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**Chapter 4**

© 2012 Pu and Liu, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Fatty Acids Stimulate Glucose Uptake by the**

**PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways**

Obesity-driven type II diabetes mellitus has become a major crisis in modern societies. In the United States, over 80% of type II diabetic patients are obese [1]. In the case of Chinese adult diabetic patients, diabetes is also significantly associated with obesity [2]. Previous investigations have focused on looking for obesity-related factors that cause insulin resistance, the failure of the body to respond to insulin, which is the hallmark of type II diabetes. The abnormal plasma fatty acid metabolism associated with diabetes mellitus [3], and the high level of obesity-related plasma free fatty acids (FFAs, also known as non-esterified fatty acids,

Natural fatty acids are carboxylic acids with saturated or unsaturated aliphatic tails which have an even number of carbon atoms from 4 to 28. When they are not incorporated into other compounds, like triglyceride or phospholipids, they are known as "free" fatty acids. When metabolized, fatty acids yield a large quantity of ATP, and thus represent an important fuel for the body, particularly for heart and skeletal muscle. They are not only essential dietary nutrients, but also function in many cellular events by activating nuclear receptors, such as the peroxisome proliferator-activated receptors (PPARs), and fatty acid binding proteins (FABPs). How FFAs induce insulin resistance is not a novel topic in pathological studies on obesityassociated type II diabetes. Many efforts have been made to uncover the underlying molecular mechanisms, but they remain elusive. It seems that FFA-induced insulin resistance occurs not via a single pathway but rather via a complicated network of pathways

Major investigatons of the mechanisms of extra FFA-induced insulin resistance have focused on the chronic effects of FFAs. However, plasma FFA concentrations are not consistent and

and reproduction in any medium, provided the original work is properly cited.

NEFA) have been identified since the 1950s as major risk factors for insulin resistance.

Jing Pu and Pingsheng Liu

http://dx.doi.org/10.5772/52456

in organs, tissues, and cells.

**1.1. Acute cellular responses to Free Fatty Acids**

**1. Introduction**

Additional information is available at the end of the chapter

## **Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways**

Jing Pu and Pingsheng Liu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52456

## **1. Introduction**

128 Protein Phosphorylation in Human Health

recognition and beyond. Nat Cell Biol 12: 836-841

and tuning. Semin Cell Dev Biol 21: 699-711

ubiquitinated substrates. Mol Cell 33: 505-516

bacteria. Nat Immunol 10: 1215-1221

receptor. Autophagy 7: 1389-1391

ubiquitinated proteins. Mol Cell 44: 279-289

response and autophagy. Sci Signal 4: pe39

elongation. Brain Res Mol Brain Res 85: 1-12

533-539

[177] Kraft C, Peter M, Hofmann K (2010) Selective autophagy: ubiquitin-mediated

[178] Sumpter R, Jr., Levine B (2010) Autophagy and innate immunity: triggering, targeting

[179] Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T (2005) p62/SQSTM1 forms protein aggregates degraded by autophagy and

has a protective effect on huntingtin-induced cell death. J Cell Biol 171: 603-614 [180] Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Overvatn A, Bjorkoy G, Johansen T (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation

of ubiquitinated protein aggregates by autophagy. J Biol Chem 282: 24131-24145 [181] Kirkin V, Lamark T, Sou YS, Bjorkoy G, Nunn JL, Bruun JA, Shvets E, McEwan DG, Clausen TH, Wild P, Bilusic I, Theurillat JP, Overvatn A, Ishii T, Elazar Z, Komatsu M, Dikic I, Johansen T (2009) A role for NBR1 in autophagosomal degradation of

[182] Thurston TL, Ryzhakov G, Bloor S, von Muhlinen N, Randow F (2009) The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated

[183] Wild P, Farhan H, McEwan DG, Wagner S, Rogov VV, Brady NR, Richter B, Korac J, Waidmann O, Choudhary C, Dotsch V, Bumann D, Dikic I (2011) Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333: 228-233 [184] Novak I, Kirkin V, McEwan DG, Zhang J, Wild P, Rozenknop A, Rogov V, Lohr F, Popovic D, Occhipinti A, Reichert AS, Terzic J, Dotsch V, Ney PA, Dikic I (2010) Nix is a

selective autophagy receptor for mitochondrial clearance. EMBO Rep 11: 45-51 [185] Ogawa M, Sasakawa C (2011) The role of Tecpr1 in selective autophagy as a cargo

pathway targets bacterial pathogens. Cell Host Microbe 9: 376-389

and phosphorylates LC3. Biochem Biophys Res Commun 395: 471-476

[186] Ogawa M, Yoshikawa Y, Kobayashi T, Mimuro H, Fukumatsu M, Kiga K, Piao Z, Ashida H, Yoshida M, Kakuta S, Koyama T, Goto Y, Nagatake T, Nagai S, Kiyono H, Kawalec M, Reichhart JM, Sasakawa C (2011) A Tecpr1-dependent selective autophagy

[187] Cherra SJ, 3rd, Kulich SM, Uechi G, Balasubramani M, Mountzouris J, Day BW, Chu CT (2010) Regulation of the autophagy protein LC3 by phosphorylation. J Cell Biol 190:

[188] Jiang H, Cheng D, Liu W, Peng J, Feng J (2010) Protein kinase C inhibits autophagy

[189] Matsumoto G, Wada K, Okuno M, Kurosawa M, Nukina N (2011) Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of

[190] Weidberg H, Elazar Z (2011) TBK1 mediates crosstalk between the innate immune

[191] Okazaki N, Yan J, Yuasa S, Ueno T, Kominami E, Masuho Y, Koga H, Muramatsu M (2000) Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal Obesity-driven type II diabetes mellitus has become a major crisis in modern societies. In the United States, over 80% of type II diabetic patients are obese [1]. In the case of Chinese adult diabetic patients, diabetes is also significantly associated with obesity [2]. Previous investigations have focused on looking for obesity-related factors that cause insulin resistance, the failure of the body to respond to insulin, which is the hallmark of type II diabetes. The abnormal plasma fatty acid metabolism associated with diabetes mellitus [3], and the high level of obesity-related plasma free fatty acids (FFAs, also known as non-esterified fatty acids, NEFA) have been identified since the 1950s as major risk factors for insulin resistance.

Natural fatty acids are carboxylic acids with saturated or unsaturated aliphatic tails which have an even number of carbon atoms from 4 to 28. When they are not incorporated into other compounds, like triglyceride or phospholipids, they are known as "free" fatty acids. When metabolized, fatty acids yield a large quantity of ATP, and thus represent an important fuel for the body, particularly for heart and skeletal muscle. They are not only essential dietary nutrients, but also function in many cellular events by activating nuclear receptors, such as the peroxisome proliferator-activated receptors (PPARs), and fatty acid binding proteins (FABPs).

How FFAs induce insulin resistance is not a novel topic in pathological studies on obesityassociated type II diabetes. Many efforts have been made to uncover the underlying molecular mechanisms, but they remain elusive. It seems that FFA-induced insulin resistance occurs not via a single pathway but rather via a complicated network of pathways in organs, tissues, and cells.

## **1.1. Acute cellular responses to Free Fatty Acids**

Major investigatons of the mechanisms of extra FFA-induced insulin resistance have focused on the chronic effects of FFAs. However, plasma FFA concentrations are not consistent and

vary widely from hour to hour, displaying waves according to nutritional state and the presence of regulators including hormones (Figure 1). The normal level of postprandial plasma FFAs is about 0.1- 0.4 mmol/L, while in obese individuals this value can reach to 0.2 - 0.6 mmol/L [4]. In healthy people the level of plasma FFAs decreases during the 2 h after a meal until it drops to nearly 0.1 mmol/L, and then rises to a concentration of about 0.3-0.4 mmol/L before the next meal. Such plasma FFA fluctuations also occur in people with metabolic disorders, but display a different pattern. In mild essential hypertensive patients, the plasma FFA concentrations at 3 and 4 h after a meal are significantly higher than that in healthy people (Figure 1, lower panel) [5]. The response of the body to acute variation in plasma FFA concentration is probably associated with the energy balance of the whole body, and requires further investigation to obtain a more in-depth understanding of the pathology of obesity-related metabolic diseases.

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 131

Previous works have reported that FFAs are able to acutely induce several cellular events in various tissues. For example, FFAs can stimulate insulin secretion in pancreatic β-cells [7, 8], leptin secretion in adipocytes [9], and glucose uptake in adipocytes and skeletal muscle cells [10, 11]. All these happen within a short interval after FFA treatment, implying that the FFAs may work as signaling molecules such as hormones, to trigger signal transduction and

During signal transduction, many intracellular signaling proteins work as molecular switches and are activated by GTP binding or phosphorylation. That FFAs acutely stimulate protein phosphorylation suggests that FFAs are able to evoke signal transduction. One study reports that arachidonic acid is able to stimulate the phosphorylation of tyrosine-containing proteins in cultured vascular endothelial and smooth muscle cells [12]; arachidonic acid-induced phosphorylation was rapid and transient, reaching a peak 0.5 min after the addition of arachidonic acid and returning to baseline by 8 min. When cyclooxygenase, lipoxygenase, and epoxygenase pathways were inhibited, phosphorylation was still detected, suggesting it was fatty acid, not its metabolites that triggered the phosphorylation. In addition, increased protein tyrosine phosphorylation was also observed after treatment with oleic, linolenic and γlinoleic acid. In another work it was reported that unsaturated fatty acids are able to stimulate protein phosphorylation by activating protein kinase C in intact hippocampal slices [13]. Oleic acid stimulated phosphorylation of several proteins of molecular weights 92,000, 58,000, 50,000, 47,000 and 44,000 Da. The 44,000 and 47,000 Da proteins were particularly sensitive to fatty acids and were phosphorylated in a dose- and time-dependent manner. Increased 32P incorporation into the 44,000 Da protein was apparent after 1 min and reached a maximum at 5 min. Phosphorylation of the 47,000 Da protein followed a similar pattern. Studies on fatty acidstimulated protein phosphorylation have shed light on the role of fatty acids as signal

During the last decade, a series of free fatty acid receptors (FFARs) has been identified, indicating that like other extracellular signal molecules, FFAs bind to their receptors on the plasma membrane to trigger signal transduction. The FFARs identified belong to a large protein family, the G protein-coupled receptors (GPCRs), which are integral membrane proteins with seven trans-membrane domains. The extracellular parts of the receptors sense external signals and activate heterotrimeric G proteins to transduce signals to downstream molecules. GPCRs are activated by various types of ligands, including ions, nucleotides, amino acids, lipids, peptides, and proteins. It is estimated that more than half of modern drugs target these receptors [14]. The known FFARs include FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), GPR84, GPR119 and

subsequent physiological events.

molecules.

