disruptions.
In the biological sciences, the system used is most commonly some living entity - a cell, tissue, or animal. Cell-free systems have their place, but because the goal is ultimately to understand processes as they happen in vivo, the researcher must go back to a live model.
| What Does "In Vivo" Mean?
"In vivo" means "in the body". It's the exact opposite of "in vitro", which means outside the body (or in the laboratory).
Unfortunately, despite having mapped out entire genomes, we still do not understand all the rules of the living systems within which we experiment. Cell lines change over time, tissue extracts are not always pure (and are affected by circulating factors present in the original animal), and trying to ensure true homogeneity (uniformity) between animals is impractical, if not impossible.
| What Is A Genome?
A genome is the complete set of genetic information of an organism including DNA and RNA.
Still, the scientific community builds upon one anothers' findings - and thanks to established protocols and ever-advancing technology, results are, for the most part, repeatable and fit nicely into accepted models, albeit with a bit of tweaking more often than not.
The field of cell signaling requires that the researcher weave an intricate tale with data from several systems, each requiring unique analytical methods - oftentimes a daunting task. Nonetheless, for the aforementioned reasons, paradoxical findings abound, and decisions regarding what experiment to pursue next must be made based upon the available data.
This month, we will use the Protein Kinase B (PKB), an enzyme immediately downstream of the PDK-1 (see part #4), to walk through a rational approach towards finding consistencies between different experiments in order to devise new directions for further clarification.
Overview Of PKB (Akt)
As stated from the very first part, the goal of slowly progressing down the insulin signaling ladder was to explain physiological effects of hormones in a manner more thoroughly than simply stating that "insulin increases glucose uptake." Along the way, the reader hopefully picked up on some widely applicable rules useable across all biological systems.
In this part, we finally have traversed far enough that we can look at the first and most well-known terminal effect of insulin - stimulation of glucose uptake.
The protein kinase B, commonly referred to as Akt, is a member of the AGC family of protein kinases, just like PDK-1. Unlike the single-isoform PDK-1, Akt has three known isoforms - 1, 2, and 3 (equivalent to PKB-alpha, beta, and gamma, respectively), with each isoform encoded on a separate gene.1
Akt1 appears to be globally expressed across cell types and species; however, Akt2 has been found predominantly in insulin-sensitive peripheral tissues, and Akt3 may be important in the brain with secondary roles in adipose (fat) tissue.1,2,5,14
Across species, Akt possesses a PH domain, which is consistent with a requirement for PI3K in order to be activated (remember that the PH domain binds the 3' phosphorylated inositol products of activated PI3K); a role of the PH domain in membrane targeting has been shown as well, and this might be a vital part of Akt regulation.18-21
Being a serine/threonine protein kinase, Akt phosphorylates and thus governs the activity of certain downstream enzymes. Currently known targets of Akt include GSK-3, FKHR, glycogen synthase, NOS, and p70S6k, giving Akt roles in a variety of cellular processes including glucose uptake and storage, nitric oxide synthesis, cell survival, and protein synthesis.1
| What Is Phosphorylation?
Phosphorylation refers to introducing phosphates into the cell that function as building blocks needed for ATP formation.
Additional novel targets that Akt might regulate include the transcriptional coactivator PGC-1 and the master regulator of lipid and cholesterol biosynthesis, the SREBP.1,6,11,12 For the purposes of this article, we will focus primarily on Akt's roles in glucose uptake (via GLUT4 translocation) in the peripheral tissues.
Since its discovery, Akt has been implicated as an important or even vital factor in insulin-stimulated glucose uptake. This function was assessed by protein overexpression studies where creation of an altered version of the endogenous protein, that is either constitutively active or inactive, is used to override endogenous kinase influences by mass action.
| What Does Endogenous Mean?
Endogenous refers to something which originates internally, or is synthesized inside the body, tissues, and/or cells.
