UNIVERSIDADE ESTADUAL DE CAMPINAS
Faculdade de Ciências Aplicadas
VANESSA DE OLIVEIRA
Hepatic Glucose Production Mediated by Omega-3 Fatty Acids: The
Role of Gαq/11 Proteins
Produção Hepática de Glicose Mediada por Ácidos Graxos
Ômega-3: O Papel das Proteínas Gα
q/11Limeira
2018
VANESSA DE OLIVEIRA
Hepatic Glucose Production Mediated by Omega-3 Fatty Acids: The Role of Gαq/11 Proteins
Produção Hepática de Glicose Mediada por Ácidos Graxos Ômega-3: O Papel das Proteínas Gαq/11
Thesis presented to the School of Applied Sciences of the University of Campinas in partial fulfillment of the requirements of the degree of Doctor in Sciences of Nutrition and Sports and Metabolism, in the area of Nutrition
Tese apresentada à Faculdade de Ciências Aplicadas da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Ciências da Nutrição e do Esporte e Metabolismo, na Área de Nutrição.
Supervisor/Orientador: Dr. Dennys Esper Correa Cintra
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA
VANESSA DE OLIVEIRA E ORIENTADA
PELO PROF. DR. DENNYS ESPER CORREA CINTRA
Limeira 2018
Ficha catalográfica
Universidade Estadual de Campinas Biblioteca da Faculdade de Ciências Aplicadas
Renata Eleuterio da Silva - CRB 8/9281
Oliveira, Vanessa de,
OL4h OliHepatic glucose production mediated by omega-3 fatty acids : the role of
Gaq/11 proteins / Vanessa de Oliveira. – Limeira, SP : [s.n.], 2018.
OliOrientador: Dennys Esper Correa Cintra.
OliTese (doutorado) – Universidade Estadual de Campinas, Faculdade de
Ciências Aplicadas.
Oli1. Gliconeogênese - Regulação. 2. Diabetes mellitus tipo 2. 3. Obesidade. 4.
Resistência à insulina. I. Cintra, Dennys Esper Correa, 1976-. II. Universidade Estadual de Campinas. Faculdade de Ciências Aplicadas. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Produção hepática de glicose mediada por ácidos graxos ômega-3
: o papel das proteínas Gaq/11
Palavras-chave em inglês:
Gluconeogenesis - Regulation Type 2 diabetes mellitus Obesity
Insulin resistance
Área de concentração: Nutrição
Titulação: Doutora em Ciências da Nutrição e do Esporte e Metabolismo Banca examinadora:
Dennys Esper Correa Cintra [Orientador] Leonardo dos Reis Silveira
Rodrigo Ferreira de Moura Adriana Souza Torsoni Moshmi Bhattacharya
Data de defesa: 27-11-2018
Programa de Pós-Graduação: Ciências da Nutrição e do Esporte e Metabolismo
FOLHA DE APROVAÇÃO Comissão Examinadora
Dr. Dennys Esper Correa Cintra Presidente
Dra. Adriana Souza Torsoni Dr. Leonardo dos Reis Silveira Membro interna Membro interno
Dra. Moshmi Bhattacharya Dr. Rodrigo Ferreira de Moura
Membro externa Membro externo
A Ata da Defesa de tese de doutorado assinada pela Comissão Examinadora consta no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade.
ACKNOWLEDGEMENTS / AGRADECIMENTOS
Foremost, I would like to express my most sincere gratitude for my Canadian / American mentor, Dr. Andy Videsh Babwah. It is hard to find enough words to express all gratitude, respect and admiration that I feel for you. Thank you for all your attention since our first contact, for welcoming me as your student and for helping me to conduct this study. Without your support, this could not be done. Thank you for your patience, your kindness, your motivation. For teaching me, for trusting me and for letting me work by your side. You are an exemplary mentor, an exceptional person, a brilliant scientist, and I am glad to have met you. I fully appreciate and value everything you´ve done for me, Andy. Thank you!!
I also would like to express my sincere gratitude to my Brazilian supervisor, Dr. Dennys Esper Corrêa Cintra. Thank you for your support, your motivation and your patience. Thank you for understanding my longing for something greater that would help us move forward. Thank you for believing me and for giving me the opportunity to develop the scientist in me. It is admirable the way you struggle for Nutrition and for free and quality education for everyone. Thank you!
I would like to thank my thesis committee examiners: Dr. Adriana Souza Torsoni, Dr. Leonardo dos Reis Silveira, Dr. Rodrigo Ferreira de Moura and Dr. Moshmi Bhattacharya. I greatly appreciate your presence and knowledge in this unique moment of my doctoral defense. Thank you for your valuable contribution.
To Dr. Moshmi Bhattacharya, I dedicate my special gratitude. Thank you for welcoming me so cozily and for being so kind to me. Thank you for supporting this study and for allowing me to work in your Lab. Thank you for your patience and for your trust. I fully admire your intelligence, and it has been a tremendous privilege to work alongside someone so nice and competent like you. I am glad to be part of your team and I hope we can continue making Science together! Thank you for everything, Moshmi!
I want to express my enormous gratitude to one of the best researchers and hardworking women I´ve ever met, Magdalena Dragan! You have no idea how much
I´ve learned from you since we first met. I am glad to have the opportunity to work by your side. You are an example for all of us. Thank you for teaching me, for being patient, for helping me and for all our great and funny moments in the Lab!! You have all my admiration and respect. Thank you for all, Magda. Cheers!!
