Metabolic, anabolic, and mitogenic insulin responses: A tissue-specific perspective for insulin receptor activators

Accepted Manuscript Metabolic, Anabolic, and Mitogenic Insulin Responses: A Tissue-Specific Perspective for Insulin Receptor Activators Daniel H. Bedinger, Sean H. Adams PII:

S0303-7207(15)30053-8

DOI:

10.1016/j.mce.2015.08.013

Reference:

MCE 9254

To appear in:

Molecular and Cellular Endocrinology

Received Date: 8 July 2015 Revised Date:

5 August 2015

Accepted Date: 9 August 2015

Please cite this article as: Bedinger, D.H., Adams, S.H., Metabolic, Anabolic, and Mitogenic Insulin Responses: A Tissue-Specific Perspective for Insulin Receptor Activators, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.08.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Metabolic, Anabolic, and Mitogenic Insulin Responses: A Tissue-Specific Perspective for Insulin Receptor Activators

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Daniel H. Bedinger1* and Sean H. Adams2 1

XOMA Corporation, Berkeley, CA; 2Arkansas Children’s Nutrition Center –and- University of Arkansas for Medical Sciences, Department of Pediatrics, Little Rock, AR

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*To whom correspondence should be sent: Daniel H. Bedinger, XOMA Corporation, 2910 Seventh St., Berkeley, CA 94710; [email protected], Phone 1-510-204-7589 Abstract

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Insulin acts as the major regulator of the fasting-to-fed metabolic transition by altering substrate metabolism, promoting energy storage, and helping activate protein synthesis. In addition to its glucoregulatory and other metabolic properties, insulin can also act as a growth factor. The metabolic and mitogenic responses to insulin are regulated by divergent post-

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receptor signaling mechanisms downstream from the activated insulin receptor (IR). However, the anabolic and growth-promoting properties of insulin require tissue-specific interrelationships between the two pathways, and the nature and scope of insulin-regulated processes

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vary greatly across tissues. Understanding the nuances of this interplay between metabolic and growth-regulating properties of insulin would have important implications for development of

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novel insulin and IR modulator therapies that stimulate insulin receptor activation in both pathway- and tissue-specific manners. This review will provide a unique perspective focusing on the roles of “metabolic” and “mitogenic” actions of insulin signaling in various tissues, and how these networks should be considered when evaluating selective pharmacologic approaches to prevent or treat metabolic disease. Keywords: Akt, ERK, Insulin, Insulin Receptor, Diabetes, Insulin-like Growth Factor

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Abbreviations: Insulin Receptor (IR), Extracellular signal-Regulated Kinase (ERK), Insulin-like Growth Factor 1 (IGF-1), Insulin-like Growth Factor Receptor (IGF-1R), Type 2 Diabetes Mellitus (T2D),

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Free Fatty Acids (FFA), Mitogen Activated Protein Kinase (MAPK), phosphatidylinositol-4,5bisphosphate-3-kinase (PI3K), Insulin Receptor Substrate (IRS), Son of Sevenless (SOS)

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Brief Outline:

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Introduction to issues and concerns with current insulin therapies. Background on Insulin: a Metabolic and Anabolic Hormone a. Metabolic actions of insulin. i. Whole body glucose homeostasis, fasting to fed transition, fuel utilization and partitioning b. Growth actions of insulin signaling. i. Shared pathways and homology with IGF receptor and IGF-2 ligand ii. Relationship between hyperinsulinism and cancer iii. Compare and contrast concept of anabolism and mitogenicity Is Mitogenic Regulation of ERK by Insulin Important in Insulin Responsive Tissues? a. Liver b. Muscle c. Adipose d. Pancreatic β-cells e. Neurons f. Endothelial Cells/Vasodilation Tissue-Specific Mitogenic Regulation by Insulin via ERK a. Liver b. Muscle c. Adipose d. Pancreatic β-cells e. Neurons f. Vascular Endothelial Cells Pathologic and Therapeutic Implications of Tissue- and Pathway-Specificity of IR Activation a. General complications of hyperinsulinemia b. Insulin, IGF, and Cancer c. Therapeutic implications for pathway specific IR modulators in metabolic disease d. Therapeutic implications for tissue-specific IR modulators Conclusion

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Introduction More than 29 million people in the United States, and many more world-wide, are

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diabetic (cdc.gov, 2014). Approximately 90-95% of diabetics have type 2 diabetes mellitus (T2D), which is characterized by insulin resistance and for much of the disease period,

hyperinsulinemia. Insulin is given to T2D patients only after they have failed several other

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therapies, but no pharmacological therapies have been demonstrated to halt the eventual

progression to insulin dependence in the face of deteriorating blood sugar control. Yet, strong

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arguments can be made that hyperinsulinemia, and by extension repeated high-dose insulin therapy, could promote peripheral tissue insulin resistance (Gavin, Roth, Neville et al., 1974,Marshall and Olefsky, 1980,Garvey, Olefsky and Marshall, 1985,Rizza, Mandarino, Genest et al., 1985) or increase risk of cancer through mitogenic actions of the hormone (Ish-Shalom, Christoffersen, Vorwerk et al., 1997,Sciacca, Le Moli and Vigneri, 2012). These observations

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highlight the need for development of new therapeutics that target specific cellular pathways or that have tissue-specific effects.

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Insulin acts as the major regulator of the fasting-to-fed metabolic transition by altering substrate metabolism, promoting energy storage, and helping activate protein synthesis

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(McGarry, 1992). In addition to its glucoregulatory and other metabolic properties, insulin can also act as a growth factor (Ish-Shalom et al., 1997). The metabolic and mitogenic responses to insulin are regulated by divergent post-receptor signaling mechanisms downstream from the activated insulin receptor (IR). However, the anabolic and growth-promoting properties of insulin require tissue-specific inter-relationships between the two pathways, and the nature and scope of insulin-regulated processes vary greatly across tissues (Taniguchi, Emanuelli and Kahn, 2006,Biddinger and Kahn, 2006). Understanding the nuances of this interplay between 4

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metabolic and growth-regulating properties of insulin would have important implications for development of novel insulin and insulin-receptor (IR) modulator therapies that stimulate insulin receptor activation in both pathway- and tissue-specific manners (Ish-Shalom et al.,

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1997,Shojaee-Moradie, Powrie, Sundermann et al., 2000,Moore, Smith, Sinha et al.,

2014,Madsbad, 2014,Kurtzhals, Schaffer, Sorensen et al., 2000,Tompkins, Brandenburg, Jones et al., 1981,Sciacca, Cassarino, Genua et al., 2010,Hansen, Kurtzhals, Jensen et al.,

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2011,Vigneri, Squatrito and Frittitta, 2012,Bedinger, Kieffer, Goldfine et al., 2015,Bedinger, Goldfine, Corbin et al., 2015). This review will provide a unique perspective focusing on the

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roles of “metabolic” and “mitogenic” actions of insulin signaling in various tissues, and how these networks should be considered when evaluating selective pharmacologic approaches to prevent or treat metabolic disease.

1. Background on Insulin: a Metabolic and Anabolic Hormone

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1.a. Metabolic actions of insulin. Insulin is secreted by pancreatic β-cells in islets of Langerhans in response to increases in blood levels of glucose and select amino acids, with a modulating role attributed to free fatty acids (FFA). Insulin secretion is modulated by both

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hormonal and neural regulation (Ahren and Holst, 2001,Chandra and Liddle, 2014,Molina,

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Rodriguez-Diaz, Fachado et al., 2014). Interestingly, maximal glucose- or amino acid-stimulated insulin release requires the presence of fatty acids (Stein, Esser, Stevenson et al., 1996,Dobbins, Chester, Stevenson et al., 1998), highlighting the close connection of this hormone with wholebody fuel metabolism. The secreted insulin travels to the liver via the hepatic portal vein, resulting in the exposure of the liver to much higher insulin levels than the systemic circulation (Rojdmark, Bloom, Chou et al., 1978). The liver has high IR levels and removes roughly half of the secreted insulin from portal blood via receptor-mediated endocytosis (Chap, Ishida, Chou et 5

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al., 1987). Insulin in the portal blood that is not cleared by the liver is then delivered into the larger systemic circulation where it can stimulate other insulin-sensitive tissues such as muscle, adipose, and the hypothalamus (Biddinger and Kahn, 2006). In times of fasting or low nutrient

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abundance, insulin levels are decreased, and the levels of insulin’s opposing pancreatic hormone, glucagon, are elevated (Unger and Cherrington, 2012). This low insulin/high glucagon state stimulates glucose-sparing metabolic functions such as the lipolysis of triglycerides and release

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of FFA in adipose tissue, the release of amino acids and lactate from muscle, and the increase in hepatic β-oxidation, ketone body production, and glucose output (McGarry and Foster, 1980).