GPR120 (Table 1).

**1.2. Free Fatty Acid Receptors**

**Figure 1.** Variation in free fatty acids (●) and insulin (○) concentrations in response to meals in healthy people (upper panel, reprinted from Frayn KN, 1998) [6] and fatty acid levels in mild essential hypertensive patients (---) and normotensive control subjects (——) (lower panel, reprinted from Singer P et al. 1985) [5].

Previous works have reported that FFAs are able to acutely induce several cellular events in various tissues. For example, FFAs can stimulate insulin secretion in pancreatic β-cells [7, 8], leptin secretion in adipocytes [9], and glucose uptake in adipocytes and skeletal muscle cells [10, 11]. All these happen within a short interval after FFA treatment, implying that the FFAs may work as signaling molecules such as hormones, to trigger signal transduction and subsequent physiological events.

During signal transduction, many intracellular signaling proteins work as molecular switches and are activated by GTP binding or phosphorylation. That FFAs acutely stimulate protein phosphorylation suggests that FFAs are able to evoke signal transduction. One study reports that arachidonic acid is able to stimulate the phosphorylation of tyrosine-containing proteins in cultured vascular endothelial and smooth muscle cells [12]; arachidonic acid-induced phosphorylation was rapid and transient, reaching a peak 0.5 min after the addition of arachidonic acid and returning to baseline by 8 min. When cyclooxygenase, lipoxygenase, and epoxygenase pathways were inhibited, phosphorylation was still detected, suggesting it was fatty acid, not its metabolites that triggered the phosphorylation. In addition, increased protein tyrosine phosphorylation was also observed after treatment with oleic, linolenic and γlinoleic acid. In another work it was reported that unsaturated fatty acids are able to stimulate protein phosphorylation by activating protein kinase C in intact hippocampal slices [13]. Oleic acid stimulated phosphorylation of several proteins of molecular weights 92,000, 58,000, 50,000, 47,000 and 44,000 Da. The 44,000 and 47,000 Da proteins were particularly sensitive to fatty acids and were phosphorylated in a dose- and time-dependent manner. Increased 32P incorporation into the 44,000 Da protein was apparent after 1 min and reached a maximum at 5 min. Phosphorylation of the 47,000 Da protein followed a similar pattern. Studies on fatty acidstimulated protein phosphorylation have shed light on the role of fatty acids as signal molecules.

### **1.2. Free Fatty Acid Receptors**

130 Protein Phosphorylation in Human Health

P et al. 1985) [5].

pathology of obesity-related metabolic diseases.

vary widely from hour to hour, displaying waves according to nutritional state and the presence of regulators including hormones (Figure 1). The normal level of postprandial plasma FFAs is about 0.1- 0.4 mmol/L, while in obese individuals this value can reach to 0.2 - 0.6 mmol/L [4]. In healthy people the level of plasma FFAs decreases during the 2 h after a meal until it drops to nearly 0.1 mmol/L, and then rises to a concentration of about 0.3-0.4 mmol/L before the next meal. Such plasma FFA fluctuations also occur in people with metabolic disorders, but display a different pattern. In mild essential hypertensive patients, the plasma FFA concentrations at 3 and 4 h after a meal are significantly higher than that in healthy people (Figure 1, lower panel) [5]. The response of the body to acute variation in plasma FFA concentration is probably associated with the energy balance of the whole body, and requires further investigation to obtain a more in-depth understanding of the

**Figure 1.** Variation in free fatty acids (●) and insulin (○) concentrations in response to meals in healthy

hypertensive patients (---) and normotensive control subjects (——) (lower panel, reprinted from Singer

people (upper panel, reprinted from Frayn KN, 1998) [6] and fatty acid levels in mild essential

During the last decade, a series of free fatty acid receptors (FFARs) has been identified, indicating that like other extracellular signal molecules, FFAs bind to their receptors on the plasma membrane to trigger signal transduction. The FFARs identified belong to a large protein family, the G protein-coupled receptors (GPCRs), which are integral membrane proteins with seven trans-membrane domains. The extracellular parts of the receptors sense external signals and activate heterotrimeric G proteins to transduce signals to downstream molecules. GPCRs are activated by various types of ligands, including ions, nucleotides, amino acids, lipids, peptides, and proteins. It is estimated that more than half of modern drugs target these receptors [14]. The known FFARs include FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), GPR84, GPR119 and GPR120 (Table 1).

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 133

in the gut. Subsequently SCFAs are released in the bloodstream and accumulate to

GPR41 is expressed abundantly in adipose tissue, enteroendocrine cells, and sympathetic ganglia. SCFAs activate GPR41 and stimulate leptin expression in mouse adipocytes and mouse primary-cultured adipocytes. Acute oral administration of propionate increases circulating leptin levels in mice [9]. Overexpression of exogenous GPR41 and knockdown of GPR41 by RNAi regulates leptin production positively and negatively. Given that leptin is a potent anorexigenic hormone that reduces food intake, propionate may inhibit food intake by increasing leptin release. The analysis of GPR41-deficient mice showed that GPR41 is expressed in enteroendocrine cells, and GPR41 deficiency is associated with reduced expression of PYY [20]. GPR41 is also abundantly expressed in sympathetic ganglia in mice and humans [19]. Studies using GPR41-/- mice and co-culturing of fetal-isolated cardiomyocytes with primary-cultured sympathetic neurons have shown that propionate promotes sympathetic outflow via GPR41, reduces intracellular cAMP concentrations and promotes ERK1/2 phosphorylation, phenomena which were not observed in sympathetic neurons from GPR41−/− mice. GPR41-mediated rise in beat rate was effectively blocked by Gallein (Gβγ blocker) and pertussis toxin (PTX) treatments, whereas NF023 (Gα(i/o) blocker) had no inhibitory effects. Knockdown of PLCβ 2/3 or ERK1/2 by RNAi significantly inhibits the propionate-induced rise in the beat rate of cardiomyocytes. These results indicate that

GPR41 activation of sympathetic neurons may involve Gβγ, PLCβ, and MAPK.

is primarily coupled to a PTX-sensitive Gi/o pathway [26].

GPR43 is highly expressed in immune cells, spleen, and bone marrow, and is also detected at low levels in the placenta, lung, liver, and adipose tissues [22]. A study on adipocytes showed that acetate and propionate can reduce lipolytic activity and thus plasma FFA level in a mouse *in vivo* model. This inhibition of lipolysis is abolished in adipocytes isolated from GPR43 knockout animals [24]. Similar to GPR41, GPR43 activation is also coupled to intracellular Ca2+ release, ERK1/2 activation, and a reduction in cAMP accumulation. Unlike GPR41, however, which signals via the Gi/o family, GPR43 signals via both the Gi/o and Gq

GPR84 mRNA is expressed mainly in bone marrow, leukocytes, the spleen and lung [25, 26]. GPR84 functions as a receptor for medium-chain FFAs with carbon chain lengths of 9–14. Capric acid (C10:0), undecanoic acid (C11:0), and lauric acid (C12:0) are the most potent agonists of GPR84. A functional study conducted in GPR84-/- mice revealed that primary stimulation of T cells with anti-CD3 results in increased IL-4, but not IL-2 or IFN-<sup>γ</sup> production, compared to wild-type mice [27]. Wang et al. reported that medium-chain FFAs act through GPR84 to amplify the stimulation of IL-12 p40 production by lipopolysaccharides in monocytes/macrophages [26]. Medium-chain FFAs induce Ca2+ mobilization and inhibit cAMP production. The activation of GPR84 by medium-chain FFAs

GPR119 in humans and rodents is expressed predominantly in the pancreas and gastrointestinal tract and also in the rodent brain [28]. The lipid signaling agent oleoylethanolamide (OEA) is an endogenous ligand of GPR119. OEA is a peripherally acting

micromolar concentrations.

pathways [21].

**Table 1.** Characteristic of FFARs.

It has been reported that GPR40 is activated by medium- and long-chain FFAs [18, 15, 16]. GPR40 is abundantly expressed in the pancreas, and is especially enriched in pancreatic βcells. When activated by FFAs, GPR40 activates G-protein, which transduces the signal leading to stimulation of insulin secretion. Using Chinese hamster ovary (CHO) cells in which GPR40 is stably expressed, Itoh et al. found that free fatty acids are able to stimulate the formation of inositol 1,4,5-trisphosphate, intracellular Ca2+ mobilization, and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 [15]. Furthermore, in 2006 Feng et al. reported that fatty acids, especially linoleic acid, are able to stimulate insulin secretion in rat β-cells by reducing the voltage-gated K+ current via GPR40 and the cAMPprotein kinase A system [36].

Unlike GPR40, the physiological ligands of GPR41 and GPR43 are short chain fatty acids (SCFAs), including acetate (C2), propionate (C3), butyrate (C4), and valerate (C5). SCFAs are generated by bacterial fermentation of undigested carbohydrates from ingested dietary fiber in the gut. Subsequently SCFAs are released in the bloodstream and accumulate to micromolar concentrations.

132 Protein Phosphorylation in Human Health

Pancreatic β

Adipose tissue [9], sympathetic ganglia [19], enteroendocrine cells [20]

Leukocyte, spleen, bone marrow, adipose tissue [22]

Immune cell, bone marrow, leukocyte, lung, lymph node, spleen [25, 26]

gastrointestinal tract, pancreas [28]

Intestinal tract, Macrophage, lung, adipose tissue [32,

Brain,

33]

**Table 1.** Characteristic of FFARs.

protein kinase A system [36].