I am profoundly grateful to Dr. Lício Augusto Velloso and Dr. Leonardo dos Reis Silveira for allowing me to conduct fundamental experiments in your laboratories. Undoubtedly the data generated helped us to better understand the observed phenomena. The time I spent in your Labs was extremily valuable to me. I fully appreciate your kindness and I hope we can extend the bonds of our collaboration in a near future.
I am also deeply grateful to Dr. Sally Radovick and Dr. Fredric Wondisford for welcoming me and giving me the incredible opportunity to join your research group. Thank you for all the support you provided for this study and for our laboratories.
My sincere thanks to all Lab members that I met and had the great opportunity to learn and work with. Especially, I would like to thank Jennifer Schaefer. Thank you for your company, for your help, for your partnership, for all the brainstorms, and more than that, for your friendship! I am sure that you will have a bright future!
I wish to acknowledge the Laboratory of Nutritional Genomics as well as the Laboratory of Molecular Biology of the Exercise for all the assistance to this study.
To the experimental animals, I dedicate all my respect and gratitude.
I want to thank my home University, The University of Campinas (UNICAMP), Brazil for providing all the support needed for my learning, for my academic and professional growth as well as for my growth as a human being. Since my first day at this exceptional University, in 2003, I felt very welcome and I knew that I had made one of the best choices of my life. I only have great things to say about “my” University. Thank you, UNICAMP for all those years of partnership!!
I also want to thank the University of Western Ontario, London, ON, Canada for welcoming me and for making me feel home during my scholarship abroad. Thanks very much for your hospitality - the world needs more Canada!
I would like to thank Rutgers, The State University of New Jersey, USA, for welcoming me and for providing all the necessary support for this study to be concluded.
My research would have been impossible without the aid and support of the Foundation for Research Support of Sao Paulo State (Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP). I would also like to thank The New Jersey Health Foundation for supporting this study.
Finalmente, porém jamais menos importante, eu gostaria de agradecer à minha família. Aos meus queridos sogros, Rosa e Jamir Benatti, à minha doce cunhada, Maíra Benatti, à dona Teddy, à minha querida tia Carmen e outros familiares que acompanharam de perto minha trajetória acadêmica, especialmente durante meu doutorado, que sempre me apoiaram e torceram por mim, a vocês, o meu muito obrigada!!
À minha mãe, obrigada Rosângela por seu carinho e por sua doação para que eu sempre tivesse o melhor. Ao meu pai, Walter, meu amigo, meu anjo da guarda, meu porto-seguro, toda a minha gratidão. Obrigada por me encorajar, por estar sempre ao meu lado, obrigada por sua sensatez, pelo seu carinho e amor. Mesmo longe, estaremos sempre juntos. Amo você!
Ao meu marido, Danilo Benatti, meu companheiro e protetor, minha profunda gratidão. Obrigada por me apoiar desde o início, mesmo sabendo que a trajetória não seria fácil! Obrigada por estar todos os dias ao meu lado, me ouvindo falar sobre experimentos, vias de sinalização, por me consolar quando um blot não sai ou quando um qPCR não funciona. Obrigada por ouvir sobre meus devaneios científicos, mesmo que lhe soem um tanto malucos! Obrigada por compreender que os deadlines roubam minhas noites de sono e que experimentos muitas vezes me
obrigam a sacrificar finais de semana e feriados. Obrigada por cuidar de mim com tanta dedicação e carinho! Você faz parte de tudo isso. Eu amo você!
Dedico eterna gratidão às minhas “filhas caninas”, Laika e Kayla, pela lealdade, pelo companheirismo, carinho e amor incondicional.
ABSTRACT
The incidence of type 2 diabetes mellitus (T2DM) is alarmingly increasing worldwide, due to obesity, its most important predisposing factor. Obesity leads to the development of insulin resistance, an elementary condition for the onset of T2DM. The liver plays a crucial role in maintaining glycemic levels by regulating gluconeogenesis (glucose de-novo synthesis) and glycogenolysis (glycogen breakdown) pathways. However, the insulin resistance in the liver impairs its ability to control such pathways, leading to excessive glucose production and release, which results in constant hyperglycemia. Besides its well recognized anti-inflammatory properties, omega-3 (ω-3) fatty acids have been described as adjuvants to the treatment of T2DM, due to their hypoglycemic effects. These fatty acids signal via G protein-coupled receptors (GPCRs), especially GPR120 (Ffar4). Once stimulated, GPR120 activates intracellular G proteins, especially Gαq and Gα11, which appear to play an important role in glycemic regulation. Therefore we hypothesized that the ablation of Gαq/11 in the liver would prevent the beneficial effects of ω-3 fatty acids on glucose homeostasis. To test our hypothesis we used male mice bearing a liver-specific conditional knockout (cKO) of Gαq and their littermate controls (Ctrls) under the background of Gα11 null mice. The conditional knockout of hepatic Gαq/11 did not impact food intake or body weight of mice fed with chow or high fat diet (HFD) for 8 weeks. Despite this, HFD fed cKO mice produced less glucose upon a pyruvate stimuli and showed improved glucose homeostasis compared to HFD fed Ctrl mice. However, after 17 weeks in HFD, cKO mice showed impaired glucose homeostasis similarly to Ctrl mice. Then, after 1 week of oral treatment (2g/kg of body weight) with oil rich in docosahexaenoic acid (DHA) (C22:4, ω-3), cKO mice showed improved glucose tolerance compared to Ctrl mice. Nevertheless, with the progression of obesity and T2DM the DHA supplementation had no longer effect in improving glucose homeostasis, since cKO mice displayed glucose intolerance and increased hepatic glucose production (HGP) similarly to Ctrl mice. No significant differences were observed on p-Akt (Ser473) or p-FoxO1 (Ser256) between Ctrl and cKO groups. However, when we investigated the effect of DHA treatment within the genotypes, we observed a decrease on p-FoxO1 (Ser256) as well as an increase on gene expression of gluconeogenic enzymes such as PcK1 (Phosphoenolpyruvate carboxykinase) and G6pc (Glucose-6-phosphatase) in the liver of Ctrl and cKO animals treated with DHA. Additionally, hepatic Kiss1 (Kisspeptin 1) and Gnas (Gαs)
gene expressions were greatly increased in Ctrl and cKO animals treated with DHA, which indicates an up-regulation of glucagon signaling. Our findings suggest that the hepatic-specific conditional knockout of Gαq/11 improves glucose homeostasis in mice fed with HFD for 8 weeks but this effect is lost with the progression of obesity and T2DM; unexpectedly, DHA treatment has a transient beneficial effect on glucose homeostasis in obese and diabetic cKO mice; when supplemented for a prolonged period, DHA stimulates other GPCR signaling pathways in the liver, which trigger mechanisms that lead to increased HGP.