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The bulk of these metabolic actions result from the activation of the canonical PI3K/Akt IR signaling pathway (Figure 1). The insulin receptor (IR) is a hetero-tetramer with two wholly extra-cellular α-subunits and two plasma membrane-spanning β-subunits that contain intracellular tyrosine kinase domains (reviewed in (De Meyts, 2008,Ebina, Ellis, Jarnagin et al.,

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1985,McKern, Lawrence, Streltsov et al., 2006,Kido, Nakae and Accili, 2001)). The specific pathways engaged downstream of the IR have been recently and thoroughly reviewed in (Taniguchi et al., 2006,Cheng, Tseng and White, 2010,Ramalingam, Oh and Thurmond,

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2013,Steelman, Chappell, Abrams et al., 2011), and are only briefly discussed here. Insulin stimulates IR autophosphorylation, leading to insulin receptor substrate (IRS) protein binding

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and phosphorylation, followed by the association and activation of phosphatidylinositol-4,5bisphosphate-3-kinase (PI3K). PI3K increases membrane abundance of phosphatidylinositol3,4,5-triphosphate (PIP3), which leads to the activation of phosphoinositide-dependent kinase 1 (PDK1) and other factors. PDK1 then activates the enzyme Akt by phosphorylating it at Thr308 (Taniguchi et al., 2006,Tan, Ng, Meoli et al., 2012). The Akt serine/threonine kinase is a key enzyme in regulating the majority of metabolic actions of insulin (Miinea, Sano, Kane et al.,

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2005,Dong, Copps, Guo et al., 2008,Shepherd, Withers and Siddle, 1998). IR signaling is terminated by dephosphorylation of IR by protein tyrosine phosphatase 1b (PTP1b) and tyrosineprotein phosphatase non-receptor type 2 (PTPN2); PI3K signaling is inhibited by activation of

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both phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5′-phosphatase-2

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(SHIP2), which dephosphorylate PIP3 (Taniguchi et al., 2006,Galic, Hauser, Kahn et al., 2005).

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Figure 1. Basic schematic of the bifurcated insulin signaling pathway. Insulin Receptor Substrate (IRS), phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K), 3-phosphoinositide dependent protein kinase-1 (PDK1), Son of Sevenless (SOS) Mitogen/Extracellular signal-regulated Kinase (MEK), Extracellular signal-Regulated Kinase (ERK), 1.b. Growth actions of insulin signaling. In addition to this role as a metabolic

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regulator, insulin also functions as a growth factor and has been considered to be the most anabolic hormone (Saltiel and Kahn, 2001,Duarte, Moreira and Oliveira, 2012,Chakrabarti, Kim, Singh et al., 2013). Despite the well-established requirement for insulin in growth, its role as proliferative or mitogenic factor is much more nuanced. In conditions where insulin levels are elevated, signaling through the IR can activate mitogenic pathways (Bedinger et al., 2015,Jensen, Hansen, De Meyts et al., 2007,Vigneri, Frasca, Sciacca et al., 2009,Entingh-Pearsall and Kahn, 2004). Phosphorylated IR and IRS-1 are bound by the Shc protein where they serve as effective 7

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adaptors for the GRB2-SOS complex, thus activating RAS and the mitogen activated protein kinase (MAPK) cascade (Hansen, Danielsen, Drejer et al., 1996,Ceresa and Pessin, 1998,McKay and Morrison, 2007). The activated MAPK, extracellular signal-regulated kinase (ERK1/2, also

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referred to as p44/p42 MAPK), is a key regulator of the mitogenic response to insulin and the IGFs (Johnson and Lapadat, 2002).

The insulin-like growth factor receptor 1 (IGF-1R) protein is highly homologous to the

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IR (55% total sequence identity), shares a nearly identical architecture, and is a potent stimulator of mitogenic cell growth (Siddle, Urso, Niesler et al., 2001,Genua, Pandini, Cassarino et al.,

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2009). The IGF-1R shares much of the same downstream signaling machinery with the IR and the resulting differences in signaling are a matter of degree, with the IGF-1R receptor being about 10-fold more efficient at stimulating mitogenic actions than the IR (Lammers, Gray, Schlessinger et al., 1989). Three protein ligands--insulin (and pro-insulin), insulin-like growth

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factor 1 (IGF-1), and insulin-like growth factor 2 (IGF-2)--are capable of binding and activating both the IR and the IGF-1R, albeit it with different affinities and potencies (Denley, Bonython, Booker et al., 2004,Belfiore and Malaguarnera, 2011). Further complicating this relationship, IR

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and IGF-1R readily form heterodimeric hybrid receptors with each other, and in tissues that express abundant levels of IGF-1R and IR, such as skeletal muscle, the hybrid receptor is the

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most abundant species (Frasca, Pandini, Vigneri et al., 2003,Bailyes, Nave, Soos et al., 1997). These heterodimeric receptors can function as IGF receptors and demonstrate relatively poor activation by insulin (Frasca et al., 2003). Still another consideration is that the IR is expressed in two different splice variant

isoforms, IR-A and IR-B (Frasca, Pandini, Scalia et al., 1999). The 12 amino acids derived from exon 11 are included in the longer IR-B isoform but not in the shorter IR-A isoform. Both IR

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isoforms are sensitive to insulin, with the IR-B having a modestly higher affinity for insulin, but in contrast to IR-B, the IR-A isoform can also be bound and activated by IGF-2 with lownanomolar potency (Frasca et al., 1999). The ratio of the two isoforms are differentially

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expressed in insulin-responsive tissues, and thus isoform specificity of an agonist could

potentially lead to tissue-specific effects (Mosthaf, Grako, Dull et al., 1990,Moller, Yokota, Caro et al., 1989,Serrano, Villar, Martinez et al., 2005,Sesti, Tullio, D'Alfonso et al., 1994,Goldstein

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and Dudley, 1990,Seino and Bell, 1989). The shared ligand and signaling structure between IR and IGF-1R plays a crucial role in embryonic development, where the IR acts as a functioning

2003,Nakae, Kido and Accili, 2001).

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IGF-2 receptor and is required for full growth of the fetus (Kitamura, Kahn and Accili,

As a result of the shared ligand binding and signaling architecture between the IR and the more mitogenic IGF-1R receptors, there has been much concern about the potential of insulin

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and its therapeutic analogs to act as tumor-promoting mitogens (Sciacca et al., 2012,Kurtzhals et al., 2000,Frasca et al., 2003,Janssen and Varewijck, 2014). There is substantial support for the notion that the hyperinsulinemia of T2DM plays an important role for increased rates of certain

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types of cancers; particularly pancreas, liver, prostate, endometrial, and breast (Belfiore and Malaguarnera, 2011,Giovannucci, Harlan, Archer et al., 2010,Rose and Vona-Davis, 2012). In

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cell models, it is clear that at high concentrations, insulin can act as a mitogen similar to IGF-1, but this effect often requires supra-physiological insulin levels to bind to and activate IGF-1Rs (Sciacca et al., 2012). Nevertheless, there are a number of tumor cell lines, some of which lack IGF-1R expression, that are sensitive to insulin as a proliferative signal in physiological concentration ranges (Skut1, LB, T-47D , ZR-75-1) (Sciacca, Mineo, Pandini et al., 2002,Sharon, Pillemer, Ish-Shalom et al., 1993,Milazzo, Giorgino, Damante et al., 1992).

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Independent of any potential mitogenic effects of insulin, it is required for normal growth and regeneration, as most tissues rely on insulin to store energy and support the protein synthesis required for growth (Saltiel and Kahn, 2001). Signaling from IRs has multiple mechanisms for

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the activation of the pro-transcriptional mechanistic target of rapamycin complex 1 (mTORC1) that is required for growth (Laplante and Sabatini, 2012). Both the metabolic Akt and the mitogenic ERK insulin-stimulated pathways converge to increase protein synthesis by

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stimulating the phosphorylation and activation of the ribosomal S6 Kinase (p70S6K) (Belfiore and Malaguarnera, 2011).