Using this approach, the majority of experiments came to the conclusion that overexpression of a constitutively active Akt1 resulted in an increase in the basal level of glucose transport equivalent to that observed when wild-type cells were stimulated with insulin.3,4,6,9
In other words, an always-active Akt1 mimicked insulin with respect to glucose uptake. These studies were performed in well-established cell lines that are widely used as models for peripheral tissues- 3T3-L1 adipocytes for white adipose tissue and L6 myotubes for skeletal muscle.
In conjunction with the elevation in glucose uptake, increases in GLUT4 translocation to the plasma membrane or to a specific cell fraction known to contain active GLUT4, were often confirmed.3,6,19,20 Thus, a model was derived where insulin leads to Akt1 activation through the IRS-PI3K-PDK pathway, and Akt1 somehow increases GLUT4 translocation to the plasma membrane.
This initial model required almost immediate reevaluation as evidence began to accumulate showing that Akt2 protein expression was higher than Akt1 in insulin-responsive tissues (adipose, muscle, liver), and that Akt2 but not Akt1, localized to GLUT4-containing vesicles5,7,18,21 - the evidence against Akt1 being the sole or even primary mediator was all the more convincing because the aforementioned studies used the same cell lines.
In fact, the pendulum began to swing in the favor of Akt2 being the more important isoform for mediating insulin's metabolic effects in peripheral tissues. Akt1 was delegated to playing a supporting role to Akt2 in this respect, although it was recognized as possibly being more important for cellular growth and differentiation.2,6 To examine this isoform specificity more closely, we will look at findings from some recent papers employing powerful gene manipulation strategies.
In Vitro Gene Knockout Approaches
With the advent of widespread utilization of gene knockout technology, an invaluable tool was available to resolve the debate over the importance of Akt1 vs. Akt2 in insulin-stimulated glucose uptake and GLUT4 translocation. Recently, Bae et al. differentiated embryonic fibroblasts into adipocytes from female mice which had either Akt1, Akt2, or both Akt1 and 2 knocked out.
The results of their study showed that Akt2 protein expression is ~2x greater in differentiated fibroblasts vs. undifferentiated ones (controls), suggesting that when cells gain an insulin-sensitive phenotype, Akt2 is upregulated and Akt1 downregulated.
Furthermore, adipocytes from the Akt2 knockout mice showed an ~30% reduction in insulin-stimulated GLUT4 translocation compared to controls and Akt1 knockouts; this decrease in GLUT4 translocation correlated to a 40% decrease in insulin-stimulated glucose transport.2 As expected, restoration of Akt2 by a viral vector restored the ability of the Akt2 knockout cells to respond to insulin.
The results of the above study provide convincing evidence that in adipocytes Akt2 is both more strongly expressed and has a more pronounced effect on insulin-stimulated GLUT4 translocation and glucose uptake than Akt1. The same group performed a parallel study using identical conditions, except the embryonic fibroblasts were differentiated into brown adipocytes.2
In this experiment, protein levels of both Akt1 and Akt2 were identical, but again, only the Akt2-knockout lineage showed significant reductions in GLUT4 translocation and glucose uptake in response to insulin.
In both experiments, protein levels of the insulin receptor, IRS-1, p85a (of PI3K), and GLUT4 were unchanged, suggesting that it is indeed Akt2 specifically that mediates the effects on glucose uptake/GLUT4 translocation. However, based upon the data from the brown adipocytes, it seems that protein expression levels do not necessarily correlate with physiological effects.
In Vivo Gene Knockout Studies: Targeted And Global
Further support for Akt2's essential role in glucose metabolism was provided by analysis of the Akt2 targeted knockout mouse.
A targeted knockout differs from traditional global knockouts in that the former allows for disruption of a gene only in certain tissues, avoiding nonspecific and often lethal effects of whole-body knockouts.
Cho et al. showed that a mouse lacking Akt2 in skeletal muscle, white adipose tissue, and liver is perfectly viable and normal except for moderate insulin resistance in both skeletal muscle and adipose tissue as assessed by glucose and insulin clamp studies.