Key words: Gluconeogenesis; Gαq/11; Omega-3 fatty acids; Obesity; Insulin
resistance; Type 2 diabetes mellitus.
RESUMO
A incidência de diabetes mellitus tipo 2 (DM2) tem aumentado assustadoramente em todo o mundo, devido à obesidade, seu fator predisponente mais importante. A obesidade, leva ao desenvolvimento da resistência à insulina, condição elementar para o surgimento do DM2. O fígado desempenha papel crucial na manutenção dos níveis glicêmicos, regulando as vias de gliconeogênese (síntese de novo de glicose) e glicogenólise (quebra do glicogênio). No entanto, a resistência à insulina no fígado prejudica a capacidade do órgão em controlar tais vias, levando à produção e liberação excessiva de glicose, o que resulta em hiperglicemia constante. Além das reconhecidas propriedades anti-inflamatórias, ácidos graxos ômega-3 (ω-3) têm sido descritos como adjuvantes ao tratamento do DM2, devido aos seus efeitos hipoglicemiantes. Esses ácidos graxos sinalizam via receptores acoplados à proteína G (GPCRs), especialmente GPR120 (Ffar4). Uma vez estimulado, o GPR120 ativa proteínas G intracelulares, especialmente Gαq e Gα11, as quais parecem desempenhar papéis importantes na regulação glicêmica. Dessa forma, nós hipotetizamos que a ablação de Gαq/11 no fígado impediria os efeitos benéficos dos ácidos graxos ω-3 na homeostase da glicose. A fim de testar nossa hipótese, nós utilizamos camundongos machos portadores de knockout condicional hepático para Gαq (cKO) e seus controles (Ctrls) sob o background de camundongos
knockout para Gα11. O knockout condicional hepático de Gαq/11 não impactou a ingestão alimentar ou o peso corporal de camundongos alimentados com ração ou dieta hiperlipídica (high fat diet - HFD) por 8 semanas. Apesar disso, quando alimentados com HFD camundongos cKO produziram menos glicose após estímulo com piruvato e apresentaram melhor homeostase da glicose em comparação aos camundongos Ctrl. No entanto, após 17 semanas em HFD, os camundongos cKO apresentaram prejuízo na homeostase da glicose, de forma semelhante aos camundongos Ctrl. Então, após 1 semana de tratamento oral com óleo rico em ácido docosahexaenóico (DHA) (C22:4, ω-3), (2g/kg de peso corporal), os camundongos cKO mostraram melhora na tolerância à glicose em comparação aos camundongos Ctrl. No entanto, com a progressão da obesidade e do DM2, tal efeito foi abolido, uma vez que os animais cKO apresentaram intolerância à glicose e aumento da produção hepática de glicose (HGP) de modo similar aos animais Ctrl. Não foi observada diferença importante em p-Akt (Ser473) ou p-FoxO1 (Ser256) entre os grupos Ctrl e cKO. No entanto, quando investigamos o efeito do tratamento com
DHA por genótipos, observamos diminuição em p-FoxO1 (Ser256), bem como aumento na expressão gênica de enzimas gliconeogênicas, tais como PcK1 (fosfoenolpiruvato carboxiquinase) e G6pc (Glicose-6-fosfatase), no fígado de animais Ctrl e cKO tratados com DHA. Além disso, as expressões gênicas de Kiss1 (Kisspeptídeo 1) e de Gnas (Gαs) foram significativamente aumentadas no fígado de animais Ctrl e cKO tratados com DHA, o que indica uma regulação positiva da sinalização de glucagon. Nossos achados sugerem que o knockout condicional de Gαq/11 no fígado melhora a homeostase da glicose em camundongos alimentados com HFD por 8 semanas, contudo, tal efeito é perdido com a progressão da obesidade e T2DM; inesperadamente, o tratamento com DHA apresentou efeito benéfico transitório na homeostase da glicose em camundongos cKO obesos e diabéticos; quando suplementado por um período prolongado, o DHA estimula outras vias de sinalização de GPCR, as quais desencadeiam mecanismos que levam ao aumento de HGP.