GPR 40 (FFAR1)

GPR 41 (FFAR3)

GPR 43 (FFAR2)

GPR 84

GPR 119

GPR 120

**Protein Tissue Expression Ligand Function Synthetic**

Insulin secretion [15]; incretin secretion [17]

5-HT secretion; PYY secretion [23]; inhibition of lipolysis [24]

Amplify IL-12 p40

Insulin secretion [29]; food intake; body weight [28]

Short C2-C4 [9] Leptin secretion [9];

Medium- and long- C8-C22 [18, 15]

Short C2-C4

Medium C9- 14C [26]

Ethanolamide

Lysophosphati dyl choline [29]

Medium- and long- C10-C22

It has been reported that GPR40 is activated by medium- and long-chain FFAs [18, 15, 16]. GPR40 is abundantly expressed in the pancreas, and is especially enriched in pancreatic βcells. When activated by FFAs, GPR40 activates G-protein, which transduces the signal leading to stimulation of insulin secretion. Using Chinese hamster ovary (CHO) cells in which GPR40 is stably expressed, Itoh et al. found that free fatty acids are able to stimulate the formation of inositol 1,4,5-trisphosphate, intracellular Ca2+ mobilization, and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 [15]. Furthermore, in 2006 Feng et al. reported that fatty acids, especially linoleic acid, are able to stimulate insulin secretion in rat β-cells by reducing the voltage-gated K+ current via GPR40 and the cAMP-

Unlike GPR40, the physiological ligands of GPR41 and GPR43 are short chain fatty acids (SCFAs), including acetate (C2), propionate (C3), butyrate (C4), and valerate (C5). SCFAs are generated by bacterial fermentation of undigested carbohydrates from ingested dietary fiber

[21]

[28],

[32]

**Agonist**

Thiazolidinedi one [16], GW9508, MEDICA16

[18]

PYY secretion [20] / Gi/o [19, 21]

[27] / Gi/o [26]

GLP-1 secretion [32] NCG21 [34],

PSN632408, PSN37569 [30], AR23145 [31]

/ Gq/11, Gi/o

GW9508 [35] Gq/11 [32]

[21]

Gs [29]

**G proteincoupling**

Gq/11, Gi [15, 16] GPR41 is expressed abundantly in adipose tissue, enteroendocrine cells, and sympathetic ganglia. SCFAs activate GPR41 and stimulate leptin expression in mouse adipocytes and mouse primary-cultured adipocytes. Acute oral administration of propionate increases circulating leptin levels in mice [9]. Overexpression of exogenous GPR41 and knockdown of GPR41 by RNAi regulates leptin production positively and negatively. Given that leptin is a potent anorexigenic hormone that reduces food intake, propionate may inhibit food intake by increasing leptin release. The analysis of GPR41-deficient mice showed that GPR41 is expressed in enteroendocrine cells, and GPR41 deficiency is associated with reduced expression of PYY [20]. GPR41 is also abundantly expressed in sympathetic ganglia in mice and humans [19]. Studies using GPR41-/- mice and co-culturing of fetal-isolated cardiomyocytes with primary-cultured sympathetic neurons have shown that propionate promotes sympathetic outflow via GPR41, reduces intracellular cAMP concentrations and promotes ERK1/2 phosphorylation, phenomena which were not observed in sympathetic neurons from GPR41−/− mice. GPR41-mediated rise in beat rate was effectively blocked by Gallein (Gβγ blocker) and pertussis toxin (PTX) treatments, whereas NF023 (Gα(i/o) blocker) had no inhibitory effects. Knockdown of PLCβ 2/3 or ERK1/2 by RNAi significantly inhibits the propionate-induced rise in the beat rate of cardiomyocytes. These results indicate that GPR41 activation of sympathetic neurons may involve Gβγ, PLCβ, and MAPK.

GPR43 is highly expressed in immune cells, spleen, and bone marrow, and is also detected at low levels in the placenta, lung, liver, and adipose tissues [22]. A study on adipocytes showed that acetate and propionate can reduce lipolytic activity and thus plasma FFA level in a mouse *in vivo* model. This inhibition of lipolysis is abolished in adipocytes isolated from GPR43 knockout animals [24]. Similar to GPR41, GPR43 activation is also coupled to intracellular Ca2+ release, ERK1/2 activation, and a reduction in cAMP accumulation. Unlike GPR41, however, which signals via the Gi/o family, GPR43 signals via both the Gi/o and Gq pathways [21].

GPR84 mRNA is expressed mainly in bone marrow, leukocytes, the spleen and lung [25, 26]. GPR84 functions as a receptor for medium-chain FFAs with carbon chain lengths of 9–14. Capric acid (C10:0), undecanoic acid (C11:0), and lauric acid (C12:0) are the most potent agonists of GPR84. A functional study conducted in GPR84-/- mice revealed that primary stimulation of T cells with anti-CD3 results in increased IL-4, but not IL-2 or IFN-<sup>γ</sup> production, compared to wild-type mice [27]. Wang et al. reported that medium-chain FFAs act through GPR84 to amplify the stimulation of IL-12 p40 production by lipopolysaccharides in monocytes/macrophages [26]. Medium-chain FFAs induce Ca2+ mobilization and inhibit cAMP production. The activation of GPR84 by medium-chain FFAs is primarily coupled to a PTX-sensitive Gi/o pathway [26].

GPR119 in humans and rodents is expressed predominantly in the pancreas and gastrointestinal tract and also in the rodent brain [28]. The lipid signaling agent oleoylethanolamide (OEA) is an endogenous ligand of GPR119. OEA is a peripherally acting

agent that reduces food intake and body weight gain in rat feeding models, suggesting that GPR119 might mediate the OEA-induced reduction of food intake [28]. Lysophosphatidyl choline (LPC) is another bioactive lipid mediator that activates GPR119 to stimulate insulin release from pancreatic islets, via Gs activation which leads to cAMP production [29].

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 135

In addition to the functions described above, FFAs are also able to acutely stimulate glucose uptake in adipocytes and skeletal muscle cells, which is directly associated with metabolic homeostasis. A few reports indicate that fatty acids have acute effects on glucose uptake, but conclusions have been inconsistent and the underlying molecular mechanisms controlling these responses are still elusive. For example, alpha-lipoic acid has been shown to enhance basal glucose uptake both in normal and ob/ob mice [10], while palmitic acid (PA) treatment was reported to inhibit insulin-stimulated but not basal glucose uptake

Although both adipocytes and skeletal muscle cells are able to ingest glucose by stimulation of FFAs, skeletal muscle consumes more than 70% of the plasma glucose, suggesting that whole body plasma glucose concentration is tightly associated to the sensitivity of muscle tissue to insulin [39, 40]. We therefore focused on the molecular mechanism of fatty acid-

**2.  When L6 cells were treated with palmitic acid (PA), the most abundant free fatty acid in the blood, glucose uptake increased rapidly in a timedependent manner, beginning from 5 min, and reaching a peak at 20 min (Figure 3, lower panel). By incubating intact PA-treated L6-GLUT4myc cells with myc antibody to detect plasma membrane-located GLUT4, we found that PA stimulates GLUT4 translocation from the cytosol to the plasma membrane (Figure 3, upper panel). The stimulatory effects of PA

Fluorescence imaging shows GLUT4 translocation to the cell surface after L6-GLUT4myc (L6) cells are treated with (upper right panel) or without (upper left panel) PA or insulin (upper middle panel). The lower panel shows that glucose uptake increases in a time-

Akt plays an important role in insulin-stimulated GLUT4 translocation and glucose uptake. Akt, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase. Akt possesses a protein domain known as the PH domain, which binds to phosphoinositides. Binding to PIP3, and phosphorylated from PIP2 by PI3 Kinase (PI3K) via its PH domain, Akt can be phosphorylated by phosphoinositide dependent kinase 1 (PDK1) at threonine 308 and/or the mammalian target of rapamycin complex 2 (mTORC2) at serine 473. In the insulin signaling pathway, the insulin receptor (IR) is activated and tyrosine is phosphorylated after binding to insulin, subsequently activating hte IRS-1/PI3K/PDK/Akt cascade, and finally increasing the level of GLUT4 in the plasma membrane (Figure 4).

**2.1. Palmitate stimulates glucose uptake, GLUT4 translocation, and**

on glucose uptake and GLUT4 translocation are similar to those of insulin.

**phosphorylation of Akt, AMPK, and ERK1/2 in L6 cells**

dependent manner when L6 cells are treated with PA.

induced glucose uptake in skeletal muscle cells [41].

[11].

**uptake**

GPR120 is highly expressed in the human and mouse intestinal tract, as well as in adipocytes, taste buds, and lungs [32, 33]. GRP120 activation by saturated FFAs with a carbon chain length of 14–18, and by unsaturated FFAs with a chain length of 16–22 has been detected [32]. Activated by medium- and long- chain fatty acids, GPR120 increases insulin secretion indirectly by stimulating the secretion of glucagon-like peptide-1 (GLP-1), the most potent insulinotropic incretin, which is coupled to the elevation of Ca2+ and activation of the ERK cascade [32]. In addition, GPR120 is also reported to function as an ω-3 FA receptor in proinflammatory macrophages and mature adipocytes that mediates the potent anti-inflammatory effects of DHA and EPA by inhibiting both the TLR and TNF inflammatory signaling pathways [37]. Chronic tissue inflammation is another important mechanism causing insulin resistance, so the effect of GPR120 on insulin sensitivity as well as on the stimulation of insulin-secretion will make it an attractive drug target for diabetestherapeutic agents.

The discovery of FFARs developed our understanding of the role of FFAs as signal molecules. Cells expressing FFARs, such as pancreatic β-cells, adipocytes, and macrophages sense FFAs and make various corresponding responses to control metabolic homeostasis (Figure 2). FFARs have thus attracted considerable attention due to their potential as valuable drug targets.

**Figure 2.** Roles of GPR40 and GPR120 in nutritional regulation. Free fatty acid receptors control metabolism through promoting the secretion or production of peptide hormones (Reprinted from Hara et al., 2011) [38].

In addition to the functions described above, FFAs are also able to acutely stimulate glucose uptake in adipocytes and skeletal muscle cells, which is directly associated with metabolic homeostasis. A few reports indicate that fatty acids have acute effects on glucose uptake, but conclusions have been inconsistent and the underlying molecular mechanisms controlling these responses are still elusive. For example, alpha-lipoic acid has been shown to enhance basal glucose uptake both in normal and ob/ob mice [10], while palmitic acid (PA) treatment was reported to inhibit insulin-stimulated but not basal glucose uptake [11].