This effect could be overcome by increasing levels of insulin, suggesting that the primary defect might be overall impairment of signaling secondary to chronic hyperglycemia/hyperinsulinemia induced by a total failure of insulin to suppress hepatic glucose output (HGO).
In further support of this latter mechanism, the researchers were unable to detect any changes in GLUT4 protein content in these tissues.12
At first glance, the results of Cho et al. and Bae et al. complement each other by showing that loss of Akt2 leads to reductions in insulin-stimulated glucose uptake absent of changes in GLUT4 protein levels, supporting a Akt2 GLUT4 translocation model. However, closer observation reveals that the two, if anything, contradict each other.
The in vivo Akt2 model of Cho et al. seems to be mildly insulin resistant secondary to a failure to suppress hepatic glucose output; if Akt2 were the primary mediator of glucose uptake/GLUT4 translocation, indelible defects should be apparent at the cellular level - the fact that increasing the concentration of insulin could overcome the mild insulin resistance argues against this.12
On the other hand, the knockout-replacement approach of Bae et al. places Akt2 solidly as a critical intermediate in insulin signaling to glucose uptake which cannot be replaced by Akt1.2 When results are inconsistent between two well-performed studies, one in vivo and the other in vitro, we must look to other similar models for clarification.
Unfortunately, for similar in vivo models, our choices are currently limited. A global Akt2 mouse knockout by another group15 does not help resolve this issue, as the phenotype of this mouse is characterized by gross lipodystrophy, precluding separation of Akt2 knockout's effect as direct (cellular), or if all physiological effects are secondary to lack of proper adipocyte development.
| What Does Lipodystrophy Mean?
An abnormal or degenerative condition of the body's fat tissue. Defective metabolism of fat.
Taken together with Cho et al.'s targeted Akt2 knockout mice, this study does serve to stress the importance of distinguishing between experimental approaches before drawing conclusions, while underlining the advantages of targeted knockout approaches when one wishes to study the effects of a protein in specific tissues.
Looking to Akt1 global knockout mice is of little help, but does strengthen the evidence in favor of Akt2's role as the principal Akt isoform for glucose metabolism.
The Akt1 global knockout mice have moderate developmental abnormalities characterized by an ~20% decrease in body weight and size - however, they are perfectly normal with respect to glucose and insulin tolerance, and show no metabolic disorders.13 Thus, this model does reinforce the notion that Akt2 does not need Akt1 to mediate its effects on glucose metabolism, but does not clarify if its role is indispensable because of hepatic glucose output suppression, versus signaling to GLUT4 translocation.
A global Akt-1/2 double knockout is a dead-end due to severe growth abnormalities, including incomplete skin, muscle, adipose, and bone development.14 The double-knockout does suggest that Akt-1 and Akt2 have some overlapping functions in development, though - whether this apparent redundancy carries over to glucose metabolism requires further investigation, although some studies suggest this to be true.2,22
With the lack of supporting targeted animal knockouts, the next step is to turn to the most recent gene manipulation method that employs real-time gene targeting.
Evolution Of Gene Knockout: siRNA And Gene Knockdown
The latest in gene silencing technology - siRNA (short-interfering-RNA) - harnesses the intrinsic ability of cells to recognize certain short double-stranded RNA sequences and use internal machinery to mark and destroy the complementary (m)RNA of said short double-stranded RNA (and therefore prevent synthesis of the mRNA-encoded protein).
This is a method cells use to counteract viral DNA, and it has proven very useful in dissecting signaling pathways because researchers can construct short but very specific double-stranded nucleotide sequences, deliver them into cells, and silence genes in a highly targeted, rapid, and efficient manner. siRNA technology avoids artifactual effects often present with overexpression approaches, and is far easier and less time-consuming than traditional methods of gene silencing.
Silencing of a gene with siRNA is referred to as "knocking-down" because the effect is transient and often does not result in total ablation of the target protein - this may at first appear to be a disadvantage, but it carries with it the potential to reduce gene expression in a controllable, dose-dependent manner. Finally, siRNA can be applied to in vivo models as well, should an in vitro experiment provide promising results.