Palavras-chave: Gliconeogênese; Gαq/11; Ácidos graxos ômega-3; Obesidade;
SUMMARY
1.0 Introduction………15
1.1 Obesity and Diabetes – The problem statement………15
1.2 Inflammation and Insulin Resistance ... 15
1.3 The role of the liver in the control of glycemic homeostasis ... 17
1.4 Insulin Signaling in the Liver ... 17
1.5 GPCRs and Omega-3 Fatty Acids in the Control of Glycemic Homeostasis………..19
1.6 The role of Gαq/11 proteins………..20
2.0 Hypothesis………...………...22
3.0 General Objectives………..……….22
3.1 Specific Objectives………...22
3.1.1 Characterization of the conditional knockout experimental (cKO) mouse and its littermate control (Ctrl)…..………...………22
3.1.2 Evaluation of Gαq/11 in the regulation of hepatic glucose production in conditional knockout experimental mice and littermate controls…..………..22
4.0 Material and Methods………...…………...23
4.1 Animal husbandry………..……….23
4.2 Generation of the conditional knockout (cKO) (Gnαqfl/fl; Gnα11-/-; Alb-Cre, experimental) and littermate control (Ctrl) (Gnαqfl/fl; Gnα11-/-) mice………..23
4.2.1 Animal Genotyping………..23
4.3 Experimental diets and ω-3 supplementation………...24
4.4 Antibodies and Reagents………...24
4.5 Intraperitoneal Glucose Tolerance Test (iGTT), Insulin Tolerance Test (ITT) and Pyruvate Tolerance Test (PTT)……….………..24
4.6 Tissue Extraction………...25
4.7 Immunoblot assays……….25
4.8 Real Time qPCR……….26
4.9 Statistical Analysis………..26
5.0 Results……….27
5.1 Characterization of the experimental model………...27
Generation of hepatic-specific condition knockout (cKO) ( Gnaqfl/fl; Gna11-/-; Alb-Cre) and littermate control (Ctrl) (Gnaqfl/fl; Gna11-/- ) mice………...27
5.2 Behavioral Parameters………...28
Food intake and body weight………...28
5.3 Physiological and molecular parameters……….…30
Hepatic-specific conditional knockout of Gαq/11 improves gluconeogenesis in mice fed with HFD for 8 weeks………...…….30
Hepatic-specific conditional knockout of Gαq/11 increases FoxO1 phosphorylation (Ser256) in mice fed with HFD for 8 weeks………...…………31
Hepatic-specific conditional knockout of Gαq/11 decreases gluconeogenesis but does not restore insulin sensitivity in obese and diabetic mice………33
Omega-3 treatment has a transient beneficial effect on glycemic homeostasis in obese and diabetic cKO mice……….………...36
6.0 Discussion……….……….45
7.0 Conclusion………….……….………48
8.0 References………..………49
1.0 INTRODUCTION
1.1 Obesity and Diabetes – The problem statement
Diabetes is one of the leading causes of morbidity and mortality worldwide (1). The number of people with diabetes increased from 108 million in 1980 to 422 million in 2014 (2). In 2015, 1.6 million deaths were directly attributed to diabetes (2). Type 1 diabetes mellitus (T1DM) (also known as juvenile or insulin dependent), is an auto-immune disease, characterized by the absence of insulin due to the collapse of pancreatic β-cells (3). Type 2 diabetes mellitus (T2DM) (also known as non-insulin dependent or adult diabetes), is the most common type of the disease and is strictly associated to the excess of body weight. It is characterized by two main phenomena: insulin resistance, which results primarily in high levels of blood glucose and hyperinsulinemia, and in a second moment, in relative insulin deficiency due to pancreatic β-cell failure (1,2,4).
In parallel to T2DM, obesity is currently considered a worldwide epidemic, which is out of control (4, 5). It arises from an imbalance between food intake and energy expenditure and can be defined by a state in which there is abnormal and excessive accumulation of fat in the white adipose tissue, capable of causing health damage (5,6). In addition to T2DM, obesity predisposes to the development of several comorbidities such as dyslipidemias, hypertension, atherosclerosis, cancer, hepatic steatosis, asthma, polycystic ovarian syndrome, neurodegenerative diseases, among others (6,7). The chronic low-grade, or subclinical inflammation seems to act as the initiator and enhancer of such metabolic disorders (9,10). This phenomenon has been pointed out as an elementary condition for the development of insulin resistance, which in turn, leads to T2DM (4,9,10,11).
1.2 Inflammation and Insulin Resistance
In the last few decades, studies have identified the excessive consumption of saturated fatty acids as the trigger factor of the low degree inflammation (11, 12, 13). As a structural component of lipopolysaccharide (LPS) of gram-negative bacteria, this type of fat can be recognized by Toll-like receptors, especially Toll-like 4 (TLR4), evolutionarily preserved in human cells of the innate immune system and adipocytes. When activated, the TLR4 triggers its pro-inflammatory cascade that culminates in the activation of the nuclear kappa B factor (Nf-κB) (12,13,14). This phenomenon results in the increase of the transcription of genes encoding pro-inflammatory
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peptides such as the tumor necrosis factor alpha (TNF-α), monocyte chemotaxis protein 1 (MCP-1), inducible nitric oxide synthase (iNOS) and interleukins 1β (IL-1β), 6 (IL-6) and 10 (IL-10), the latter being recognized as anti-inflammatory, responsible for containing the advance of inflammation (14,15,16).
In the same way that TLR4 receptors recognize lipid structures present in invasive microorganisms, saturated fatty acids from the diet can also be recognized. Consequently, the immune system is activated, regardless the presence of an invasive microorganism (12-16), since these substances are widely spread in foods such as coconut oil, palm kernel, milk and milk products, meats and chocolate (15, 17).
In the low-degree inflammation, pro-inflammatory proteins interfere at specific spots of signaling pathways such as the leptin and insulin hormones (18). The resistance of these hormones signaling, in either the central nervous system (CNS) and peripheral organs, seems to be the initial step for the genesis of metabolic diseases, since they are responsible for the control of numerous functions throughout the organism (18, 19).