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The complexities and context-specificity of the insulin signaling pathway highlight both the challenges and potential value of pathway-specific and tissue-selective activation of the IR. In addition to the complex nature of IR signaling in healthy physiology, it is also important to consider the pathway-specific signaling defects of insulin action present in insulin resistant type

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2 diabetics (Cusi, Maezono, Osman et al., 2000,Defronzo, 2009,Frojdo, Vidal and Pirola, 2009,Rask-Madsen and Kahn, 2012). To place these questions to better context, tissue-specific

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considerations of the metabolic and mitogenic pathways are outlined below.

2. Tissue-Specific Metabolic Regulation by Insulin via Akt

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The metabolic processes regulated by IR signaling are largely the result of signaling

through the PI3K kinase signaling branch, predominately downstream from Akt (Shepherd et al., 1998,Cusi et al., 2000,Lazar, Wiese, Brady et al., 1995).

In addition to the Akt-mediated

actions, activation of PI3K by IR results in some non-Akt mediated activation, primarily through the cAbl, PKC-λ, and PKC-ζ effectors (reviewed in (Genua et al., 2009,Sajan, Jurzak, Samuels et al., 2014,Sajan, Standaert, Bandyopadhyay et al., 1999)) and may be involved in modulating

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signaling pathway sensitivity and signal termination. A number of key aspects of metabolic IR action in important insulin-responsive tissues are described in this section. 2.a. Liver: The liver serves as a central regulator of whole-body metabolism. It is

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responsible for the majority of glucose synthesis (gluconeogenesis), lipid packaging, storage, and distribution, a large fraction of ketogenesis from FFA, and is a major site of glycogen storage. In the absence of IR stimulation, the liver increases β-oxidation, ketone production, and glucose

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output (McGarry, 1992). Insulin is a potent inhibitor of hepatic glucose production (Brown, Tompkins, Juul et al., 1978,Rizza, Mandarino and Gerich, 1981,Peak, Rochford, Borthwick et

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al., 1998), although the effects of insulin on direct hepatic regulation of glucose output are dependent to a large extent on relative glucagon levels (Unger and Cherrington, 2012,Cherrington, Edgerton and Sindelar, 1998). The transition from glycogen utilization to glycogen synthesis is rapidly stimulated by insulin (Cersosimo, Garlick and Ferretti,

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2000,Chiasson, Atkinson, Cherrington et al., 1980), and glucokinase levels elevate quickly (Nouspikel and Iynedjian, 1992). Many of the other effects of insulin on hepatocytes are less rapid and involve altered transcription patterns, such as the down-regulation of genes that

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promote gluconeogenesis and hepatic glucose release. For example, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression are reduced by insulin

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via Akt-mediated phosphorylation and nuclear exclusion of FoxO1 (Nakae, Biggs, Kitamura et al., 2002,Oh, Han, Kim et al., 2013,Gabbay, Sutherland, Gnudi et al., 1996). Insulin also decreases VLDL release from the liver via FoxO1-mediated mechanisms (Kamagate and Dong, 2008,Chirieac, Chirieac, Corsetti et al., 2000), and increases the expression of genes that promote fatty acid synthesis, largely via up-regulation of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) that increases the levels of fatty acid synthase (FAS)

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and acetyl-CoA carboxylase-1 (ACC1) (Li, Brown and Goldstein, 2010,Thorn, Sekar, Lavezzi et al., 2012). In hyperinsulinemic and insulin resistant type 2 diabetes, the ability of insulin to suppress hepatic gluconeogenesis is reduced via a desensitization of the Akt/FoxO1 axis, but the

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SREBP-1c-driven fatty acid production is maintained, leading to both hyperglycemia and

hyperlipidemia (Rask-Madsen and Kahn, 2012,Li et al., 2010). Mice with liver-specific deletion of the IR (LIRKO) are severely glucose-intolerant and hyperinsulinemic (Michael, Kulkarni,

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Postic et al., 2000).

A response to very low insulin levels, as would be seen in type 1 diabetes mellitus, is

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increased adipose tissue lipolysis coupled to a triggering of hepatic FFA oxidation and production of ketones; this process can lead to pathological ketoacidosis (Kreisberg, 1978). The rate of ketogenesis is inhibited by IR via the Akt-mediated decrease in expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) gene (Nadal, Marrero and

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Haro, 2002). Relatively low levels of insulin are sufficient to prevent toxic levels of ketone production, both through hepatic regulation and a decrease in adipose tissue lipolysis (Brown et al., 1978). Insulin also protects hepatocytes from injury and inflammatory damage by inhibiting

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iNOS production in an Akt-dependent manner (Harbrecht, Nweze, Smith et al., 2012). Thus, the IR-mediated PI3K/Akt signaling in hepatocytes is essential for whole-body glucose and lipid

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homeostasis as well as the preservation of hepatocyte health (Michael et al., 2000). 2.b. Muscle: Insulin stimulates glucose transport and glycogen synthesis in the muscle.

The uptake by muscle accounts for the disposal of the majority of prandial glucose in humans (DeFronzo and Tripathy, 2009,Marette, Burdett, Douen et al., 1992). Insulin stimulates glucose uptake and glycogen synthesis in muscle via the PI3K/Akt-mediated signaling axis (Taniguchi et al., 2006). In addition to insulin and other signaling factors, exercise and stretch can also activate

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Akt in muscle (Sakamoto, Aschenbach, Hirshman et al., 2003,Sakamoto and Goodyear, 2002,Bodine, Stitt, Gonzalez et al., 2001). Despite the importance of insulin-stimulated muscle glucose uptake, mice with muscle-

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specific knockout of the IR (MIRKO) show normal glucose tolerance, albeit it with increased adiposity (Cariou, Postic, Boudou et al., 2004,Kim, Michael, Previs et al., 2000,Bruning,

Michael, Winnay et al., 1998). MIRKO mice also have elevated triglycerides and FFA levels

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which suggests that insulin-induced uptake and utilization of lipids is an important point of

regulation for whole-body lipid balance (Bruning et al., 1998). The lack of overt hyperglycemia

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or severe muscle dysfunction in MIRKO mice may be partially explained by the insulinindependent activation of glucose uptake by muscle that is induced by contraction. The high metabolic demand of muscle contraction activates AMP-activated protein kinase (AMPK), and stimulates Glut4 insertion into the plasma membrane, thus stimulating insulin-independent

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glucose transport (Ryder, Chibalin and Zierath, 2001,Mauvais-Jarvis, Virkamaki, Michael et al., 2000,Wojtaszewski, Higaki, Hirshman et al., 1999,Hayashi, Hirshman, Kurth et al., 1998,Sakamoto, McCarthy, Smith et al., 2005). The increase in lipids and adiposity in the

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MIRKO mice suggest that IR signaling in muscle affects whole body fuel partitioning and utilization, but their euglycemia suggests that IR action in muscle may not be required for

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glucose homeostasis due to other variables that can become engaged. It also should be noted that the role of glucose uptake in muscle is likely a larger contributor to glucose homeostasis in humans than it is in mice, therefore the phenotype of a human with a muscle specific IR deletion could be quite different (Nandi, Kitamura, Kahn et al., 2004). 2.c. Adipose: Adipose tissue is another major insulin-responsive metabolic tissue and is estimated to take up 5-15% of an oral glucose load (Biddinger and Kahn, 2006). Insulin inhibits

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lipolysis in adipocytes and stimulates Glut4 translocation and glucose uptake (Chakrabarti et al., 2013,Bryant, Govers and James, 2002,Arner and Langin, 2007). Insulin also regulates transcription in adipocytes: genes regulating fatty acid synthesis are up-regulated (Kim, Sarraf,

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Wright et al., 1998,Rosen, Walkey, Puigserver et al., 2000), as is the leptin gene (Saladin, De Vos, Guerre-Millo et al., 1995).

Mice with the IR deleted from fat tissue (FIRKO) are lean and insulin-sensitive, and this

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phenotype is very interesting in that it demonstrates that IR signaling in the adipose tissue, at least in mice, is not absolutely necessary to maintain health and glucose homeostasis (Bluher,

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Michael, Peroni et al., 2002,Bluher, Kahn and Kahn, 2003). Most surprisingly, FIRKO mice have fairly normal blood FFA levels, which suggests that the lack of insulin signaling in adipocytes is not allowing runaway lipolysis to occur (Bluher et al., 2002). While no clear explanation is immediately apparent, this lack of enhanced lipolysis may be related to a decrease

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in sympathetic stimulation to the small adipose depots. One of insulin’s mechanisms to prevent lipolysis is to counter the pro-lipolytic signaling coming from β-adrenergic stimulation of protein kinase A (PKA) and the subsequent activation of hormone sensitive lipase (HSL). IR-stimulated

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Akt activates phosphodiesterase-3B (PDE3B) which then reduces the levels of cAMP, inactivating PKA (Cheng et al., 2010,Arner and Langin, 2007). Another possible explanation for

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the lack of enhanced lipolysis is that the presence of a small level of residual IR in the FIRKO adipose tissue is sufficient to moderate lipolysis, but not sufficient to stimulate glucose and lipid uptake. The FIRKO phenotype suggests that activation of the adipose IR is not necessary for glucose homeostasis in mice, but it is not clear that a human with a total lack of IR signaling in adipose would respond in the same fashion. Also, not all of the metabolic adaptations of the FIRKO mice have been identified.