Currently, there are two excellent siRNA studies that specifically look at the effects of Akt1 vs. Akt2 in glucose metabolism.16,26 Jiang et al. found that siRNA directed towards Akt1 resulted in a 20-30% decrease in insulin-stimulated glucose transport, while siRNA against Akt2 reduced insulin-stimulated glucose transport (ISGT) by 50-58%; combined knockdown of both had an additive effect, resulting in ~80% inhibition of ISGT.16
Further supporting Akt2's dominant role in ISGT in 3T3-L1 adipocytes, knockdown of Akt2 was only 70% effective, while that of Akt1 was nearly 100%, indicating that incomplete silencing of Akt2 had an approximately 2-fold greater impact on ISGT when compared to almost total Akt1 knockdown.
However, when one compares the degree of kinase activity inhibition in relation to the magnitude of ISGT decline, Akt1's contribution to ISGT is significant; the near-complete depletion of Akt1 activity resulted in a 10-20% reduction in total Akt kinase activity, and the incomplete silencing of Akt2 dropped total Akt kinase activity by ~60%.
While these results parallel the effects on ISGT very nicely, an even more important observation is that a 20% decline in total Akt kinase activity by siRNA-Akt1 leads to a 20-30% decrease in ISGT - this is comparable to the 1:1 relationship between inhibition of total Akt kinase activity and ISGT inhibition for Akt2.
In other words, when one looks at the % inhibition of ISGT in relation to the % inhibition of total Akt kinase activity for each isoform, they are the same.
Katome et al. performed a similar experiment using the same 3T3-L1 adipocyte cell line, although their cells had a modified GLUT4 protein that allowed for purification.26 In their experiments, siRNA directed against Akt1 decreased ISGT from ~6x to ~4x, versus a drop to ~5x for siRNA directed against Akt1.
Again, there is a tight correlation between inhibition of total Akt kinase activity and inhibition of ISGT with respect to siRNA against Akt1.26 Total Akt kinase activity inhibition by siRNA against Akt2 was not performed, unfortunately; however, like Jiang et al., Katome et al. observed a greater effect by silencing Akt2 in 3T3-L1 adipocytes with respect to ISGT and insulin-stimulated GLUT4 translocation - as well as a lesser but still significant contribution from Akt1.
It is likely that total protein expression levels can explain at least some of the differences, given the many findings from in vitro studies showing that constitutively active Akt1 can mimic insulin's effects on glucose uptake and GLUT4 translocation in 3T3-L1 cells.6,9,18,19
A consistent observation in overexpression of constitutively active Akt isoforms (mostly Akt1) is that basal activity of Akt (and glucose transport) in these cells equals that of insulin-stimulated activity in control cells, but does not increase further upon insulin stimulation,6,9,26 or only increases a small percentage (in a muscle cell line, though).3
Conversely, dominant-negative overexpression studies and siRNA knockdown findings both report no alterations in basal (without insulin) total Akt kinase activity (and glucose transport), but show varying degrees of blockade of insulin-stimulated total Akt kinase activity.6,9,16,26
Finally, there appears to be alternate an Akt-independent pathway (or pathways) for insulin-stimulated glucose uptake in 3T3-L1 adipocytes, evidenced by persistent increases in glucose uptake when insulin is added even when siRNA towards Akt has entirely erased the insulin-stimulated kinase activity,26 or total Akt activity has been reduced by 80% with a dominant-negative mutant.22
Drawing from the results of in vitro studies and some animal knockout models, it is reasonable to conclude that Akt2 is the primary mediator of insulin-stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes, with a lesser but nonetheless important role for Akt1 as well - this role of Akt1 seems to increase in importance as Akt2 activity is lost, even though Akt1 cannot completely replace Akt2.