Due to the redundancy of the inflammatory process, pathways such as TLR4, TNF-α and IL-1β are constantly activated leading to the activation of serine kinases such as IKK (inhibitor of kappa kinase) and JNK (c-Jun N-terminal kinase) (10, 11, 14, 19). Such proteins negatively interfere in the insulin signaling by phosphorylating the insulin receptor substrate 1 (IRS-1) in 307 serine residues (Ser307) which prevents its phosphorylation in tyrosine by the insulin receptor (IR). This process recognized as insulin resistance, impairs the hormone signal transduction in several tissues of the organism, which predisposes the individual to T2DM (14, 20, 21).
The diminished insulin sensitivity in insulin-dependent tissues leads to an imbalance in glycemic homeostasis (20, 21, 22). Regarding the periphery, insulin resistance results in decreased glucose uptake by skeletal muscle and white adipose tissue as well as increased lipolysis with concomitant reduction of lipogenesis, also in adipose tissue. In muscle and liver, occurs reduction of protein synthesis and glycogenesis (22). The hepatic tissue, fundamental in the control of glycemia, intensifies its glucose production through gluconeogenesis, contributing to the perpetuation of the hyperglycemic and hyperinsulinemic state (23). Thus, insulin resistance or its deficiency, results in a profound metabolic dysregulation, leading to enhanced glucose and lipid blood levels in both fasting and fed states (23, 24).
1.3 The role of the liver in the control of glycemic homeostasis
Glucose homeostasis in the body is dynamically controlled by the supply of nutrients and intracellular hormonal signaling, which regulates the use and production of glucose in a tissue-specific manner (25, 26). Among the tissues that contribute to the maintenance of circulating glucose normal levels, are the liver, skeletal and cardiac muscle, adipose tissue and the brain. After a meal rich in carbohydrates, approximately 33% of glucose is uptaken by the liver, another 33% by skeletal muscle and adipose tissue and then the remaining glucose is uptaken by the brain, kidneys and red cells. In this scenario, insulin and glucagon act as major counter-regulatory hormones that orchestrate signals from the periphery in order to maintain euglycemia (25, 26).
The liver plays a central role in the maintenance of glycemic homeostasis, being the main responsible for glycogen storage as well as glucose production (gluconeogenesis) (26). In the postprandial state, when nutrient supply is abundant in the body, insulin is secreted by pancreatic β-cells. The action of insulin in the liver results in glycogen synthesis and reduction of gluconeogenesis. When nutrients become scarce, insulin levels decrease and pancreatic α cells release glucagon, which promotes increased hepatic glucose production (HGP) (23,25). The HGP occurs through two mechanisms: glycogen breakdown and de novo glucose synthesis (gluconeogenesis) from precursors such as glycerol, pyruvate, lactate and alanine (23, 24, 25).
The liver is insulin-independent for glucose uptake, which is mediated by GLUT2 vesicles. Therefore, the main function of insulin in the liver is to control the glycidic metabolism by stimulating glycogen synthesis (glycogenesis) and inhibiting glucose production (gluconeogenesis) (26).
1.4 Insulin Signaling in the Liver
The insulin receptor (IR), has 2 allosteric α subunits and 2 catalytic β subunits which have intrinsic tyrosine kinase activity. After insulin binds to the α subunit, allosteric inhibition is suppressed and then the β subunits auto-phosphorylate into tyrosine residues (Tyr 1158, 1162, 1163). Activated β subunits phosphorylate their substrates, IRS1/2, in tyrosine residues (Tyr 1222). Then, IRS1/2 associates with the regulatory subunit p85 of PI3-k, activating its catalytic subunit p110α. PI3-k, in turn, mediates the phosphorylation of phosphatidyl inositol (4,5) bi-phosphate (PIP2) in
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phosphatidyl-inositol (3,4,5) tri-phosphate (PIP3). PIP3 recruits PDK1 (phosphoinositide-dependent kinase 1) and Akt / PKB (protein kinase B) to the cell membrane, where PDK1 phosphorylates Akt in threonine 308 (Thr 308). The mTORC2 protein, activated by PIP3 catalyzes the phosphorylation of Akt in serine 473 (Ser473). The Akt protein has nodal function in insulin signaling, driving the hormone signal through several intracellular pathways (25, 26, 27).
The Akt protein is able to propagate the insulin signal to the nucleus where it then phosphorylates the FoxO1 - Forkhead-box containing gene, O subfamily in serine residue 256. Thus, FoxO1 is exported to the cytoplasm and degraded by the proteasome (26,27). This reduces FoxO1´s activity in transcribing gluconeogenic genes, such as phosphoenolpyruvate carboxykinase enzyme (PEPCK), which converts oxaloacetate to phosphoenolpyruvate in the gluconeogenic pathway and glucose phosphatase enzyme (G-6Pase), responsible for converting glucose 6-phosphate in free glucose in the liver, allowing the extrusion of glucose to the exterior of the cell. Thus, when the insulin signaling pathway is activated in the liver, the gluconeogenic activities are supressed (27, 28).
Insulin regulates glycogen metabolism essentially through phosphorylation and control of the activity of enzymes that catalyze the synthesis or lysis of glycogen. The glycogen synthase (GS) enzyme can be deactivated by multiple residues phosphorylation. This event can be mediated by different kinases such as cyclic adenosine monophosphate (cAMP), cAMP-dependent protein kinase (PKA) and glycogen synthase kinase-3 (GSK-3). The GSK-3 is the main responsible for the deactivation of GS enzyme. In liver and muscle, Akt phosphorylates GSK-3α in serine 21 (Ser21) and GSK-3β in serine 9 (Ser9) residues, reducing its catalytic activity. Thus, GS is no longer inhibited, resulting in increased glycogen production (glycogenesis) (25, 27, 28).