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2.d. Pancreatic β-cells: While pancreatic β-cells are not key sites of glucose utilization or storage, nor producers of metabolic fuel from a whole-body perspective, they are an important insulin-responsive tissue. Insulin signaling is required to maintain proper β-cell mass and first-

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phase insulin secretion (Kulkarni, Bruning, Winnay et al., 1999,Cheng, Lam, Wu et al.,

2012,Goldfine and Kulkarni, 2012,Folli, Okada, Perego et al., 2011). Insulin secretion by the βcells, through an autocrine mechanism, stimulates both insulin and β-glucokinase gene

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transcription (Leibiger, Leibiger, Moede et al., 2001), both of which serve to support a robust

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first phase insulin secretion. Absence of insulin signaling in β-cells also impairs mitochondrial function by reducing expression and phosphorylation of Bcl-2-associated death promoter proteins (BADs) (Liu, Okada, Assmann et al., 2009). Insulin-stimulated alterations in gene expression are regulated by multiple signaling pathways, all of which are downstream from PI3K (Leibiger et al., 2001). IR signaling in the β-cell is clearly required for maintenance of insulin

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secretion and proper metabolic control.

2.e. Neurons: The IR is widely-expressed in the brain, with the highest levels in the olfactory bulb, cortex, hippocampus, hypothalamus, and cerebellum. IGF-1R is highest in the

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cortex, hippocampus, thalamus, and lower in the cerebellum, olfactory bulb and hypothalamus

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(Kleinridders, Ferris, Cai et al., 2014). IR expressing neurons of the brain and periphery (Waldbillig and LeRoith, 1987) directly respond to insulin by promoting satiety and hormonal regulation. Insulin signaling in the CNS was recently reviewed by Durate et. al. (Duarte et al., 2012). Mice with neuronal-specific IR deletion (NIRKO) have elevated food consumption and increased body mass (Bruning, Gautam, Burks et al., 2000,Obici, Zhang, Karkanias et al., 2002) as well as impaired sympathetic counter-regulatory response to hypoglycemia (Diggs-Andrews, Zhang, Song et al., 2010). In rats, reduction in hypothalamic IR levels also increased food intake

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markedly and impaired the insulin-stimulated decrease in hepatic glucose production (Obici, Feng, Karkanias et al., 2002). Insulin stimulation increases the expression of proopiomelanocortin (POMC) and cocaine-and-amphetamine-related transcript (CART), and

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inhibits the expression of neuropeptide-Y (NPY) and Agouti-related peptide (AgRP) in the

arcuate nucleus (Kleinridders et al., 2014,Bruning et al., 2000,Corp, Woods, Porte et al., 1986). The important role of nervous system regulation by insulin has also been demonstrated with

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hepatic vagotomy, as blockade of vagal control reduces the insulin-mediated drop in hepatic glucose production (Pocai, Lam, Gutierrez-Juarez et al., 2005). Although there are some neurons

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that express insulin-sensitive Glut4 transporters, glucose uptake by most neurons is generally considered to be either insulin-independent or only indirectly regulated by insulin. Insulin is actively transported into the cerebrospinal fluid (CSF) by receptor-mediated mechanisms and some of the regions of the brain where insulin action is key, such as the arcuate nucleus of the

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hypothalamus, are adjacent to circumventricular organs with fenestrated capillaries that may allow for passive diffusion of insulin (Duarte et al., 2012,Schaeffer, Hodson and Mollard, 2014). The activation of the IR in the CNS appears to be a critical site of insulin action in the

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maintenance of metabolic homeostasis. The role of insulin signaling in peripheral neurons is also emerging as an additional metabolic derangement in insulin resistance and diabetes, and

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insulin sensitivity and signaling may be required to maintain the health of peripheral neurons (Dunn and Adams, 2014).

2.f. Endothelial Cells/Vasodilation: Insulin is not a key regulator of either glucose

transport or metabolism in endothelial cells (Vicent, Ilany, Kondo et al., 2003,Frasca, Pandini, Malaguarnera et al., 2007), but insulin does regulate a number of other functions including an anti-apoptotic effect and increased expression of eNOS (Vincent, Barrett, Lindner et al., 2003).

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The increased eNOS expression is regulated by FoxO proteins downstream of Akt (Fisslthaler, Benzing, Busse et al., 2003,Tsuchiya, Tanaka, Shuiqing et al., 2012). It has been hypothesized that a significant portion of the insulin-stimulated increase in glucose uptake by skeletal muscle

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in vivo is due to an increase in capillary recruitment and the NO-mediated vasodilatory effects of

insulin (Vincent et al., 2003,Vincent, Clerk, Lindner et al., 2004,Kubota, Kubota, Kumagai et al., 2011,Kubota, Kubota and Kadowaki, 2013,Mather, Laakso, Edelman et al., 2000). The actions

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of insulin in the vascular endothelium have been reviewed (Cersosimo and DeFronzo,

2006,Muniyappa, Iantorno and Quon, 2008). Thus, activation of IR in vascular endothelium has

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a fairly modest, but positive effect on metabolic homeostasis.

3. Is Mitogenic Regulation of ERK by Insulin Important in Insulin Responsive Tissues? The dose-response for ERK activation by insulin is five- to ten-fold less sensitive than

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Akt activation in most insulin-responsive tissues and cell lines (Bedinger et al., 2015,Jensen et al., 2007,Berlato and Doppler, 2009,Huang, Thirone, Huang et al., 2005). Experimental measurement of ERK activation typically requires relatively high concentrations of insulin (> 1

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nM), suggesting that this pathway is subject to much less amplification from the IR than is the Akt pathway, since the latter can be readily activated by concentrations of below 100 picomolar

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(Tan et al., 2012). Nevertheless, biochemically detectable levels of ERK activation are induced by insulin in a number of cell and tissue types at physiological insulin levels (vide infra). So the question arises: what are the likely effects of insulin-mediated ERK activation in vivo? Is ERK activation by insulin required or beneficial for healthy physiology? Are there opportunities or risks for using IR agonists that do not activate ERK? To consider these questions, a survey of several tissues is presented.

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3.a. Liver: The adult liver expresses far more insulin than IGF-1 receptors (IGF-1R is expressed at less than 20% that of IR) (Frasca et al., 2003,Desbois-Mouthon, Wendum, Cadoret et al., 2006). Also, the liver responds directly to growth hormone rather than IGF-1 (Sjogren,

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Liu, Blad et al., 1999). Thus, in the healthy liver, even at high concentrations, insulin acts via the IR rather than the IGF-1R. Lower doses of insulin, at best show a modest or inconsistent pERK response. For context, studies demonstrating insulin-stimulated ERK activation employ

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lethal insulin doses (from 10 U/kg (Wu and Williams, 2012) to 5 U/mouse (Bard-Chapeau,

Hevener, Long et al., 2005)). Thus, the importance of insulin-stimulated ERK activation in the

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liver has not been clearly established, even though activation by other means may be relevant. Liver-specific knockout of ERK-2 results in both a reduction of the endoplasmic reticulum (ER) calcium pump SERCA2, and increased ER stress in hepatocytes (Kujiraoka, Satoh, Ayaori et al., 2013). The phenotype of the whole-body ERK-1-/- mice was somewhat different in that the

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livers were smaller than wild type, but this difference was largely due to decreased lipid content rather than ER stress-related atrophy, suggesting functional differences between these two ERK isoforms (Pages, Guerin, Grall et al., 1999,Jager, Corcelle, Gremeaux et al., 2011). These data

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highlight that ERK signaling is important for the functioning of hepatocytes, but does not demonstrate that insulin-stimulated activation of this pathway is required to maintain hepatocyte

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health. Other growth factors and signaling systems are more robust activators of ERK in hepatocytes, such as leptin, HGF, and norepinephrine (Moon, Chamberland and Mantzoros, 2012,Melien, Nilssen, Dajani et al., 2002,Michalopoulos and DeFrances, 1997). Interestingly, hepatic over-activation of ERK leads to systemic insulin resistance with fasting hyperglycemia and decreased hepatic fatty acid oxidation, while decreasing ERK expression in mice with dietinduced obesity (DIO) or in a genetically obese background (ob/ob) has positive effects on

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glucose homeostasis and insulin sensitivity (Jager et al., 2011,Jiao, Feng, Li et al., 2013,Souza Pauli, Ropelle, de Souza et al., 2014,Zheng, Zhang, Pendleton et al., 2009). These observations

player in the hepatic metabolic insulin response.