134 Protein Phosphorylation in Human Health

therapeutic agents.

valuable drug targets.

et al., 2011) [38].

agent that reduces food intake and body weight gain in rat feeding models, suggesting that GPR119 might mediate the OEA-induced reduction of food intake [28]. Lysophosphatidyl choline (LPC) is another bioactive lipid mediator that activates GPR119 to stimulate insulin

GPR120 is highly expressed in the human and mouse intestinal tract, as well as in adipocytes, taste buds, and lungs [32, 33]. GRP120 activation by saturated FFAs with a carbon chain length of 14–18, and by unsaturated FFAs with a chain length of 16–22 has been detected [32]. Activated by medium- and long- chain fatty acids, GPR120 increases insulin secretion indirectly by stimulating the secretion of glucagon-like peptide-1 (GLP-1), the most potent insulinotropic incretin, which is coupled to the elevation of Ca2+ and activation of the ERK cascade [32]. In addition, GPR120 is also reported to function as an ω-3 FA receptor in proinflammatory macrophages and mature adipocytes that mediates the potent anti-inflammatory effects of DHA and EPA by inhibiting both the TLR and TNF inflammatory signaling pathways [37]. Chronic tissue inflammation is another important mechanism causing insulin resistance, so the effect of GPR120 on insulin sensitivity as well as on the stimulation of insulin-secretion will make it an attractive drug target for diabetes-

The discovery of FFARs developed our understanding of the role of FFAs as signal molecules. Cells expressing FFARs, such as pancreatic β-cells, adipocytes, and macrophages sense FFAs and make various corresponding responses to control metabolic homeostasis (Figure 2). FFARs have thus attracted considerable attention due to their potential as

**Figure 2.** Roles of GPR40 and GPR120 in nutritional regulation. Free fatty acid receptors control metabolism through promoting the secretion or production of peptide hormones (Reprinted from Hara

release from pancreatic islets, via Gs activation which leads to cAMP production [29].

Although both adipocytes and skeletal muscle cells are able to ingest glucose by stimulation of FFAs, skeletal muscle consumes more than 70% of the plasma glucose, suggesting that whole body plasma glucose concentration is tightly associated to the sensitivity of muscle tissue to insulin [39, 40]. We therefore focused on the molecular mechanism of fatty acidinduced glucose uptake in skeletal muscle cells [41].

## **2. Mechanism study on Free Fatty Acid acute stimulation of glucose uptake**

## **2.1. Palmitate stimulates glucose uptake, GLUT4 translocation, and phosphorylation of Akt, AMPK, and ERK1/2 in L6 cells**

A rat skeletal muscle cell line L6 with stable expression of myc-tagged GLUT4 (L6) was used to study the acute effects of fatty acids. When L6 cells were treated with palmitic acid (PA), the most abundant free fatty acid in the blood, glucose uptake increased rapidly in a timedependent manner, beginning from 5 min, and reaching a peak at 20 min (Figure 3, lower panel). By incubating intact PA-treated L6-GLUT4myc cells with myc antibody to detect plasma membrane-located GLUT4, we found that PA stimulates GLUT4 translocation from the cytosol to the plasma membrane (Figure 3, upper panel). The stimulatory effects of PA on glucose uptake and GLUT4 translocation are similar to those of insulin.

Fluorescence imaging shows GLUT4 translocation to the cell surface after L6-GLUT4myc (L6) cells are treated with (upper right panel) or without (upper left panel) PA or insulin (upper middle panel). The lower panel shows that glucose uptake increases in a timedependent manner when L6 cells are treated with PA.

Akt plays an important role in insulin-stimulated GLUT4 translocation and glucose uptake. Akt, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase. Akt possesses a protein domain known as the PH domain, which binds to phosphoinositides. Binding to PIP3, and phosphorylated from PIP2 by PI3 Kinase (PI3K) via its PH domain, Akt can be phosphorylated by phosphoinositide dependent kinase 1 (PDK1) at threonine 308 and/or the mammalian target of rapamycin complex 2 (mTORC2) at serine 473. In the insulin signaling pathway, the insulin receptor (IR) is activated and tyrosine is phosphorylated after binding to insulin, subsequently activating hte IRS-1/PI3K/PDK/Akt cascade, and finally increasing the level of GLUT4 in the plasma membrane (Figure 4).

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 137

after 3 h. When treated with different concentrations of PA, Akt phosphorylation increased with PA concentration, beginning from 0.2 mM. Such time- and dose-dependent responses to PA treatment in cells match the characteristics of signal transduction, and so it is possible that a signal transduction cascade initiated by PA leads to the activation of Akt. To further verify the stimulatory effect of PA on Akt activation, we treated rat skeletal muscle tissue with PA. Rats were anesthetized and perfused with 2 mM PA. Skeletal muscle strips were collected and then incubated *in vitro* with PA. Similar to the results from cells, Akt phosphorylation also increased in PA-treated skeletal muscle tissue, suggesting that this

**Figure 5.** Palmitate acutely stimulates AMPK and Akt phosphorylation in a time- and dose-dependent

To investigate the putative PA-mediated signaling pathway, we tested the activity of other molecules and found that AMP-activated protein kinase (AMPK) and extracellular signalrelated kinase (ERK1/2) can also be activated by acute PA treatment. AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, which together make a functional enzyme that plays a role in cellular energy homeostasis [43]. AMPK is activated by an elevated AMP/ATP ratio and undergoes a conformational change of its γ subunit to expose the active site (Thr172) on the catalytic subunit α that is phosphorylated by the upstream kinase AMPK kinase (AMPKK) [44]. Upon activation, AMPK decreases energy consumption by inhibiting fatty acid and protein synthesis and enhances energy production by stimulating fatty acid oxidation and glucose transport to increase cellular energy levels. While it is known that AMPKα is phosphorylated at Thr258 and Ser485, its upstream kinases still need further study [45]. ERK1/2 belongs to the mitogen-activated protein kinase (MAPK) family, a widely conserved family of serine/threonine protein kinases. The ERK1/2 (p44/42 MAPK) signaling pathway responds to various extracellular stimuli including mitogens, growth factors, and cytokines [46]. Upon activation by MEK1 and MEK2 by phosphorylation of its Thr202 and Tyr204 residues,

respectively, ERK1/2 phosphorylates downstream targets, forming a signal cascade.

acute response of PA may be physiologically relevant.

manner [41].

**Figure 3.** Palmitate stimulates GLUT4 translocation and glucose uptake [41].

**Figure 4.** Insulin signaling pathway for glucose uptake stimulation (Reprinted from Frøsig and Richter, 2009) [42].

In our study, PA stimulated Akt phosphorylation at serine 473 in a time- and dosedependent manner (Figure 5). During PA treatment, Akt phosphorylation was detected after 10 min, peaked at 45 min, then decreased dramatically after 1 h, and became nondetectable after 3 h. When treated with different concentrations of PA, Akt phosphorylation increased with PA concentration, beginning from 0.2 mM. Such time- and dose-dependent responses to PA treatment in cells match the characteristics of signal transduction, and so it is possible that a signal transduction cascade initiated by PA leads to the activation of Akt. To further verify the stimulatory effect of PA on Akt activation, we treated rat skeletal muscle tissue with PA. Rats were anesthetized and perfused with 2 mM PA. Skeletal muscle strips were collected and then incubated *in vitro* with PA. Similar to the results from cells, Akt phosphorylation also increased in PA-treated skeletal muscle tissue, suggesting that this acute response of PA may be physiologically relevant.

136 Protein Phosphorylation in Human Health

Richter, 2009) [42].

**Figure 3.** Palmitate stimulates GLUT4 translocation and glucose uptake [41].

**Figure 4.** Insulin signaling pathway for glucose uptake stimulation (Reprinted from Frøsig and

In our study, PA stimulated Akt phosphorylation at serine 473 in a time- and dosedependent manner (Figure 5). During PA treatment, Akt phosphorylation was detected after 10 min, peaked at 45 min, then decreased dramatically after 1 h, and became nondetectable

**Figure 5.** Palmitate acutely stimulates AMPK and Akt phosphorylation in a time- and dose-dependent manner [41].

To investigate the putative PA-mediated signaling pathway, we tested the activity of other molecules and found that AMP-activated protein kinase (AMPK) and extracellular signalrelated kinase (ERK1/2) can also be activated by acute PA treatment. AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, which together make a functional enzyme that plays a role in cellular energy homeostasis [43]. AMPK is activated by an elevated AMP/ATP ratio and undergoes a conformational change of its γ subunit to expose the active site (Thr172) on the catalytic subunit α that is phosphorylated by the upstream kinase AMPK kinase (AMPKK) [44]. Upon activation, AMPK decreases energy consumption by inhibiting fatty acid and protein synthesis and enhances energy production by stimulating fatty acid oxidation and glucose transport to increase cellular energy levels. While it is known that AMPKα is phosphorylated at Thr258 and Ser485, its upstream kinases still need further study [45]. ERK1/2 belongs to the mitogen-activated protein kinase (MAPK) family, a widely conserved family of serine/threonine protein kinases. The ERK1/2 (p44/42 MAPK) signaling pathway responds to various extracellular stimuli including mitogens, growth factors, and cytokines [46]. Upon activation by MEK1 and MEK2 by phosphorylation of its Thr202 and Tyr204 residues, respectively, ERK1/2 phosphorylates downstream targets, forming a signal cascade.

Similar to Akt, AMPK in L6 cells is also activated acutely by PA. AMPK phosphorylation (Thr172) starts as early as 5 min after PA treatment and reaches a peak at 20 min. After 1 h, the signal cannot be detected (Figure 5, upper panel). In addition, PA-induced phosphorylation of AMPK is also dose-dependent. Unlike Akt and AMPK, ERK1/2 is phosphorylated for a shorter duration, increasing after 5 min and returning to basal level after 15 min (Figure 6).

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 139

**Figure 7.** PA-induced glucose uptake is decreased when Akt activity is blocked by an Akt inhibitor (left

**Figure 8.** PA-induced glucose uptake and Akt phosphorylation is decreased when AMPK activity is reduced by an AMPK inhibitor (left panel) or AMPK dominant-negative construct (right panel) [41].

panel) or RNAi (right panel) [41].

**Figure 6.** Palmitate acutely stimulates ERK1/2 phosphorylation in a time-dependent manner [41].

#### **2.2. Akt, AMPK and ERK1/2 are involved in PA-stimulated glucose uptake**

To test if Akt, AMPK, and ERK1/2 are involved in PA-stimulated glucose uptake, tools such as inhibitors, dominant negative constructs, and short interference RNA (siRNA) were used to inhibit their protein activity or expression levels. Western blotting and glucose uptake assay results showed that all of these proteins participate in signal transduction.