However, in the context of obesity and insulin resistance, the liver loses its ability to respond to hormonal stimulation, which results in decreased glycogenesis and increased hepatic gluconeogenesis, perpetuating the hyperglycemic state in the body (29).
1.5 GPCRs and Omega-3 Fatty Acids in the Control of Glycemic Homeostasis
Several studies have shown that saturated fatty acids present on a large scale in western diet can activate pro-inflammatory pathways such as TLR-4 and TNF-α (12,13,14). Thus, frequent consumption of a high-fat diet contributes not only to weight gain but also to the maintenance of low-degree inflammation leading to insulin resistance and T2DM. On the other hand, long chain unsaturated fatty acids (LCUFA), like those of the omega-3 (ω-3 or n-3) family, are well recognized due to their anti-inflammatory properties (30, 31, 32). Among them, EPA (pentahexaenoic acid, C 20:5, ω-3) and DHA (docosahexaenoic acid, C 22:6, ω-3) have marine origin and are found in viscera and oil extracted from deep and cold water fish. Besides these, ALA (α-linolenic acid, C 18: 3, ω-3), is present in vegetable sources, such as soybean and canola oils, as well as flaxseed and chia seed oils. A great number of health benefits have been attributed to these nutrients, such as triglyceride level reduction, atherosclerosis prevention, insulin sensitivity and glycemic homeostasis improvement, hepatic steatosis reduction and recovery of food intake control (30 – 33).
In an elegant study published in 2010, Oh DY et al. (34) described the mechanism by which long chain fatty acids exert their functions. ω-3 fatty acids, as well as other long chain unsaturated fatty acids, such as oleic (C 18:1, ω-9) and palmitoleic (C 16:1, ω-7), signal via G protein coupled receptors (GPCRs), particularly GPR120 (Ffar4) and GPR40 (Ffar1) (34). The GPR40 is mainly expressed in pancreatic islets, and mediates the ability of long chain fatty acids to potentiate glucose-stimulated insulin secretion (35). In duodenal I cells, GPR40 has been shown to induce the secretion of cholecystokinin, a neuropeptide inducing postprandial satiety (36). The GPR120, in turn, is widely found in various cell types such as adipocytes, macrophages, enterocytes, and pancreatic islets and is also expressed in skeletal muscle and liver. Recent studies have shown that the GPR120 is a multifactorial protein involved in the improvement of many aspects of metabolic homeostasis such as insulin sensitivity, macrophagic function, hepatic steatosis and hormonal secretion by pancreatic islets and entero-endocrine cells (34, 37, 38).
The GPCRs are seven transmembrane receptors and mediate the activation of intracellular G proteins: the subunits α, β and γ. As sensitized by specific ligants as ω-3 fatty acids, GPCRs, undergo an intracellular rearrangement of helices 6 and 3,
20
leading to the activation of Galpha subunits by means of GDP dissociation and GTP binding via GEF (guanine exchange factor) (39). Then, the activated Gα subunit dissociates from the βγ dimer (40,46). Deactivation of this mechanism occurs upon the GTP hydrolysis at the nucleotide binding site in the Gα subunit, a process modulated by the regulator of G-protein signaling (RGS), promoting the re-association of the heterotrimer (αβγ) (39, 40). The de-sensitization of GPCRs occurs through two steps involving the phosphorylation of GRK-activated receptors (G protein-coupled receptor kinase) which serves as a target for the arrestin proteins, and subsequent binding to β-arrestin-2. Such binding facilitates endocytosis of the GPCRs mediated by clathrins. This process may lead to the degradation or re-sensitization and return of these receptors to the plasma membrane (40, 41).
Oh et al. (2010) (34), have shown that when recruited to the intracellular domain of the GPR120, the β-arrestin-2 attracts TAB1,2,3 proteins, disrupting TLR-4 and TNF-α inflammatory pathways. Cintra DE et al. (2012) (31) also demonstrated a similar mechanism in hypothalamic neurons, which reestablished the food intake control in obese animals when treated with ω-3 or ω-9. Nevertheless, these studies as well as other literature evidences, suggest that long chain unsaturated fatty acids acting via GPR120 or 40 appear to exert their benefits on glycemic homeostasis by activating other signaling pathways regardless the improvement of the inflammatory process (35, 42, 43, 44).
1.6 The role of Gαq/11 proteins
As previously mentioned, GPCRs are transmembrane proteins, able to conduct chemical signals across the cell membrane and mediate the activation of intracellular G proteins (α, β and γ subunits) (40, 46).
Gα subunits have been classified into 5 different families: Gαs, Gαi, Gαq, Gα12/13 and the recently discovered Gαv (45). The Gαs and Gαi families regulate adenylate cyclase activity, while Gαq activates phospholipase C-β (PLC-β) and Gα12/13 can activate small families of GTPases (46).
The Gαq family comprises 4 members: Gαq and Gα11 are ubiquitously expressed, Gα14 is found in the kidneys, liver and lung and Gα15/16 (orthologous of mice and humans, respectively), is expressed only in hematopoietic cells. The Gαq and Gα11 subunits share 88% homology in their amino acid sequence and, in face of such similarity, Gαq fully compensates Gα11, although the opposite has not been
observed (47, 48). Gαq activates PLC-β which in turn hydrolyzes phosphatidyl-inositol 4,5-biphosphate (PIP2), releasing diacylglycerol (DAG) and 1,4,5-inositol-triphosphate (IP3). DAG activates isoforms of PKC (protein kinase C) while IP3 diffuses through the cytosol to the endoplasmic reticulum (ER), where it binds to its receptor in calcium channels on the ER membrane, inducing massive release of calcium (Ca2+) to the cytosol (40, 45, 46).