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suggest that while ERK has important regulatory roles in metabolic homeostasis, it is not a key

Liver cells are typically quiescent, but upon injury or resection they proliferate rapidly (Michalopoulos and DeFrances, 1997,Mohammed and Khokha, 2005). Either in the absence of

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portal insulin or in LIRKO mice, hepatocytes atrophy (Michael et al., 2000,Michalopoulos and DeFrances, 1997). However, insulin by itself is not a proliferative signal, and other signals and

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growth factors such as TNFα and HGF must also be present to induce hepatocyte proliferation (Desbois-Mouthon et al., 2006,Mohammed and Khokha, 2005). In models of liver injury by resection, hepatocyte expression of IGF-1R is up-regulated and mitogenic IGF-1R signaling contributes to the proliferative repair response (Desbois-Mouthon et al., 2006,Caro, Poulos,

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Ittoop et al., 1988).

Despite the fact that insulin is not independently mitogenic in hepatocytes, it does stimulate the release of IGF-1 from the liver and decreases hepatic expression of insulin-like

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growth factor-binding proteins (IGFBPs) which act to limit IGF-1 bioavailability (Maddux, Chan, De Filippis et al., 2006,Griffen, Russell, Katz et al., 1987,Baxter, 2014,Lewitt and Baxter,

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1991). The increased production of IGF-1and decreased IGFBP levels induced by insulin act to raise whole-body IGF-1 activity, establishing a mechanism by which hepatic insulin stimulation could indirectly regulate mitogenic outcomes. The mechanism by which insulin stimulates IGF1 production by hepatocytes is unlikely to be mediated by the ERK pathway, as only modest elevations of insulin are required to stimulate IGF-1 expression (Griffen et al., 1987) and in vitro

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studies with rat hepatocytes show that increases in IGF-1 mRNA expression are mediated by PI3K rather than ERK (Shoba, Newman, Liu et al., 2001). The hepatic production of IGF-1 may also indirectly affect insulin sensitivity. The liver

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is the main source of circulating IGF-1 levels and IGF-1 stimulates negative feedback

mechanisms which reduce the secretion of growth hormone (GH). One action of GH is that it decreases whole-body insulin sensitivity, especially in muscle, and IGF-1 decreases GH

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secretion through hypothalamic feedback inhibition. Through this mechanism of reducing GH levels, IGF-1 has been proposed to have insulin-sensitizing effects (Yakar, Liu, Fernandez et al.,

3.b. Muscle:

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2001,Clemmons, 2004).

In skeletal muscle, ERK signaling can be activated at the upper

physiological levels of insulin (Cusi et al., 2000,Dufresne, Bjorbaek, El-Haschimi et al., 2001). Similar to the relationship described for the liver, the ability of myocytes to signal via ERK is

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crucial for muscle growth and regeneration (Haddad and Adams, 2004), but a role for insulininduced ERK activation in this process has not been established and is not required for insulin’s metabolic actions (Wojtaszewski, Lynge, Jakobsen et al., 1999). Importantly, constitutive ERK

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activation, rather than insulin-stimulated ERK activation in muscle is evident in various models of insulin resistance (Plomgaard, Bouzakri, Krogh-Madsen et al., 2005). ERK activation in

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muscle has multiple mechanisms of reducing insulin sensitivity including inhibitory serine phosphorylation of IRS-1 (Gual, Le Marchand-Brustel and Tanti, 2005,Copps and White, 2012), decreased microtubule stability (Asrih, Pellieux, Papageorgiou et al., 2011), and serine 273 phosphorylation of PPARγ (Banks, McAllister, Camporez et al., 2014). Except in cases of severe insulin resistance, insulin infusion at tolerable doses in both humans and animals leads to clear induction of Akt phosphorylation in muscle, however the

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ERK1/2 phosphorylation response is much more variable and subject to large individual-toindividual variations in background phosphorylation, even in the fasted state (Cusi et al., 2000,Ruiz-Alcaraz, Lipina, Petrie et al., 2013). Muscle has substantial expression of IGF-1R

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and IGF-1R/IR heteroreceptors, with roughly 20% of the muscle IR/IGF-1R content expressed as homodimeric IR (Frasca et al., 2003,Fernandez, Kim, Yakar et al., 2001), making the muscle responsive to the IGF-1 ligand. Given the high level of IGF-1R expression in muscle, much of

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the observed ERK activation, especially in the many studies that use supraphysiological insulin levels, can likely be attributed to insulin signaling through either the IGF-1R receptor or the

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hybrid receptor.

Insulin action and ERK phosphorylation also intersect in the context of exercise. Both high insulin dose and exercise can activate ERK signaling in muscle (Goodyear, Chang, Sherwood et al., 1996). Fluckey et. al showed that exercise-related ERK activation is required

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for insulin-stimulated increases in protein synthesis (Fluckey, Knox, Smith et al., 2006,Fluckey, Vary, Jefferson et al., 1996). Thus, the activation of ERK, via exercise rather than insulin, allows insulin to drive significant increases in protein synthesis via mTORC1, downstream from

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Akt activation. Activation of the Akt/mTORC1 pathway is also a necessary factor in the development of muscle hypertrophy and prevention of atrophy (Bodine et al., 2001).

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Another action of Akt, the phosphorylation and nuclear exclusion of FoxO1, plays a role

in preventing atrophy and muscle catabolism by decreasing the expression of ubiquitin ligases (Sandri, 2008). Insulin promotes (and may be required for) muscle hypertrophy, but is also incapable of inducing hypertrophy in the absence of other signals that activate ERK. In contrast, stimulation of muscle by IGF-1 is sufficient to stimulate hypertrophy in the absence of resistance exercise (Haddad and Adams, 2004,Adams and McCue, 1998). IGF-1 administration has been

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shown to slow atrophy in denervated muscle model (Stitt, Drujan, Clarke et al., 2004). However, IGF-1 over-expression alone is not sufficient to totally prevent atrophy in unloaded muscle models (Criswell, Booth, DeMayo et al., 1998,Adams, 2002).

Following resistance exercise

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and during muscle regeneration after injury, muscle IGF-1 levels are elevated in a growth

hormone-independent manner (Adams, 2002). Muscle regeneration and hypertrophy involves the differentiation, proliferation, and integration of muscle satellite cells, which increases both

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the DNA and protein content of myofibrils, and IGF-1-mediated ERK activation is a key driver of this process (Adams, 2002). In summary, there is no evidence of a key role for insulin-

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induced ERK activation in muscle homeostasis, metabolic regulation, or repair, but there are independent yet convergent pathways between insulin and ERK that impact these outcomes. 3.c. Adipose: ERK-1 signaling has been shown to promote adipogenesis (Prusty, Park, Davis et al., 2002), and ERK-1 knockout mice have decreased adipose mass (Bost, Aouadi,

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Caron et al., 2005). However, total leptin deficiency (ob/ob mouse) overrides this requirement, and the ob/ob ERK-1-/- double-knockout mice display obesity (Jager et al., 2011). Both lean and obese ERK-1-/- mice showed markedly improved glucose and insulin tolerance, suggesting that

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ERK-1 activation, in general, decreases insulin sensitivity (Banks et al., 2014). Furthermore, inhibitor studies demonstrated that ERK activation is not required for GLUT4 translocation and

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glucose uptake in adipocytes (Reusch, Bhuripanyo, Carel et al., 1995). In 3T3-L1 adipocytes, Weisse et al. (Wiese, Mastick, Lazar et al., 1995) studied the ability of insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) to stimulate ERK and PI3K signaling pathways and compared these outcomes to stimulation of glucose uptake, lipid synthesis, and glycogen synthesis. They found that high dose insulin (100 nM) could induce a modest increase in ERK activation; but PDGF and EGF were more potent stimulators. PGDF, but not EGF,

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activated PI3K at a maximal levels similar to insulin; however, only insulin was able to induce glucose uptake, lipid synthesis, and glycogen synthesis. This demonstrated that activation of either of the upstream signaling factors, Akt or ERK, alone is not sufficient for the metabolic

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actions of insulin in fat cells, and that specific properties of the IR drive activation of the metabolic effects (Wiese et al., 1995).