We applied API-2, an Akt selective inhibitor, to block Akt activity. As a result, API-2 abolished Akt phosphorylation as well as significantly decreasing PA-induced glucose uptake (Figure 7, left panel). In addition, siRNA duplexes were nucleofected into L6 cells; compared to the negative control (N.C.), total Akt expression level was efficiently downregulated. Glucose uptake assays showed that PA-induced glucose uptake decreased when Akt expression decreased due to RNAi (Figure 7, right panel). Together, these data suggest that PA induces glucose uptake in skeletal muscle cells via Akt activation.

To study the role of AMPK in PA-induced glucose uptake, we used AMPK inhibitor Compound C, an myc-tagged AMPK dominant negative (AMPK-DN) plasmid, and siRNA targeting AMPK catalytic subunits α1 and α2. Since AMPK and Akt activation was observed sequentially in PA-treated cells, we also examined the relationship between AMPK and Akt. AMPK inhibitor Compound C suppressed AMPK activity and decreased PA-induced Akt phosphorylation and glucose uptake (Figure 8, left panel). Similar results were obtained when AMPK-DN was nucleofected into L6 cells. Furthermore, an siRNA duplex mixture targeting AMPK α decreases PA-stimulated Akt phosphorylation in L6 cells (Figure 8, right panel), consistent with the inhibitor and AMPK-DN experiments.

In contrast, when AMPK agonist AICAR was used to stimulate AMPK phosphorylation, Akt was also stimulated rapidly in a time-dependent manner (Figure 9), suggesting that it is possible to stimulate Akt via AMPK activation in L6 cells. These data suggest that PAstimulated AMPK phosphorylation may contribute to regulating Akt activity and is involved in PA-induced glucose uptake.

after 15 min (Figure 6).

Similar to Akt, AMPK in L6 cells is also activated acutely by PA. AMPK phosphorylation (Thr172) starts as early as 5 min after PA treatment and reaches a peak at 20 min. After 1 h, the signal cannot be detected (Figure 5, upper panel). In addition, PA-induced phosphorylation of AMPK is also dose-dependent. Unlike Akt and AMPK, ERK1/2 is phosphorylated for a shorter duration, increasing after 5 min and returning to basal level

**Figure 6.** Palmitate acutely stimulates ERK1/2 phosphorylation in a time-dependent manner [41].

**2.2. Akt, AMPK and ERK1/2 are involved in PA-stimulated glucose uptake**

assay results showed that all of these proteins participate in signal transduction.

that PA induces glucose uptake in skeletal muscle cells via Akt activation.

panel), consistent with the inhibitor and AMPK-DN experiments.

involved in PA-induced glucose uptake.

To test if Akt, AMPK, and ERK1/2 are involved in PA-stimulated glucose uptake, tools such as inhibitors, dominant negative constructs, and short interference RNA (siRNA) were used to inhibit their protein activity or expression levels. Western blotting and glucose uptake

We applied API-2, an Akt selective inhibitor, to block Akt activity. As a result, API-2 abolished Akt phosphorylation as well as significantly decreasing PA-induced glucose uptake (Figure 7, left panel). In addition, siRNA duplexes were nucleofected into L6 cells; compared to the negative control (N.C.), total Akt expression level was efficiently downregulated. Glucose uptake assays showed that PA-induced glucose uptake decreased when Akt expression decreased due to RNAi (Figure 7, right panel). Together, these data suggest

To study the role of AMPK in PA-induced glucose uptake, we used AMPK inhibitor Compound C, an myc-tagged AMPK dominant negative (AMPK-DN) plasmid, and siRNA targeting AMPK catalytic subunits α1 and α2. Since AMPK and Akt activation was observed sequentially in PA-treated cells, we also examined the relationship between AMPK and Akt. AMPK inhibitor Compound C suppressed AMPK activity and decreased PA-induced Akt phosphorylation and glucose uptake (Figure 8, left panel). Similar results were obtained when AMPK-DN was nucleofected into L6 cells. Furthermore, an siRNA duplex mixture targeting AMPK α decreases PA-stimulated Akt phosphorylation in L6 cells (Figure 8, right

In contrast, when AMPK agonist AICAR was used to stimulate AMPK phosphorylation, Akt was also stimulated rapidly in a time-dependent manner (Figure 9), suggesting that it is possible to stimulate Akt via AMPK activation in L6 cells. These data suggest that PAstimulated AMPK phosphorylation may contribute to regulating Akt activity and is

**Figure 7.** PA-induced glucose uptake is decreased when Akt activity is blocked by an Akt inhibitor (left panel) or RNAi (right panel) [41].

**Figure 8.** PA-induced glucose uptake and Akt phosphorylation is decreased when AMPK activity is reduced by an AMPK inhibitor (left panel) or AMPK dominant-negative construct (right panel) [41].

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 141

Having shown that the two pathways work independently in PA-induced glucose uptake in L6 cells, we investigated signaling molecules upstream of the intersection. When we used PI3 Kinase (PI3K) -specific inhibitor LY294002 to treat L6 cells, PA-induced glucose uptake was totally abolished (Figure 11, upper panel), suggesting that PI3K may control these two pathways. Indeed, LY294002 could abolish PA-stimulated AMPK, Akt, and ERK1/2 phosphorylation (Figure 10, lower right and Figure 11, lower panel). Results from the above experiments indicate that, in skeletal muscle cell lines and tissues, acute PA-stimulated glucose uptake occurs via activation of the PI3K/AMPK/Akt and PI3K/ERK1/2 pathways

**Figure 11.** PI3K-specific inhibition abolishes PA-induced glucose uptake and phosphorylation of Akt

**Figure 12.** Schematic diagram of PA-induced signal pathway stimulation of acute glucose uptake in

leading to GLUT4 translocation (Figure 12).

and AMPK [41].

skeletal muscle cells [41].

**Figure 9.** AMPK agonist AICAR stimulates Akt phosphorylation in a time-dependent manner [41].

The role of ERK1/2 in PA-stimulated signal transduction was examined by using the MEK1/2 inhibitors PD98056 and U0126. While both inhibitors decreased basal and PAinduced ERK1/2 phosphorylation, U0126 was more potent (Figure 10, upper left). When ERK1/2 activity was inhibited by U0126, PA-induced glucose uptake was reduced significantly (Figure 10, upper right). These data suggest that PA-stimulated ERK1/2 phosphorylation may contribute to PA-induced glucose uptake. To determine the relationship between ERK1/2 and the AMPK/Akt pathway, AMPK α1/α2 siRNA transfected cells were used to test ERK1/2 activity; ERK1/2 phosphorylation increased at the same rate as in N.C. cells after PA treatment (Figure 10, lower left). In addition, the Akt inhibitor API-2 did not affect PA-induced ERK1/2 phosphorylation, and MEK1/2 inhibitors PD98056 and U0126 did not affect PA-induced Akt phosphorylation (Figure 10, lower right). These data suggest that ERK1/2 contributes to PA-stimulated signal transduction independently from the AMPK/Akt pathway, consistent with the partial decrease in PA-induced glucose uptake by inhibition of either Akt, AMPK, or ERK1/2.

**Figure 10.** MEK1/2 inhibitors decrease PA-induced ERK1/2 phosphorylation and glucose uptake, but do not affect Akt activity; AMPK and Akt activity inhibition does not affect ERK1/2, while PI3K inhibitor does [41].

Having shown that the two pathways work independently in PA-induced glucose uptake in L6 cells, we investigated signaling molecules upstream of the intersection. When we used PI3 Kinase (PI3K) -specific inhibitor LY294002 to treat L6 cells, PA-induced glucose uptake was totally abolished (Figure 11, upper panel), suggesting that PI3K may control these two pathways. Indeed, LY294002 could abolish PA-stimulated AMPK, Akt, and ERK1/2 phosphorylation (Figure 10, lower right and Figure 11, lower panel). Results from the above experiments indicate that, in skeletal muscle cell lines and tissues, acute PA-stimulated glucose uptake occurs via activation of the PI3K/AMPK/Akt and PI3K/ERK1/2 pathways leading to GLUT4 translocation (Figure 12).

140 Protein Phosphorylation in Human Health

by inhibition of either Akt, AMPK, or ERK1/2.

inhibitor does [41].

**Figure 9.** AMPK agonist AICAR stimulates Akt phosphorylation in a time-dependent manner [41].

The role of ERK1/2 in PA-stimulated signal transduction was examined by using the MEK1/2 inhibitors PD98056 and U0126. While both inhibitors decreased basal and PAinduced ERK1/2 phosphorylation, U0126 was more potent (Figure 10, upper left). When ERK1/2 activity was inhibited by U0126, PA-induced glucose uptake was reduced significantly (Figure 10, upper right). These data suggest that PA-stimulated ERK1/2 phosphorylation may contribute to PA-induced glucose uptake. To determine the relationship between ERK1/2 and the AMPK/Akt pathway, AMPK α1/α2 siRNA transfected cells were used to test ERK1/2 activity; ERK1/2 phosphorylation increased at the same rate as in N.C. cells after PA treatment (Figure 10, lower left). In addition, the Akt inhibitor API-2 did not affect PA-induced ERK1/2 phosphorylation, and MEK1/2 inhibitors PD98056 and U0126 did not affect PA-induced Akt phosphorylation (Figure 10, lower right). These data suggest that ERK1/2 contributes to PA-stimulated signal transduction independently from the AMPK/Akt pathway, consistent with the partial decrease in PA-induced glucose uptake

**Figure 10.** MEK1/2 inhibitors decrease PA-induced ERK1/2 phosphorylation and glucose uptake, but do not affect Akt activity; AMPK and Akt activity inhibition does not affect ERK1/2, while PI3K

**Figure 11.** PI3K-specific inhibition abolishes PA-induced glucose uptake and phosphorylation of Akt and AMPK [41].

**Figure 12.** Schematic diagram of PA-induced signal pathway stimulation of acute glucose uptake in skeletal muscle cells [41].

## **2.3. Palmitate stimulates Akt phosphorylation via binding to the plasma membrane**

As shown in Figure 12, how PA activates PI3K is still unknown. According to our current understanding of signal transduction, we speculate that PA may bind to a protein on the cell plasma membrane to trigger signal transduction. We performed fatty acid binding assays to test this hypothesis.