Several studies have demonstrated a number of events related to the activation of the Gαq and Gα11 subunits, such as gonadotropin releasing hormone (GnRH) secretion via GPR54, which is the kisspeptin (KISS1) receptor (KISS1R) (48), appetite regulation via melanocortin-4 receptors (MC4Rs) in the paraventricular nucleus of the hypothalamus (50), platelet aggregation (51) and glucose metabolism (42). In addition to these functions, the GPCR-Gαq/11 signaling pathway has been suggested to regulate a number of metabolic processes related to obesity and type 2 diabetes (52, 53). Olefsky et al. (53) proposed that the stimulation of Gαq-coupled endothelin-1 receptor (ETA) is capable of activating the catalytic β1-α subunit of PI3-k via Gαq in a 3T3-L1 adipocyte lineage. The acute treatment of these cells with endothelin-1 increased the activity of the p110α portion in the same proportion as insulin does, inducing the translocation of GLUT4 to the membrane, resulting in increased glucose uptake. However, no increase in Akt phosphorylation was detected. Similarly, Olefsky´s group demonstrated that the adenoviral expression of Gαq (Q209L) increased PI3-k activity in both immunoprecipitated p110α and p110γ. The researchers found that modest stimulation of p110α is sufficient to induce GLUT4 translocation to the membrane with subsequent increase in glucose uptake into 3T3-L1 adipocytes, even with negligible effect on Akt phosphorylation (42)
Although there is strong evidence for the role of the GPCRs-Gαq/11 signaling pathway and omega-3 fatty acids in the control glucose homeostasis, the mechanisms involved in this process are not fully elucidated. The constant hyperglycemia that permeates the condition of insulin resistance and T2DM is largely due to the imbalance between glycogen synthesis and hepatic gluconeogenesis, which enhance the production and exportation of glucose into the bloodstream (23, 29).
Given the recognized role of ω-3 fatty acids as important adjuvants in the treatment of metabolic disorders, the study of the action of these nutrients mediated
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by the GPCRs-Gαq/11 pathway in the liver may bring new possibilities for therapeutic interventions in the context of obesity and of diabetes.
2.0 HYPOTHESIS
Besides the anti-inflammatory properties, ω-3 fatty acids acting via Gαq/11 coupled signaling could exert beneficial effects in the control of hepatic glucose production (gluconeogenesis) and glucose homeostasis, and that the hepatic-specific conditional knockout of Gαq and the ablation of Gα11 subunit, would prevent such actions.
3.0 GENERAL OBJECTIVE
To evaluate the role of Gαq/11 coupled signaling in the regulation of hepatic glucose production and glucose homeostasis in Gα11 null mice bearing a hepatic-specific conditional knockout of Gαq.
3.1 SPECIFIC OBJECTIVES
3.1.1 Characterization of the conditional knockout experimental (cKO) mouse and its littermate control (Ctrl).
A - To generate the experimental mice bearing a conditional deletion of Gαq in the liver, under the background of null Gα11 mice, and their littermate controls;
B - To characterize the expression (mRNA and protein) of the Ffar4 (GPR120) and
Gnaq (Gαq) in the liver of the cKO and Ctrl mice;
C - To measure body weight and food intake of cKO and Ctrl mice fed with chow or HFD;
D - To evaluate the glucose tolerance, insulin sensitivity and hepatic glucose production (gluconeogenesis) of cKO and Ctrl mice fed with chow or HFD;
E - To investigate the activation of insulin signaling proteins, such as Akt and FoxO1, as well as the gene expression of gluconeogenic enzymes, such as PEPCK (Pck1) and G6P (G6pc) in the liver of Ctrl and cKO mice fed with chow or HFD.
3.1.2 Evaluation of Gαq/11 in the regulation of hepatic glucose production in
A – To evaluate the glucose tolerance, insulin sensitivity and hepatic glucose production of Ctrl and cKO mice fed with HFD and treated with ω-3 fatty acids (docosahexaenoic acid – DHA) via orogastric route (gavage);
B - To evaluate the role of Gαq/11-coupled signaling in mediating the effects of ω-3 fatty acids administered via orogastric route;
C - To investigate the activation of insulin signaling proteins, such as Akt and FoxO1, as well as the gene expression of gluconeogenic enzymes, such as PEPCK (Pck1) and G6P (G6pc) in the liver of Ctrl and cKO mice fed with HFD and treated with ω-3 fatty acids via orogastric route.
4.0 MATERIAL AND METHODS
4.1 Animal husbandry
Animal studies were approved by the Animal Care Committee of The University of Western Ontario, Canada and of Rutgers – The State University of New Jersey, USA, according to guidelines established by the Canadian and American Council on Animal Care, respectively. The mice were maintained under a 12 h light / dark cycle, and provided with standard rodent chow or high fat diet and water ad libitum.
4.2 Generation of the conditional knockout (cKO) (Gnαqfl/fl; Gnα11-/-; Alb-Cre,
experimental) and littermate control (Ctrl) (Gnαqfl/fl; Gnα11
-/-) mice.
The parental lines, Gnαqfl/fl;Gnα11-/-
(Offermanns et al., 1998) (22) mice, were generated as previously described; the Alb-Cre line was purchased from The Jackson Laboratory (Bar Harbor, ME USA). Parental lines, both maintained on the C57BL/6J genetic background, were crossed to each other to generate Gnaqfl/+;Gna11-/-;Alb-Cre F1 mice. These were backcrossed to the parental line, Gnαqfl/fl;Gnα11-/-, to generate an F2-segregating population of Gnαqfl/fl;Gnα11
-/-; Alb-Cre (experimental genotype bearing a liver-specific conditional deletion of Gnαq under the background of whole-body deletion of Gnα11 and subsequently referred to as cKO) and Gnαqfl/fl; Gnα11
(control genotype subsequently referred to as Ctrl).