This compensation could explain the failure of Cho et al. to see any primary effect of targeted Akt2 knockout on insulin signaling, although they did show that Akt2 is indispensable in controlling insulin-mediated suppression of hepatic glucose output.12
However, 3T3-L1 adipocytes may have an Akt-independent mechanism for facilitation of ISGT/GLUT4 translocation based upon the findings of Katome et al.26 - whatever this pathway is, it is unable to recapitulate the magnitude of ISGT elevation that Akt1 - and especially Akt2 - mediates.
In Vivo Non-Genomic Studies
Given the abundant in vitro and animal knockout studies emphasizing Akt's essential role in insulin signaling to glucose uptake, it would be reassuring to see if this is manifested in normal in vivo models as well. The most common approach towards answering this question is to induce insulin resistance in the animal and compare tissue biopsies taken immediately after treatment, for analysis of Akt activity/phosphorylation versus a control animal.
Unfortunately, while inhibition in early steps in insulin signaling - IRS-1/2 tyrosine phosphorylation and associated PI3K activity - are often observed, the effects on Akt are not consistent with in vitro overexpression or gene knockout/knockdown studies.10,11,17,24
In normal rats, lipid infusion is able to inhibit IRS-1/2 tyrosine phosphorylation and lower Akt1 activity by 41% in muscle strips, but causes no changes in Akt2 activity.10 In Zucker rats, a model for human obesity, Akt activity shows tissue-specific responses to insulin.
Comparing lean vs. obese Zuckers reveals that the liver of obese rats actually experiences an upregulation of Akt1 by 37%, while decreasing Akt2 activity 35%; muscle experiences 30% decreases for both Akt1 and 2 activation, but white adipose tissue shows a reciprocal Akt-1 vs. 2 response where activation of Akt1 decreases and that of Akt2 increases (both ~25%).17
In contrast to 3T3-L1 adipocytes, the % ISGT inhibition in relation to Akt kinase activity inhibition in obese Zucker adipose tissue is greater, such that the ~50% drop in Akt kinase activity results in a 70% decrease in ISGT.
This could be explained by adipose cell insulin resistance, as the obese rats did exhibit a much larger adipocyte cell size, which is known to be inversely related to insulin sensitivity.17 Of course, such an answer does not help with determination of Akt's role in glucose metabolism.
Currently, there are few human studies that look closely at the relationship between Akt and insulin resistance, and they too have failed to provide any convincing links.8,23,25
Obesity, itself, shows a poor correlation between Akt and glucose disposal in skeletal muscle even though obesity + type II diabetes results in a 2-fold greater impairment of ISGT.23 In a lipid infusion study with lean, normal humans, treatment leads to a 24% impairment in whole-body glucose disposal but no differences in total or phospho-Akt levels;25 glucose infusion increased Akt phosphorylation several-fold, but again this effect was the same between glucose only vs. glucose + lipid.
Finally, skeletal muscle biopsies between lean and obese nondiabetic humans revealed that in lean subjects, there was an increase in phosphorylation of all three Akt isoforms upon insulin infusion, whereas obese patients showed only increases in Akt1 phosphorylation.8
This last study is somewhat consistent with a previous study that found a positive correlation between insulin-stimulated Akt 1 and 2 phosphorylation and glucose disposal rate in lean but not obese nondiabetic or diabetic subjects.25
Based upon the in vivo human data, the best we can conclude is that Akt phosphorylation in skeletal muscle does seem to correlate positively with insulin stimulation, and presumably, glucose disposal; however, we cannot make the logical leap that lack of Akt activity leads to insulin resistance and/or glucose intolerance.
It is worth noting that the human studies looked mainly at skeletal muscle biopsies, whereas most of the research on Akt and glucose uptake has been performed in adipose tissue or in 3T3-L1 adipocyte lines, and the most striking phenotype of Akt2 knockout was observed in mouse liver.
Furthermore, it is difficult to evaluate the importance of a protein when comparing studies that completely knock out or otherwise inhibit function to a severe extent and then assess the consequences, versus studies that use a model/approach known to cause insulin resistance first, and then look at the percent change in Akt activity (which, as seen above, is invariably reduced to a much lesser extent, if at all, compared to gene/protein manipulation).