Pre-adipocytes have few IR, but as they undergo adipogenesis, IR levels greatly increase

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to the point where they exceed IGF-1R concentrations (Entingh-Pearsall and Kahn, 2004). In cell culture studies, high concentrations of insulin act through the IGF-1R to stimulate ERK

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activation and promote the early proliferative phase of adipogenesis (Rosen and MacDougald, 2006). As adipogenesis progresses and IR expression increases, ERK-1 activation becomes less important and metabolic insulin signaling becomes dominant (Rosen and MacDougald, 2006). In vivo, the high levels of IGF ligands during embryonic and postnatal growth and development

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provide the IGF-1R stimulation required to activate ERK and drive adipogenesis, and as a result FIRKO mice with IR absent in adipose tissue still develop fat pads. 3.d. Pancreatic β-cells:

β-cell mass can expand and regenerate in response to injury or

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metabolic changes. Increases in the number of β-cells appears to depend both the differentiation from a variety of potential precursor cells (Habener and Stanojevic, 2012,Assmann, Hinault and

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Kulkarni, 2009) as well as through a balance of apoptosis and replication of adult β-cells (Assmann et al., 2009,Dor, Brown, Martinez et al., 2004), however the relative extent of these processes in adult humans is still being evaluated. In embryonic and postnatal development, IGF-1 and IGF-2 play either modest or supporting roles in the expansion and development of βcell mass (Kulkarni, 2005,Cantley, Choudhury, Asare-Anane et al., 2007,Otani, Kulkarni, Baldwin et al., 2004), although β-cell mass appears to be mediated through the IR (Otani et al.,

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2004,Kulkarni, Holzenberger, Shih et al., 2002,Okada, Liew, Hu et al., 2007). The IR-mediated growth effects of β-cells are regulated by the PI3K/Akt/FoxO1 and PI3K/mTORC1/P70S6K signaling pathways (Okada et al., 2007,Pende, Kozma, Jaquet et al., 2000). As mentioned above,

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insulin acts via a paracrine secretion and response mechanism to increase the expression of

insulin and glucokinase, and this action is IR/PI3K mediated, not involving ERK (Leibiger et al., 2001). However, this observation does not suggest that ERK signaling plays no role in β-cell

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biology. Rather, when elevated blood glucose levels trigger insulin secretion and associated calcium flux, calcineurin activates ERK1/2 which stimulates insulin expression (Duan and Cobb,

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2010,Gibson, Lawrence, Gibson et al., 2006). Nevertheless, chronic stimulation of ERK1/2 causes a desensitization effect and reduced insulin expression (Lawrence, McGlynn, Park et al., 2005). Under optimal conditions, ERK1/2 plays a role as a nutrient-sensing regulator of insulin secretion in an IR-independent manner in pancreatic β-cells.

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Given the modest phenotype of IR and IGF-1R KO in mouse β-cells (Kulkarni, 2005,Kido, Nakae, Hribal et al., 2002), it is clear that other stimulators of β-cell growth and maintenance are the dominant drivers of β-cell proliferation and anti-apoptotic response.

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Hepatocyte growth factor (HGF) is one such factor and acts as a potent stimulator of β-cell proliferation (Garcia-Ocana, Takane, Syed et al., 2000). Other factors such as parathyroid

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hormone related protein (PTHrP,), GH, glucagon like peptide-1 (GLP-1), and prolactin all may be able to contribute to the stimulation of β-cell growth (reviewed in (Assmann et al., 2009,Lee and Jun, 2014,Bernal-Mizrachi, Kulkarni, Scott et al., 2014,Kulkarni, Mizrachi, Ocana et al., 2012)). These growth factors stimulate β-cell proliferation through a number of signaling pathways including activation of various PKC isoforms (δ,ε,ζ,λ) and PI3K for PTHrP and GLP-1 (Friedrichsen, Neubauer, Lee et al., 2006,Vasavada, Wang, Fujinaka et al., 2007), and

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JAK2/STAT5 for prolactin and GH (Kondegowda, Mozar, Chin et al., 2012,Choi, Cai and Woo, 2011). β-cell progression through the G1/S checkpoint is dependent on cyclin D1 and cdk4 in mice, and cdk4 and/or 6 in humans (Cozar-Castellano, Fiaschi-Taesch, Bigatel et al., 2006), and

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is regulated by the WNT/β-catenin system (Bernal-Mizrachi et al., 2014,Aly, Rohatgi, Marshall et al., 2013). Thus, insulin-stimulated ERK activation may not play a significant role in the growth or maintenance of pancreatic β-cell mass.

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3.e. Neurons: ERK activation is a key signaling component for many functions in

neurons including survival (Frebel and Wiese, 2006), memory (Shiflett and Balleine, 2011),

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synaptic plasticity (Thomas and Huganir, 2004,Choi, Cho, Hoyt et al., 2008,Yang, Wei, Liu et al., 2013), and transcriptional regulation (Wiegert and Bading, 2011). Much of the effect of IGF signaling in the CNS is ERK-mediated (Choi et al., 2008,Wang, Qin, Gong et al., 2014), but the dominant actions of insulin in the CNS are PI3K-dependent (Kleinridders et al., 2014). There

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are several in vitro studies that support a role for ERK in insulin-stimulated effects in neurons (Mayer and Belsham, 2009,O'Malley and Harvey, 2007,Kim, DiVall, Deneau et al., 2005), but these experiments utilized a supraphysiological insulin concentration (10 nM) capable of

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activating IR/IGF-1R hybrid receptors (Pandini, Frasca, Mineo et al., 2002). While the functions of brain IR largely involve satiety and metabolic control, IGF-1R is required for normal brain

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development and somatotropic functions (Kappeler, De Magalhaes Filho, Dupont et al., 2008). Thus, the activation of ERK by insulin stimulation of IR has not been demonstrated to be a factor in the activity or health of neurons in vivo. 3.f. Vascular Endothelium: Chronic hyperinsulinemia is a risk factor for atherosclerosis and hypertension (reviewed in (DeFronzo, 2010)), and with insulin resistance, the generally beneficial PI3K/Akt-mediated effects of insulin action are impaired and ERK activation is

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increased (Cusi et al., 2000,Groop, Forsblom and Thomas, 2005,Gogg, Smith and Jansson, 2009). Hyperlipidemia is a common result of insulin resistance, and VLDL are elevated (Muniyappa et al., 2008,Otero, Stafford and McGuinness, 2014), typically coincident with a pro-

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inflammatory state: both outcomes serve to either worsen or accelerate the progression of

atherosclerotic lesions (Cersosimo and DeFronzo, 2006,Muniyappa et al., 2008). The MAPKs, ERK and c-Jun N-terminal kinase (JNK), decrease the activity of the PI3K/Akt insulin signaling

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pathway in part by serine phosphorylation of IRS-1 (Muniyappa et al., 2008). Since the antiatherogenic signals in insulin-sensitive endothelium are mediated by the Akt/FoxO pathway

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(Tsuchiya et al., 2012,Montagnani, Golovchenko, Kim et al., 2002,Wang, Goalstone and Draznin, 2004), inhibition of this pathway is deleterious.

Hypertension also has associations with ERK-mediated effects. Angiotensin II is a driver of an ERK-mediated decrease in eNOS production (Cersosimo and DeFronzo, 2006,Andreozzi,

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Laratta, Sciacqua et al., 2004), and Angiotensin II also reduces insulin sensitivity by stimulating IRS-2 serine phosphorylation by PKCβ (Park, Li, Rask-Madsen et al., 2013), which decreases activation of the PI3K/Akt pathway. Upper physiological levels of insulin have been shown to

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stimulate ERK-mediated vasoconstriction and eNOS reductions in ex vivo tissue or cell models (Eringa, Stehouwer, van Nieuw Amerongen et al., 2004). These data highlight the inter-

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relationship between insulin resistance and hypertension, and implicate ERK activation as playing a role.