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 143

Could the postulated cell membrane protein which binds to FFAs and triggers signal transduction to stimulate glucose uptake in skeletal muscle cells be a G-protein coupled receptor? FFA binding assays suggest that PA initiates signal transduction via a protein(s) on the cell surface, and meanwhile in the FFA-stimulated signal cascade ERK1/2 pathways were involved, which also appeared in some known FFAR signal pathways. Therefore, a GPCR on the plasma membrane of skeletal muscle cells may be the FFA receptor we have

PA is a long-chain fatty acid with 16 carbons. We tested other long-chain fatty acids such as C18:1 (oleic acid), C18:2 (Linoleic acid), and C18:0 (Stearic acid) in addition to PA to examine their effects of stimulating AMPK and Akt phosphorylation. All of these fatty acids activated AMPK and Akt in a time- and dose-dependent manner. It is thus likely that our

The known long-chain FFARs include GPR40 and GPR120, both of which are related to insulin secretion. Oh et al. found that GPR120 agonists DHA and GW9508 enhance glucose uptake by activating the PI3K-Akt pathway and GLUT4 translocation in 3T3-L1 adipocytes. The stimulatory effect of DHA and GW9508 was blocked when GPR120 or Gαq/11 was depleted by siRNA knockdown [37], indicating that FFAs stimulate glucose uptake in adipocytes via the FFA receptor GPR120. However, neither GPR120 nor GPR40 is expressed in muscles. In the Oh *et al*. study, DHA and GW9508 did not enhance glucose uptake in L6 skeletal muscle cells. We therefore conclude that known long-chain FFARs GPR40 and GPR120 are not our postulated FFA receptor. In 2005, Gaël Jean-Baptiste *et al*. described the GPCRs expressed in skeletal muscle tissue, but none of them are FFA receptors [47]. Our postulated receptor might therefore be a novel FFAR whose function is to stimulate glucose uptake in skeletal muscle tissue. Like GPR40 and GPR120, our postulated FFAR is also related to metabolic homeostasis, and so it is likely to be a potential drug target for the treatment of diabetes. Identification of this FFAR is one of

Another significant implication of our results is that PA plays two opposing roles in skeletal muscle. Under chronic treatment it inhibits insulin-stimulated glucose uptake by blocking Akt phosphorylation, while it enhances glucose uptake by activating Akt when cells are exposed to PA for a short time. What is the relationship between the long-term and shortterm effects of FFAs on glucose uptake? Our results show that phosphorylation of Akt is only stimulated when the concentration of PA reached a certain level (at or above 0.2 mmol/L in C2C12 cells). We thus conclude that phosphorylation of Akt may require high concentrations of fatty acids under physiological situations. Akt phosphorylation is not detectable 3 h after fatty acid treatment (Figure 5, upper panel). In addition, when fatty acids are withdrawn, the Akt phosphorylation signal disappears after 3 h in C2C12 cells (data not shown), suggesting that fatty acid-induced phosphorylation and dephosphorylation of Akt can be completed within one cycle of a postprandial FFA wave. It is therefore possible that

postulated FFAR may function using long-chain FFAs as its ligands.

**3. Future work**

postulated.

our goals.

L6 cells were incubated with PA at low temperature (4°C) to facilitate PA binding to the cell surface while preventing its internalization, then washed with buffer to remove unbound PA, or with BSA solution to remove not only unbound but also some membrane-bound PA by competitive binding, Cells were then transferred to 37°C to recover cellular activity. The amount of PA which binds to the cell surface was measured by adding trace amounts of 3Hlabeled PA to the solution. Results showed that after washing, with either buffer or BSA solution, very little PA remained (Figure 13, upper panel). When these cells were transferred to 37°C, Western blot results showed that Akt phosphorylation took place at a similar level to that in cells kept at 37°C, and increased in a time-dependent manner in buffer-washed cells, while p-Akt was not detectable in BSA solution-washed cells (Figure 13, lower panel). These results suggest that the amount of cell surface-bound PA was sufficient to activate Akt; intracellular PA accumulation was not required. Moreover, based on lipid analysis by TLC, fatty acids were the main component of total lipids during cell treatment with fatty acid and the 10 min incubation at 37°C. These results indicate that it is fatty acids rather than their metabolites that trigger signal transduction .

**Figure 13.** Cell plasma-bound PA stimulates Akt phosphorylation [41].

## **3. Future work**

142 Protein Phosphorylation in Human Health

their metabolites that trigger signal transduction .

**Figure 13.** Cell plasma-bound PA stimulates Akt phosphorylation [41].

**membrane**

test this hypothesis.

**2.3. Palmitate stimulates Akt phosphorylation via binding to the plasma**

As shown in Figure 12, how PA activates PI3K is still unknown. According to our current understanding of signal transduction, we speculate that PA may bind to a protein on the cell plasma membrane to trigger signal transduction. We performed fatty acid binding assays to

L6 cells were incubated with PA at low temperature (4°C) to facilitate PA binding to the cell surface while preventing its internalization, then washed with buffer to remove unbound PA, or with BSA solution to remove not only unbound but also some membrane-bound PA by competitive binding, Cells were then transferred to 37°C to recover cellular activity. The amount of PA which binds to the cell surface was measured by adding trace amounts of 3Hlabeled PA to the solution. Results showed that after washing, with either buffer or BSA solution, very little PA remained (Figure 13, upper panel). When these cells were transferred to 37°C, Western blot results showed that Akt phosphorylation took place at a similar level to that in cells kept at 37°C, and increased in a time-dependent manner in buffer-washed cells, while p-Akt was not detectable in BSA solution-washed cells (Figure 13, lower panel). These results suggest that the amount of cell surface-bound PA was sufficient to activate Akt; intracellular PA accumulation was not required. Moreover, based on lipid analysis by TLC, fatty acids were the main component of total lipids during cell treatment with fatty acid and the 10 min incubation at 37°C. These results indicate that it is fatty acids rather than Could the postulated cell membrane protein which binds to FFAs and triggers signal transduction to stimulate glucose uptake in skeletal muscle cells be a G-protein coupled receptor? FFA binding assays suggest that PA initiates signal transduction via a protein(s) on the cell surface, and meanwhile in the FFA-stimulated signal cascade ERK1/2 pathways were involved, which also appeared in some known FFAR signal pathways. Therefore, a GPCR on the plasma membrane of skeletal muscle cells may be the FFA receptor we have postulated.

PA is a long-chain fatty acid with 16 carbons. We tested other long-chain fatty acids such as C18:1 (oleic acid), C18:2 (Linoleic acid), and C18:0 (Stearic acid) in addition to PA to examine their effects of stimulating AMPK and Akt phosphorylation. All of these fatty acids activated AMPK and Akt in a time- and dose-dependent manner. It is thus likely that our postulated FFAR may function using long-chain FFAs as its ligands.

The known long-chain FFARs include GPR40 and GPR120, both of which are related to insulin secretion. Oh et al. found that GPR120 agonists DHA and GW9508 enhance glucose uptake by activating the PI3K-Akt pathway and GLUT4 translocation in 3T3-L1 adipocytes. The stimulatory effect of DHA and GW9508 was blocked when GPR120 or Gαq/11 was depleted by siRNA knockdown [37], indicating that FFAs stimulate glucose uptake in adipocytes via the FFA receptor GPR120. However, neither GPR120 nor GPR40 is expressed in muscles. In the Oh *et al*. study, DHA and GW9508 did not enhance glucose uptake in L6 skeletal muscle cells. We therefore conclude that known long-chain FFARs GPR40 and GPR120 are not our postulated FFA receptor. In 2005, Gaël Jean-Baptiste *et al*. described the GPCRs expressed in skeletal muscle tissue, but none of them are FFA receptors [47]. Our postulated receptor might therefore be a novel FFAR whose function is to stimulate glucose uptake in skeletal muscle tissue. Like GPR40 and GPR120, our postulated FFAR is also related to metabolic homeostasis, and so it is likely to be a potential drug target for the treatment of diabetes. Identification of this FFAR is one of our goals.

Another significant implication of our results is that PA plays two opposing roles in skeletal muscle. Under chronic treatment it inhibits insulin-stimulated glucose uptake by blocking Akt phosphorylation, while it enhances glucose uptake by activating Akt when cells are exposed to PA for a short time. What is the relationship between the long-term and shortterm effects of FFAs on glucose uptake? Our results show that phosphorylation of Akt is only stimulated when the concentration of PA reached a certain level (at or above 0.2 mmol/L in C2C12 cells). We thus conclude that phosphorylation of Akt may require high concentrations of fatty acids under physiological situations. Akt phosphorylation is not detectable 3 h after fatty acid treatment (Figure 5, upper panel). In addition, when fatty acids are withdrawn, the Akt phosphorylation signal disappears after 3 h in C2C12 cells (data not shown), suggesting that fatty acid-induced phosphorylation and dephosphorylation of Akt can be completed within one cycle of a postprandial FFA wave. It is therefore possible that Akt phosphorylation and dephosphorylation occur again and again as the concentration of FFA increases and decreases. Plasma FFA concentration starts to rise from 2 h after a meal (Figure 1) and continues to rise until the next meal due to the release of FFA from adipocytes during fasting. Based on our findings, when increasing plasma FFA reaches a certain level it stimulates Akt phosphorylation and glucose uptake. In obesity patients, elevated plasma FFA probably reaches the FFA level triggering Akt phosphorylation earlier during a plasma FFA wave, leading to abnormal glucose uptake. Many abnormal fatty acid cycles may contribute to the development of insulin resistance by disturbing glucose homeostasis. This Yin-Yang balance of PA in skeletal muscle is likely to be physiologically significant, and the possibility of its involvement in the development of insulin resistance needs to be investigated further.

Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 145

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[12] Buckley B. J. & Whorton A. R. (1995). Arachidonic acid stimulates protein tyrosine

[13] Chen S. G. & Murakami K. (1995). Synergistic activation by cis-fatty acid and diacylglycerol of protein kinase C and protein phosphorylation in hippocampal slices.

[14] Wise A., Gearing K. & Rees S. (2002). Target validation of G-protein coupled receptors.

[15] Itoh Y., Kawamata Y., Harada M., Kobayashi M., Fujii R., Fukusumi S., Ogi K., Hosoya M., Tanaka Y. & Uejima H. (2003). Free fatty acids regulate insulin secretion from

concentrations. Journal of Clinical Endocrinology & Metabolism, 63, 481-484. [5] Singer P., Godicke W., Voigt S., Hajdu I. & Weiss M. (1985). Postprandial hyperinsulinemia in patients with mild essential hypertension. Hypertension, 7, 182-

diabetes and obesity. Diabetes Care, 23, 118-124.