With all of these caveats in place, it is perhaps best to place our faith in the in vitro and animal gene knockout/knockdown models for assessment of Akt's importance - or lack of - in insulin-mediated glucose uptake.
| What Does Caveat Mean?
A warning; a note of caution.
In summary, based upon the in vivo non-knockout studies, it appears that loss of Akt activity is not an inducer of insulin resistance, and a failure to detect decreases in Akt activity is not necessarily indicative of normal glucose tolerance. However, in vitro and animal knockout/knockdown studies provide solid evidence that Akt is necessary, although probably not solely sufficient, for normal/optimal responses to insulin with respect to glucose metabolism.
The generation of targeted Akt-1 and Akt-1+2 double-knockout animals will be necessary to conclusively link in vitro and in vivo knockout with in vivo non-knockout findings. Furthermore, additional studies using siRNA in liver and muscle cells will prove helpful in understanding tissue-specific effects of Akt isoforms, particularly with the dramatic effect Akt2 has in the Akt2 targeted knockout model,16 as impaired HGO is a major contributor to worsening of the metabolic syndrome.
As we reach the end of the insulin signaling pathway and begin to focus on physiological effects, the literature becomes far less conclusive with respect to the importance of specific proteins. Biological systems are messy, but if one knows how to critically search the literature and carefully integrate in vitro findings with in vivo models, rational conclusions can be drawn and future experiments required to conclusively demonstrate the validity of said conclusions become readily apparent.
Oftentimes, results from cell lines and animal models are criticized as being of limited relevance to humans. However, given the obvious limitation to what we can ethically manipulate in humans, we must resort to these less-than-optimal model systems.
When the overwhelming majority of evidence from several models using different approaches all show similar findings, though, it is reasonable to conclude that the results can be applied more generally as well.
In the case of Akt, it is safe to say that Akt 1 and 2 are both essential mediators of insulin-stimulated glucose uptake in adipose tissue and skeletal muscle, although they clearly do not act alone. Akt's effects on the liver are promising but require further support before we can conclusively state that it is absolutely vital for controlling hepatic glucose output.
Also, we need to look at the other kinases that mediate insulin-stimulated glucose production under various in vivo to ascertain whether Akt's absolute contribution to ISGT varies.
Optimally, we will be able to define conditions in vitro that correlate closely with Akt vs. other pathways involved in ISGT, and then move to in vivo models to see if our predictions hold. If so, this approach holds great promise for targeted amelioration of metabolic perturbations with distinct etiologies.
In the next part, by a similar approach, we will look at other molecules immediately downstream of PDK-1 which may also mediate insulin-stimulated glucose uptake and try to explain how it is possible for overexpression of Akt isoforms to mimic insulin's effect on glucose transport and GLUT4 translocation, and still leave room for other factors.
Redundancy in signaling is a theme we have encountered many times in upstream signaling, and we will attempt to unearth its purpose in mediation of terminal physiological effects as well!
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- Bae, S. S., Cho, H., Mu,. J. & Birnbaum, M. J. (2003) Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B. J Biol Chem, 278(49): 49539-49536.
- Hajduch. E., Alesi, D. R., Hemmings, B. A. & Hundal, H. S. (1998) Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes, 47(7): 1006-13.
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- Kohn, A. D., Summers, S. A., Birnbaum, M. J. & Roth, R. A. (1996) Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem, 271(49): 31372-8.
- Tirosh, A., Potashnik. R., Bashan, N. & Rudich, A.(1999) Oxidative stress disrupts insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. A putative cellular mechanism for impaired protein kinase B activation and GLUT4 translocation. J Biol Chem, 274(15): 10595-602.
- Brozinick, J. T. Jr., Roberts, B. R. & Dohm, G. L. (2003) Defective signaling through Akt-2 and -3 but not Akt-1 in insulin-resistant human skeletal muscle: potential role in insulin resistance. Diabetes, 52(4): 935-41.