The role of IGF-1 and IGF-1R in atherosclerosis is complex. The stimulation of

proliferative and migration effects by IGF/IGF-1R signaling may add to the development of plaques (Clemmons, 2007,Zhu, Zhao, Witte et al., 2001), but the anti-apoptotic effects of this signaling may prevent necrosis and rupture (Gao, Wassler, Zhang et al., 2014,von der Thusen,

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Borensztajn, Moimas et al., 2011). Low circulating levels of IGF-1 and higher IGFB-3 levels correlate as an independent risk factor for ischemic heart disease (Juul, Scheike, Davidsen et al., 2002,Colao, Spiezia, Di Somma et al., 2005). ERK and PI3K both mediate IGF-1-mediated

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growth of vascular smooth muscle cells (VSMC), which could potentially lead to increases in the size of atherosclerotic plaques and in neo-intimal formation (Niu, Li, Hakim et al., 2007). Thus, the aggregate of results indicates that some level of IGF signaling is advantageous to support the

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integrity of the vascular intima, but excessive levels of mitogenic signaling would accelerate

ERK activation for vascular health.

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atherosclerotic progression. There is no evidence to support the requirement of IR mediated

4. Pathologic and Therapeutic Implications of Tissue- and Pathway-Specificity of IR Activation

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4.a. General complications of hyperinsulinemia: There is evidence to suggest that hyperinsulinemia precedes or initiates the process of insulin resistance (Corkey, 2012), and there are many studies demonstrating that insulin induces insulin resistance both in vitro and in vivo

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(Gavin et al., 1974,Marshall and Olefsky, 1980,Garvey et al., 1985,Del Prato, Leonetti, Simonson et al., 1994,Flores-Riveros, McLenithan, Ezaki et al., 1993,Garvey, Olefsky and

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Marshall, 1986,Kozka, Clark and Holman, 1991,Ricort, Tanti, Van Obberghen et al., 1995,Kim, McLean, Philip et al., 2011). Hyperinsulinemia likely contributes to many of the co-morbidities of the insulin-resistant state including atherosclerosis, microvascular complications, hyperlipidemia, and general dysregulation of nutrient flux (DeFronzo, 2010,Otero et al., 2014). These effects are especially important, as hyperinsulinemia can be a long-term condition that may be present for many years or decades before the development of diabetes (Lundgren,

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Bengtsson, Blohme et al., 1990). Simply giving more insulin to already hyperinsulinemic patients will improve blood glucose levels, but may exacerbate other complications of hyperinsulinemia, including insulin resistance itself.

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4.b. Insulin, IGF, and Cancer: There are substantial data supporting the association of hyperinsulinemia, especially in T2DM patients, with increased rates of certain types of cancers (Belfiore and Malaguarnera, 2011,Giovannucci et al., 2010,Rose and Vona-Davis,

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2012,Goodwin, Ennis, Pritchard et al., 2002). This observation raises a number of questions: Does hyperinsulinemia directly lead to the development or increase the risk of malignant cells?

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Is insulin stimulating other factors that lead to the increase in cancer? Does insulin therapy itself lead to an increased risk of cancer? And lastly, is there a potential for IR-based modulator therapies to circumvent any of these potential risks?

In some neoplasms, IR expression is greatly increased and insulin can act as a

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proliferative stimulant in its own right (Frasca et al., 2003,Law, Habibi, Hu et al., 2008). This appears to be particularly true in some breast and endometrial cancers (Belfiore and Malaguarnera, 2011,Frasca et al., 2003,Law et al., 2008,Wang, Zhang, Zhao et al., 2013), and

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experimental overexpression of IR in certain tumor cell lines increases the proliferative response to insulin (Wang et al., 2013). It is important to note that ERK, and not Akt, is responsible for

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stimulating cell cycle progression in tumor cells (Weng, Smith, Brown et al., 2001,Djiogue, Nwabo Kamdje, Vecchio et al., 2013), and therefore signals derived from the IGF-1R or IGF1R/IR hybrid are much better at stimulating proliferation than signals coming from the IR. Many neoplasms and tissues can proliferate in response to higher levels of insulin via cross-talk with IGF-1R and IGF-1R/IR hybrid receptors, but these levels of insulin are not typically achieved in humans even under insulin therapy conditions, except near the site of insulin or insulin analog

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injections, where lipohypertrophy is fairly common, necessitating the constant rotation of injection sites (Hauner, Stockamp and Haastert, 1996). Despite the likely limited role of insulin acting as a direct mitogen to stimulate the

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proliferation of most types of tumors, secondary effects of insulin and the IR on tumors may also occur. Insulin stimulates increased levels of total and free IGF-1 (Griffen et al., 1987,Baxter, 2014,Lewitt and Baxter, 1991), as well as a variety of other potential regulators of proliferation

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and differentiation, such as cytokines and growth factors (Gong, Dou and Liang, 2014) including leptin, VEGF, and IL-6 (Rose and Vona-Davis, 2012,Mistry, Digby, Desai et al., 2007). Insulin

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stimulates the production of VEGF from both the endothelium and adipocytes (as reviewed in (Rose and Vona-Davis, 2012)) and this may support the growth of neoplasms and metastasis by aiding the angiogenic process. Levels of free IGF-1 and IGF-2 are correlated with increased incidence of breast tumors (Espelund, Cold, Frystyk et al., 2008) and colorectal cancer (Erarslan,

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Coskun, Turkay et al., 2014), and have been shown to induce increased metastasis and severity of colorectal (Wu, Yakar, Zhao et al., 2002) and mammary tumors (Wu, Cui, Miyoshi et al., 2003,Belardi, Gallagher, Novosyadlyy et al., 2013). Many tumors secrete high levels of IGF-2,

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and contain the IR-A form of the IR, which binds well to IGF-2 (Frasca et al., 1999), thus causing a strong paracrine stimulation of proliferation (Livingstone, 2013). The relationship

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between insulin-induced elevations of free IGF growth factor levels and the proliferative actions of IGFs could be a mechanism by which hyperinsulinemia increases carcinogenesis risk. Furthermore, reductions in growth hormone signaling lead to increased longevity and reduced rates of cancer, potentially through reduced circulating levels of IGF-1 (Anisimov and Bartke, 2013).

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Insulin has been shown to increase the expression of estradiol and estrogen receptor in breast cancer cells and adipocytes (Rose and Vona-Davis, 2012). Hyperinsulinemic postmenopausal women have adipose-derived estrogen and this expression is believed to be insulin-

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mediated via both a PI3K- and MAPK-dependent mechanism. The relative contribution of

increased estrogen signaling induced by hyperinsulinemia in breast cancer is controversial, as hyperinsulinemia is correlated with the incidence of both ER-positive and ER-negative breast

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cancers (all reviewed in (Rose and Vona-Davis, 2012)). Therefore, it is extremely difficult to estimate, let alone quantitate, the extent to which insulin-stimulated increases in growth factor

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levels could be responsible for the development of neoplasms.

Despite both the relatively strong clinical correlations and the experimental evidence that suggest hyperinsulinemia contributes to cancer risk, studies have not been able to definitively demonstrate that the clinical use of exogenous insulin clearly increases the risk of cancers (Garg,

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Hirsch and Skyler, 2009), and cancer rates between insulin and the various approved analogs do not appear to be different (Grimaldi-Bensouda, Cameron, Marty et al., 2014). Another factor that affects the clinical relevance of insulin-induced cancer is that the most commonly prescribed

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drug in T2DM, metformin, appears to negate any increase in cancer rates seen with insulin therapy (Anisimov and Bartke, 2013). Based on these observations, it would seem that the

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therapeutic risk of specific IR agonists in terms of cancer progression is modest, especially for those agonists that more selectively activate the “metabolic” (Akt) but only minimally trigger “mitogenic” (ERK) pathways. Furthermore, any IR modulator therapy should be evaluated for IGF-1R activation, as a high level of IGF-1R activation would most likely increase the oncogenic risk (Hansen et al., 2011). It follows that IR modulators with high specificity toward the IR only would be desirable in this regard.