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pancreatic β cells through GPR40. Nature, 422, 173-176.

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## **4. Conclusion**

Free fatty acids (FFAs) function as signal molecules by activating their receptors in the cell plasma membrane to evoke signal transduction by a series of protein phosphorylation events, eventually leading to physiological events. Some of the known cell responses to FFAs are directly or indirectly related to metabolic homeostasis, so the study of FFAtriggered signal transduction will help us to understand the development of metabolic disorders and to design strategies for therapy. FFA receptors have become attractive drug targets for metabolic diseases. We have investigated the mechanism of long-chain fatty acid palmitate-induced glucose uptake in skeletal muscle cells and found that the two independent PI3K/AMPK/Akt and PI3K/ERK1/2 pathways are responsible for this process. Our results also provide supporting evidence that palmitate triggers signal transduction via a cell surface protein(s) that is probably a novel FFA receptor whose identity still remains to be determined.

## **Author details**

Jing Pu

*Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA*

Pingsheng Liu\* *National Laboratory of Biomacromolecules, Institute ofBiophysics, Chinese Academy of Sciences, Beijing, China*

## **Acknowledgement**

This work was supported by grants from the Ministry of Science and Technology of China (2006CB911001 and 2009CB919003), and the National Natural Science Foundation of China (30871229).

<sup>\*</sup> Corresponding Author

#### **5. References**

144 Protein Phosphorylation in Human Health

needs to be investigated further.

**4. Conclusion**

be determined.

Pingsheng Liu\*

(30871229).

Corresponding Author

 \*

**Acknowledgement**

Jing Pu

**Author details**

Akt phosphorylation and dephosphorylation occur again and again as the concentration of FFA increases and decreases. Plasma FFA concentration starts to rise from 2 h after a meal (Figure 1) and continues to rise until the next meal due to the release of FFA from adipocytes during fasting. Based on our findings, when increasing plasma FFA reaches a certain level it stimulates Akt phosphorylation and glucose uptake. In obesity patients, elevated plasma FFA probably reaches the FFA level triggering Akt phosphorylation earlier during a plasma FFA wave, leading to abnormal glucose uptake. Many abnormal fatty acid cycles may contribute to the development of insulin resistance by disturbing glucose homeostasis. This Yin-Yang balance of PA in skeletal muscle is likely to be physiologically significant, and the possibility of its involvement in the development of insulin resistance

Free fatty acids (FFAs) function as signal molecules by activating their receptors in the cell plasma membrane to evoke signal transduction by a series of protein phosphorylation events, eventually leading to physiological events. Some of the known cell responses to FFAs are directly or indirectly related to metabolic homeostasis, so the study of FFAtriggered signal transduction will help us to understand the development of metabolic disorders and to design strategies for therapy. FFA receptors have become attractive drug targets for metabolic diseases. We have investigated the mechanism of long-chain fatty acid palmitate-induced glucose uptake in skeletal muscle cells and found that the two independent PI3K/AMPK/Akt and PI3K/ERK1/2 pathways are responsible for this process. Our results also provide supporting evidence that palmitate triggers signal transduction via a cell surface protein(s) that is probably a novel FFA receptor whose identity still remains to

*Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health*

This work was supported by grants from the Ministry of Science and Technology of China (2006CB911001 and 2009CB919003), and the National Natural Science Foundation of China

*and Human Development, National Institutes of Health, Bethesda, MD, USA*

*National Laboratory of Biomacromolecules, Institute ofBiophysics,*

*Chinese Academy of Sciences, Beijing, China*

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Fatty Acids Stimulate Glucose Uptake by the PI3K/AMPK/Akt and PI3K/ERK1/2 Pathways 147

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146 Protein Phosphorylation in Human Health

[16] Kotarsky K., Nilsson N.E., Flodgren E., Owman C. & Olde B. (2003). A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs. Biochemical

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**Section 2**

**Protein Phosphorylation in Transcription,**

**pre-mRNA Splicing and DNA Damage**

**Protein Phosphorylation in Transcription, pre-mRNA Splicing and DNA Damage**

148 Protein Phosphorylation in Human Health

1319-27.

Reviews, 68, 320-344.

1528-1536.

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by site-directed mutagenesis. Journal of Biological Chemistry, 278, 28434.

England Journal of Medicine, 322, 223-228.

insulin signaling. Obesity, 17, S15-S20.

**Chapter 5**

© 2012 Calvo and García, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

**RNA Polymerase II Phosphorylation**

RNA polymerases (RNAPs) are among the most important cellular enzymes. They are present in all living organisms from Bacteria and Archaea to Eukarya and are responsible for DNA-dependent transcription. Although in Bacteria and Archaea there is only one RNAP, Eukarya possess up to three RNAPs in animals (I, II and III) and five in plants (IV and V) [1-2]. All of the RNAPs are evolutionarily related and have common structural and functional properties. The minimally conserved structural organization is represented by the bacterial enzyme, which contains only 4 subunits (''), whereas Archaea and Eukarya RNAPs are composed of 12 subunits (Rpb1-Rpb12) [3]. In prokaryotes, one RNAP transcribes all of the genes into all of the RNAs, however, in eukaryotes, this is achieved by three RNAPs. RNAPI transcribes genes that encode for 18S and 28S ribosomal RNAs; RNAPIII transcribes short genes, such as tRNAs and 5S ribosomal RNA, and RNAPII transcribes all protein-coding genes and genes for small noncoding RNAs (e.g., small nuclear RNAs (snRNAs) that are involved in splicing). The largest catalytic subunits of all three eukaryotic polymerases share homology among themselves and with the largest subunit of bacterial polymerase [4]. Solely the largest subunit of RNAPII (Rpb1) contains an unusual evolutionarily conserved carboxy-terminal domain (CTD) [5], which is subjected to numerous post-translational modifications of extraordinary importance in gene expression regulation [6-8].RNAPII transcription plays a central role in gene expression and is highly regulated at many steps, such as initiation, elongation and termination. Furthermore, phosphorylation of the Rpb1 CTD is known to regulate all of the transcription steps and coordinate these steps with other nuclear events. Prior to mRNA biosynthesis, RNAPII proceeds through several steps, such as promoter recognition, preinitiation complex (PIC) assembly, open complex formation, initiation and promoter escape. This sequence of events is initiated by the binding of gene-specific activators and coactivators, which results in the recruitment of basal transcription machinery (i.e., general transcription factors (GTFs):

**and Gene Expression Regulation**

Additional information is available at the end of the chapter

Olga Calvo and Alicia García

http://dx.doi.org/10.5772/48490

**1. Introduction**

## **RNA Polymerase II Phosphorylation and Gene Expression Regulation**

Olga Calvo and Alicia García

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48490

## **1. Introduction**

RNA polymerases (RNAPs) are among the most important cellular enzymes. They are present in all living organisms from Bacteria and Archaea to Eukarya and are responsible for DNA-dependent transcription. Although in Bacteria and Archaea there is only one RNAP, Eukarya possess up to three RNAPs in animals (I, II and III) and five in plants (IV and V) [1-2]. All of the RNAPs are evolutionarily related and have common structural and functional properties. The minimally conserved structural organization is represented by the bacterial enzyme, which contains only 4 subunits (''), whereas Archaea and Eukarya RNAPs are composed of 12 subunits (Rpb1-Rpb12) [3]. In prokaryotes, one RNAP transcribes all of the genes into all of the RNAs, however, in eukaryotes, this is achieved by three RNAPs. RNAPI transcribes genes that encode for 18S and 28S ribosomal RNAs; RNAPIII transcribes short genes, such as tRNAs and 5S ribosomal RNA, and RNAPII transcribes all protein-coding genes and genes for small noncoding RNAs (e.g., small nuclear RNAs (snRNAs) that are involved in splicing). The largest catalytic subunits of all three eukaryotic polymerases share homology among themselves and with the largest subunit of bacterial polymerase [4]. Solely the largest subunit of RNAPII (Rpb1) contains an unusual evolutionarily conserved carboxy-terminal domain (CTD) [5], which is subjected to numerous post-translational modifications of extraordinary importance in gene expression regulation [6-8].RNAPII transcription plays a central role in gene expression and is highly regulated at many steps, such as initiation, elongation and termination. Furthermore, phosphorylation of the Rpb1 CTD is known to regulate all of the transcription steps and coordinate these steps with other nuclear events. Prior to mRNA biosynthesis, RNAPII proceeds through several steps, such as promoter recognition, preinitiation complex (PIC) assembly, open complex formation, initiation and promoter escape. This sequence of events is initiated by the binding of gene-specific activators and coactivators, which results in the recruitment of basal transcription machinery (i.e., general transcription factors (GTFs):

© 2012 Calvo and García, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) and RNAPII to promoters [9-11]. Basal transcription factors position RNAPII on promoters to form the PIC but also function at later steps, such as promoter melting and initiation site selection. Thereafter, initiation proceeds, and RNAPII leaves the promoter during promoter clearance and proceeds into processive transcript elongation. Finally, when the gene has been fully transcribed, transcriptional termination occurs, and RNAPII is released and recycled to reinitiate a new round of transcription [12-14].

RNA Polymerase II Phosphorylation and Gene Expression Regulation 153

animals, plants, yeast, and in many protists [5, 36-37], and it has been hypothesized that the CTD structure has originated through amplifications of a repetitive DNA sequence and that the number of repeats appears directly correlated with genomic complexity (Figure 1A; [38]). For example, mouse and human CTDs contain 52 repeats [35, 39-40]; the Drosophila CTD contains 45 repeats [41]; 25-27 repeats are found in the yeast CTD (Figure 1A; [4]); and 15 repeats are found in protozoan CTDs [5, 38]. Although the CTD is completely dispensable for *in vitro* transcription, it is required for efficient RNA processing [17, 42]. In fact, the CTD is essential for cell viability because its deletion is lethal in mice, *Drosophila* and yeast, and

Original studies showed that two RNAPII forms can be differentiated in SDS-PAGE gels because of the different mobility of Rpb1 [43]. These two forms were termed RNAPIIA and RNAPIIO, and they differ in the extent of CTD phosphorylation. RNAPIIA is hypophosphorylated [44], and RNAPIIO is hyperphosphorylated [45]. Moreover, both forms, IIA and IIO, are functionally distinct because the IIA form is preferentially recruited to the promoter and associated with preinitiation complexes [46], whereas RNAPIIO functions during elongation, is highly phosphorylated [44] and thus requires dephosphorylation to stimulate its recruitment into the PIC complexes and to reinitiate a new round of transcription [47]. We currently know that this earlier two-step transcription cycle

partial truncations or site-specific mutations cause specific growth defects [5, 42].