- Ueki, K., Yamamoto-Honda, R., Kaburagi, Y., Yamauchi, I., Tobe, K., Burgering, B. M., Coffer, P. J., Komuro, I., Akanuma, Y., Yazaki, Y. & Kadowaki, T. (1998) Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem, 273(9): 5315-22.
- Kim, Y. B., Shulman, G. I. & Kahn, B. B. (2002) Fatty acid infusion selectively impairs insulin action on Akt1 and protein kinase C lambda/zeta but not on glycogen synthase kinase-3. J Biol Chem, 277(36): 32915-22.
- Kim, Y. B., Peroni, O. D., Franke, T. F. & Kahn, B. B. (2000) Divergent regulation of Akt1and Akt2 isoforms in insulin target tissues of obese Zucker rats. Diabetes, 49: 847-56.
- Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B. III., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I. & Birnbaum, M. J. (2001) Insulin resistance and a Diabetes -Mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB-beta). Science, 292: 1728-31.
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- Jiang, Z. Y., Zhou, Q. L., Coleman, K. A., Chouinard, M., Boese, Q. & Czech, M. P. (2003) Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. PNAS, 100(13): 7569-74.
- Sajan, M. P., Standaert, M. L., Miura, A., Kahn, R. C. & Farese, R. V. (2005) Tissue-specific difference in activation of atypical protein kinase C and protein kinase B in muscle, liver, and adipocytes of insulin receptor substrate-1 knockout mice. Mol Endocrinol, 18(10): 2513-21.
- Calera, M. R., Martinez, C., Liu, H., Jack, A. K., Birnbaum, M. J. & Pilch, P. F. (1998) Insulin increases the association of Akt-2 with Glut4-containing vesicle. J Biol Chem, 273(13): 7201-4.
- Docluzeau, P. H., Fletcher, L. M., Welsh, G. I. & Tavare, J. M. (2002) Functional consequence of targeting protein kinase B.Akt to GLUT4 vesicles. J Cell Sci, 115(Pt 14): 2857-66.
- van Dam, E. M., Grovers, R. & James, D. E. (2005) Akt activation is required at a late stage of insulin-induced GLUT4 translocation to the plasma membrane. Mol Endocrinol, 19(4): 1067-77.
- Calera, M. R., Martinez, C., Liu, H., Jack, A. K., Birnbaum, M. J. & Pilch, P. F. (1998) Insulin increases the association of Akt-2 with Glut4-containing vesicles. J Biol Chem, 273(13): 7201-4.
- Kitamura, T., Ogawa, W., Sakuge, H., Hino, Y., Koruda, S., Takata, M., Matsumoto, M., Maeda, T., Kinoshi, H., Kikkawa, U. & Kasuga, M. (1998) Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol, 18(7): 3708-17.
- Kim, Y. B., Nikoulina, S. E., Ciaraldi, T. P., Henry, R. R. & Kahn, B. B. (1999) Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest, 104(6): 733-41.
- Nadler, S. T., Stoehr, J. P., Rabaglia, M. E., Schueler, K. L., Birnbaum, M. J. & Attie, A. D. (2001) Normal Akt/PKB with reduced PI3K activation in insulin-resistant mice. Am J Physiol Endocrinol Metab, 281(6): E1249-54.
- Kruszynska, Y. T., Worrall, D. S., Ofrecio, J., Frias, J. P., Macaraeg, G. & Olefsky, J. M. (2002) Fatty acid-induced insulin resistance: decreased muscle PI3K activation but unchanged Akt phosphorylation. J Clin Endocrinol Metab, 87(1): 226-34.
- Katome, T., Obata, T., Matsushima, R., Masuyama, N., Cantley, L. C., Gotoh, Y., Kishi, K., Shiota, H. & Ebina, Y. (2003) Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of Akt/protein kinase B isoforms in insulin action. J Biol Chem, 278(30): 28312-23.