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4.c. Therapeutic implications for pathway- or tissue-specific IR modulators in metabolic disease: From the data reviewed herein, the clearest way to have a safe and effective IR agonist for use in metabolic disease would be to engineer it in such a way as to either preferentially or

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solely activate the Akt signaling pathway, rather than the ERK pathway. Pathway-biased

therapeutic agonists for seven transmembrane receptors have been developed (Rajagopal, Ahn, Rominger et al., 2011). These molecules preferentially induce specific conformations in the

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receptors that allow for different G-proteins to associate, and thus agonists can have vastly

different signaling outputs from the same receptor. However, no agonists with that extent of bias

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have been described for receptor tyrosine kinase receptors such as the IR. This result is likely due to the somewhat less conformation-sensitive, phosphorylation-dependent signaling architecture. Recently there have been two reports of IR agonist molecules that preferentially activate the Akt signaling pathway over the ERK pathway, the peptide S597 (Jensen et al., 2007)

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and the human monoclonal antibody XMetA (Bhaskar, Goldfine, Bedinger et al., 2012). Interestingly, both molecules are partial agonists and stimulate less IR autophosphorylation compared to insulin. The property of partial agonism itself is likely the driver of both

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compounds’ apparent pathway specificity, because as previously reviewed, the Akt pathway is

2015).

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the preferred (or much more sensitive) activation pathway for IR stimulation (Bedinger et al.,

There is no obvious benefit to stimulating ERK activation with an IR agonist in the

treatment of metabolic diseases such as T2DM. It is clear that ERK has a number of beneficial actions, from learning and plasticity of neurons, recovery of injury in the liver, and maintenance of insulin production by the pancreatic β-cell but, as reviewed herein, none of these positive ERK mediated properties appears to be mediated by the IR. On the contrary, the activation of

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ERK in the context of insulin resistance and hyperinsulinemia appears to drive a number of pathological processes including desensitization of the Akt pathway in liver, muscle, and adipose tissue, as well as exacerbating hyperlipidemia and hypertension. Furthermore, activation of the

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ERK pathway may lead to mitogenic stimulation and promote the growth of neoplastic cells. New IR agonists could be designed with enhanced specificity to the homodimeric IR and not the IGF-1R or IGF-1R/IR heteroreceptors, further decreasing mitogenic potential. The ability to

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activate the metabolic functions of the IR without stimulating these ERK-mediated pathologies would be beneficial.

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4.d. Therapeutic implications for tissue-specific IR modulators: The role of IR signaling in tissues varies, and there have been several reports of IR agonists with preferential tissue action, although they have all been biased to the liver, probably due to the highly fenestrated sinusoidal structure that allows direct access of macromolecules in the blood to the hepatocytes (Shojaee-

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Moradie et al., 2000,Moore et al., 2014,Bedinger et al., 2015). The accessible nature of the hepatocyte IR to agonist therapy may be fortuitous as the liver is a compelling tissue to target with a selective insulin therapy. The production of glucose by the liver is the main driver of

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elevated fasting blood glucose in T2DM (DeFronzo, 2004). In this condition, suppression of hepatic glucose output by insulin is impaired, and IR actions in other tissues that help regulate

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glucose production, such as decreased gluconeogenic substrate delivery (adipose and muscle tissue) and vagal control (neurons) appear to be dysfunctional (Defronzo, 2009,Cherrington et al., 1998). Action in the liver by a tissue-specific IR agonist may not be sufficient for complete metabolic control in T2DM or pre-diabetes, but could have great utility as part of the pharmacological armamentarium. Furthermore, a liver-specific agent would have promise to

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improve blood glucose control while avoiding the weight gain associated with insulin therapy in some people. While this review has made extensive reference to the phenotypes of tissue-specific IR

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knockout mice, there are a number of things to consider when interpreting those data. First, in humans glucose uptake in muscle is believed to play a larger role in glucose homeostasis relative to the mouse; the latter is believed to have higher dependence on liver regulation (Nandi et al.,

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2004). Despite this limitation, the IR deficient mice provide useful insights into many facets of insulin action. The IR depletion studies in mice suggest that neither skeletal muscle nor adipose

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tissue IR expression is an absolute requirement to maintain normal blood glucose (Bruning et al., 1998,Bluher et al., 2002). The adipose IR knockout mouse had a beneficial metabolic profile, and IR stimulation in the adipose tissue has both potential positive and negative effects in terms of metabolic health. IR stimulation would attenuate lipolysis and reduce blood FFA levels,

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which is in most cases would have a positive effect in T2DM or pre-diabetes, but it would also increase the uptake of glucose and lipids into the adipose tissue, promoting energy storage and adipose expansion. Avoiding activation of the IR in adipose tissue by a therapeutic IR agonist

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could potentially be beneficial, in that it would avoid the increased adiposity associated with insulin therapy, while an IR agonist that did not activate the adipose tissue may poorly regulate

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lipolysis. If a patient has sufficient endogenous insulin to adequately moderate lipolysis, then an IR agonist therapy that does not target adipose tissue may benefit the patient by decreasing blood glucose without promoting excessive adipose gain. Insulin stimulation of muscle glucose uptake is a powerful glucose-lowering mechanism.

IR agonists with ability to activate muscle IR would most likely have a more potent ability to lower blood glucose than an agonist that could not activate the muscle, with the caveat that

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prolonged or excessive stimulation of muscle glucose uptake would increase the likelihood of fasting hypoglycemia (Moore et al., 2014). The necessity of muscle insulin action by an IR agonist would thus be determined by the extent of glucose reduction needed by a patient, and

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their level of insulin resistance.

The IRs of the liver, the brain, and the pancreas appear to be the most essential for

glycemic control. Whole body IR knockout mice (IRKO) do not survive more than a few days

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due to keto-acidosis (Kitamura et al., 2003). When IR is re-expressed in the liver, brain, and βcells of IRKO mice, they are viable and only moderately more prone to diabetes than normal

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mice (Okamoto, Nakae, Kitamura et al., 2004). Stimulation of IR in neurons is required for proper control of satiety and metabolism. It remains to be demonstrated if non-insulin IR agonists can access IRs in the CNS, but IR agonists may be able to gain access to the CSF via the same IR-mediated active transport mechanisms as insulin. Also, some of the IR-responsive

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regions of the brain, such as the arcuate nucleus of the hypothalamus and the hindbrain, are adjacent to circumventricular organs with fenestrated capillaries (Duarte et al., 2012,Schaeffer et al., 2014). It is almost certain that the ability to target IR in neurons would be beneficial to the

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efficacy of an IR agonist by promoting satiety and helping control hepatic glucose production. In most type 2 diabetics, the β-cells of the pancreas secrete significant levels of insulin,

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and although it may be insufficient for whole body glucose homeostasis, the local concentrations of insulin bathing the β-cell should be high enough to fully activate IR. Therefore, despite the important role for IR activation in maintaining a proper insulin response, an IR agonist may not require activity on β-cell IRs in order to be an effective therapy for T2DM. In light of the theory that hyperinsulinemia begets insulin resistance, it is intriguing to speculate that a therapeutic

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regimen that reduces β-cell hypersecretion—when coupled to IR activation in liver—would have positive effects on metabolism in the pre-diabetic or T2DM states. The importance of an IR agonist signaling in the vasculature is somewhat harder to

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predict. In general, increased insulin metabolic (Akt) signaling in the blood vessels should lead to increased vasodilation and some benefits for persons at risk for hypertension. Yet, this would have to be balanced against the potential for mitogenic activation that would carry risks of

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Conclusion

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exacerbating atherosclerosis and hypertension.

The ability to design IR agonists and/or modulators that have both variable potency and different levels of stimulation of key signaling pathways and receptor subsets is steadily progressing. These agonists include both modified insulins, synthetic peptides, and monoclonal

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antibodies. Preferential tissue activation is also moving forward by engineering properties into these compounds such as varying the molecular weight, altering solubility, and manipulating binding to other factors and transport proteins. It is hoped that this comprehensive review of the

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tissue- and pathway-specific details of insulin biology will serve as a resource to aid in the development of safer and more effective next-generation therapies for T2DM and insulinopenic

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diabetes.

Acknowledgements: The authors would like to thank Ira Goldfine M.D. for helpful suggestions and editing of this manuscript.

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Funding: This review was inspired by work where SHA received funding from the American Diabetes Association, NIH-NIDDK, and by a Cooperative Research and Development Agreement (CRADA 58-3K95-1-1497) between XOMA Corp. and United States Department of Agriculture-

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Agricultural Research Service (USDA-ARS).

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Highlights Section for: MCE-D-15-00369 “Metabolic, Anabolic, and Mitogenic

Insulin Responses: A Tissue-Specific Perspective for Insulin Receptor Activators” We review tissue-specific insulin responses and the relevant activation pathways.

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Some insulin responses are beneficial to the treatment of diabetes, others are not. Selective insulin receptor (IR) agonists may differentially target certain tissues. Selective IR agonists may differentially target certain signaling pathways.

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Selective IR modulators may be beneficial for the treatment of diabetes.