**Figure 1.** Human and *Saccharomyces cerevisiae* Rpb1-CTDs.

During its passage across a gene, RNAPII must overcome challenges. Initially, the polymerase needs to escape from the promoter, and the synthesis of the pre-mRNAs must be tightly coupled to its subsequent processing (i.e., capping, splicing, and polyadenylation). Then, initiation factors must be exchanged for elongation factors [15], which are thought to increase the transcription rate and RNAPII processivity. In fact, recently, there has been an extraordinary increase in the number of proteins known to influence transcription elongation by avoiding transcriptional arrest, facilitating chromatin passage and mRNA processing [16-21], allowing mRNA packaging into a mature ribonucleoprotein (mRNP) and controlling mRNP quality and mRNA export [13, 22-28]. Therefore, the discovery of all of these factors has provided further evidence that the elongation phase is also highly regulated in eukaryotic cells and strictly coordinated with other nuclear processes [12-14].

## **2. RNAPII CTD phosphorylation: The CTD code**

During the last two decades, gene expression studies have provided further evidence that many steps in gene expression, originally considered distinct and independent, are, in fact, highly coordinated, linked and regulated in a complex web of connections [29-30]. The central coordinator that directs this regulatory network (i.e., from transcription initiation to termination and with pre-mRNA processing) in combination with many other nuclear functions is RNAPII, and the carboxy-terminal domain (CTD) of its largest subunit is of remarkable importance. CTD phosphorylation regulates and coordinates the entire transcription cycle with pre-mRNA processing, mRNA transport and with chromatin remodeling and modification [13]. The CTD, therefore, has a critical integrating role in essentially all of the mRNA biogenesis steps, thus, it is subject to a dynamic regulation during the transcription cycle (i.e., [21, 31-32]). Therefore, RNAPII phosphorylation is one of the key processes in the regulation of transcription specifically and gene expression in general; consequently, deciphering the mechanisms that underlie RNAPII phosphorylation regulation has become one of the most studied issues in the field of gene expression.

RNAPII is comprised of 12 subunits (Rpb1-12) that are structurally and functionally conserved from yeast to mammals [33-34]. In 1985, the largest subunit of RNAPII, Rpb1, from mouse and *Saccharomyces cerevisiae,* was cloned [4, 35], and its sequence revealed that it contained a highly conserved carboxy-terminal domain (CTD). This domain has been extensively studied since then and, although it is a simple repetition in tandem of the heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (YSPTSPS); Figure 1), the CTD has an extremely complex functionality. The consensus sequence is present in animals, plants, yeast, and in many protists [5, 36-37], and it has been hypothesized that the CTD structure has originated through amplifications of a repetitive DNA sequence and that the number of repeats appears directly correlated with genomic complexity (Figure 1A; [38]). For example, mouse and human CTDs contain 52 repeats [35, 39-40]; the Drosophila CTD contains 45 repeats [41]; 25-27 repeats are found in the yeast CTD (Figure 1A; [4]); and 15 repeats are found in protozoan CTDs [5, 38]. Although the CTD is completely dispensable for *in vitro* transcription, it is required for efficient RNA processing [17, 42]. In fact, the CTD is essential for cell viability because its deletion is lethal in mice, *Drosophila* and yeast, and partial truncations or site-specific mutations cause specific growth defects [5, 42].

**Figure 1.** Human and *Saccharomyces cerevisiae* Rpb1-CTDs.

152 Protein Phosphorylation in Human Health

transcription [12-14].

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) and RNAPII to promoters [9-11]. Basal transcription factors position RNAPII on promoters to form the PIC but also function at later steps, such as promoter melting and initiation site selection. Thereafter, initiation proceeds, and RNAPII leaves the promoter during promoter clearance and proceeds into processive transcript elongation. Finally, when the gene has been fully transcribed, transcriptional termination occurs, and RNAPII is released and recycled to reinitiate a new round of

During its passage across a gene, RNAPII must overcome challenges. Initially, the polymerase needs to escape from the promoter, and the synthesis of the pre-mRNAs must be tightly coupled to its subsequent processing (i.e., capping, splicing, and polyadenylation). Then, initiation factors must be exchanged for elongation factors [15], which are thought to increase the transcription rate and RNAPII processivity. In fact, recently, there has been an extraordinary increase in the number of proteins known to influence transcription elongation by avoiding transcriptional arrest, facilitating chromatin passage and mRNA processing [16-21], allowing mRNA packaging into a mature ribonucleoprotein (mRNP) and controlling mRNP quality and mRNA export [13, 22-28]. Therefore, the discovery of all of these factors has provided further evidence that the elongation phase is also highly regulated in eukaryotic cells and strictly coordinated with other nuclear processes [12-14].

During the last two decades, gene expression studies have provided further evidence that many steps in gene expression, originally considered distinct and independent, are, in fact, highly coordinated, linked and regulated in a complex web of connections [29-30]. The central coordinator that directs this regulatory network (i.e., from transcription initiation to termination and with pre-mRNA processing) in combination with many other nuclear functions is RNAPII, and the carboxy-terminal domain (CTD) of its largest subunit is of remarkable importance. CTD phosphorylation regulates and coordinates the entire transcription cycle with pre-mRNA processing, mRNA transport and with chromatin remodeling and modification [13]. The CTD, therefore, has a critical integrating role in essentially all of the mRNA biogenesis steps, thus, it is subject to a dynamic regulation during the transcription cycle (i.e., [21, 31-32]). Therefore, RNAPII phosphorylation is one of the key processes in the regulation of transcription specifically and gene expression in general; consequently, deciphering the mechanisms that underlie RNAPII phosphorylation

regulation has become one of the most studied issues in the field of gene expression.

RNAPII is comprised of 12 subunits (Rpb1-12) that are structurally and functionally conserved from yeast to mammals [33-34]. In 1985, the largest subunit of RNAPII, Rpb1, from mouse and *Saccharomyces cerevisiae,* was cloned [4, 35], and its sequence revealed that it contained a highly conserved carboxy-terminal domain (CTD). This domain has been extensively studied since then and, although it is a simple repetition in tandem of the heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (YSPTSPS); Figure 1), the CTD has an extremely complex functionality. The consensus sequence is present in

**2. RNAPII CTD phosphorylation: The CTD code**

Original studies showed that two RNAPII forms can be differentiated in SDS-PAGE gels because of the different mobility of Rpb1 [43]. These two forms were termed RNAPIIA and RNAPIIO, and they differ in the extent of CTD phosphorylation. RNAPIIA is hypophosphorylated [44], and RNAPIIO is hyperphosphorylated [45]. Moreover, both forms, IIA and IIO, are functionally distinct because the IIA form is preferentially recruited to the promoter and associated with preinitiation complexes [46], whereas RNAPIIO functions during elongation, is highly phosphorylated [44] and thus requires dephosphorylation to stimulate its recruitment into the PIC complexes and to reinitiate a new round of transcription [47]. We currently know that this earlier two-step transcription cycle model that is based on the two RNAPII forms is overly simple. Different phosphorylated forms of RNAPII are specific and characteristic of the different steps that occur during the transcription cycle [48], and the correct progression of RNAPII through the transcription cycle is dependent on changes in the CTD phosphorylation status. Differential CTD phosphorylation promotes the exchange of initiation and elongation factors during promoter clearance [15], the exchange of elongation and 3'-end processing factors at termination [49], as well as RNAPII recycling [50] and, moreover, links pre-mRNA processing and other nuclear events with transcription [17, 42]. Therefore, the CTD phosphorylation cycle is very complex. It is widely known that the three serines (i.e., Ser2, Ser5, and Ser7) [7, 51], the tyrosine [52-53] and the threonine [54] in each repeat can be phosphorylated. Additionally, both proline residues can be isomerized by a prolyl isomerase [55]. Moreover, glycosylation of serines and threonine can also occur [8], and in human cells, the CTD can be methylated at some of the degenerate repeat sites [56]. The multitude of possible CTD modifications, especially Ser phosphorylations, in combination with the numerous repetitions, gives rise to a wide range of variations (i.e., phosphorylation patterns) that have been termed the "CTD code" (Figure 2) [6-8].

RNA Polymerase II Phosphorylation and Gene Expression Regulation 155

phosphorylation patterns either indirectly or directly by contacting phosphorylated residues. Among the CTD-associated factors are export and histone modifier factors and

**2.1. Ser2P and Ser5P, and to a lesser extent, Ser7P, are the main determinants of**

To determine precisely which serine residues are phosphorylated in a particular repeat has been challenging because of the numbers of phospho-acceptor amino acid residues and consensus motif repetitions (Figure 1). However, studies involving chromatin immunoprecipitation with specific monoclonal antibodies have provided evidence that differential phosphorylation of the Ser residues coincides with the temporal and spatial recruitment of different factors [8, 32, 48, 66-67]. In fact, these antibodies have been largely used to decipher and characterize the role of CTD phosphorylation during the transcription cycle and in gene expression regulation [8, 32, 68]. Antibodies that selectively recognize either Ser2 or Ser5 phosphorylation (i.e., Ser2P or Ser5P, respectively) were the first residues to be described [66]; phosphorylation of these two residues has been extensively studied, and they have been considered as the two main determinants of the CTD code [6]. It is widely known that CTD phosphorylation switch from Ser5 to Ser2 during the course of transcription and is subject to a dynamic regulation during the whole transcription cycle [69-71]. The level of Ser5 phosphorylation peaks early in the transcription cycle and remains constant or decreases as RNAPII progresses to the 3′ end of the gene (Figure 3); [48, 67, 72]). In contrast, Ser2 phosphorylation is the predominant modification in the coding and 3′-end gene regions and occurs simultaneously with productive elongation [31, 48, 73]. On the other hand, de-phosphorylation of Ser5 occurs during the initiation-elongation transition and throughout the entire elongation step, whereas Ser2 de-phosphorylation occurs at the end of transcription to recycle the polymerase and reinitiate a new round of transcription. Therefore, reversible phosphorylation/de-phosphorylation of the CTD plays a significant