4.2.1 Animal Genotyping
Toe or tail snips (0.2 cm) were placed into 75 µL of Alkaline lysis buffer and heated at 95 C for 1 h. After heating, samples were cooled down at 4 C for 5 min. Then, 75 µL of neutralizing buffer was added and mixed to samples by vortex. For
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the detection of CRE enzyme, forward Cre5’ (5’-CGA CCA AGT GCA AGC AAT GCT-3’) and reverse Cre3’ (5’-GGT GCT AAC CAG CGT TTT CGT-3’) were used at 10 µM. For PCR reaction, Thermo Scientific Dream Taq Hot Start Green PCR Master Mix (2X) from Thermo Fisher was used according to the manufacture’s instructions. DNA samples were loaded onto 1.5% agarose gel with ethidium bromide. Nucleic acids bands were detected at Chemidoc Touch Imaging System (Bio-Rad).
4.3 Experimental diets and ω-3 supplementation
Experimental animals and their littermate controls were fed with chow or HDF according to the American Institute of Nutrition, AIN93-G. Chow was obtained from Labdiet (Prolab RMH3000 5P00) and the high fat diet was obtained from ENVIGO TD.06414, and from Research Diets Inc, D12492, modified to have 60% of total energy from fat. The fat content of these high fat diets comes from lard and soybean oil (31% and 3% of total content (g) of the diet respectively).
Mice were fed with the referred diets for at least 8 weeks from weaning, for the development of obesity and insulin resistance in mice fed with HFD.
Obese and insulin resistant mice were treated via orogastric route (gavage) with oil rich in DHA (1,000 mg in 1.5 mL) at 2 g/kg of body weight, which corresponded to 70 mg (100µL) of DHA, daily, for 3 weeks. The energy content of the DHA dose was negligible. DHA was obtained from General Nutrition Center (GNC) Inc., New Jersey, USA.
4.4 Antibodies and Reagents
Unless otherwise stated, all reagents and materials were obtained from Fisher Scientific (Ottawa, Canada; New Hampshire, USA). These include acrylamide, nitrocellulose membrane (0.22 or 0.45 µM), chemiluminescent reagents, protease and phosphatase inhibitors and TRIS buffer. Monoclonal antibodies (as outlined in the Methods) were obtained from Cell Signaling Technology (Boston, USA), Abcam (Cambridge, MA, USA) e Santa Cruz Biotechnology (Santa Cruz, CA)
4.5 Intraperitoneal Glucose Tolerance Test (iGTT), Insulin Tolerance Test
For the ITT, after 6 hours fasting, tail puncture was held for the first blood collection for basal dosage of glucose, which was equivalent to time zero (t0) of the test. Insulin (Humulin R U-100) (1 U/Kg of body weight) was injected via intraperitoneal (i.p.) and blood samples were collected from the tail at 15, 30, 45, 60 and 90 minutes to determine the blood glucose levels.
For the iGTT, after 6 hours fasting, tail puncture was held for the first blood collection for basal dosage of glucose, which was equivalent to time zero (t0) of the test. After this, 25% glucose solution (2 g/Kg of body weight) was injected intraperitoneally and blood glucose levels were measured after 15, 30, 60, 90 and 120 minutes, upon the injections.
For the PTT, after 12 h overnight fasting, tail puncture was held for the first blood collection for basal dosage of glucose, which was equivalent to time zero (t0) of the test. Next, a pyruvate solution (2 g/kg of body weight) was injected into the peritoneal cavity and blood glucose levels were measured after 15, 30, 60, 90 and 120 minutes upon the injections.
4.6 Tissue Extraction
On the last day of the experiment, after 6 hours of fasting, animals were anesthetized, euthanized and fragments of liver were collected for analysis of tissue content and gene expression of the proteins involved in Gαq/11 coupled signaling, in insulin signaling (Akt; FoxO1) and gluconeogenesis (PEPCK; G6p). The fragments of the liver were homogenized in lysis buffer (RIPA) containing protease and phosphatase inhibitors. Then, the homogenates were centrifuged for 20 minutes at 16,000 rcf, 4 °C. Protein concentrations of the samples were determined using the method of Bradford, Pierce (55) or DC Protein Assay (Bio-Rad), adapted from the method of Lowry (56).
For further analysis by qPCR, the tissue fragments were homogenized in specific buffer (RLT added with β-mercaptoethanol) from Qiagen RNasy Plus Mini Kit, according to the specificities of the method.
4.7 Immunoblot assays
Samples of total protein extract were loaded onto polyacrylamide gel for electrophoresis separation (SDS-PAGE). The separated proteins were transferred to nitrocellulose membrane in semi-dry transfer apparatus (Bio-Rad). After
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preincubation of the membrane with blocking solution to minimize nonspecific binding (10% skim milk powder, TRIS-saline buffer and 0.1% Tween 20) for 1 hour at room temperature and continuous stirring, the membranes were incubated overnight, under continuous agitation at 4 °C with specific primary antibody. On the following day, the membranes were incubated with the secondary antibody, relative to the primary, for 1 hour in continuous agitation at room temperature. The signal was detected by chemiluminescence with a specific kit (Super Signal West Dura Extended Duration Substrate (Thermo Scientific) and a VersaDoc or Chemidoc Touch Imaging System (Bio-Rad). The bands were quantified by optical densitometry by using the Bio-Rad Image Lab Software, version 6.0.1.
4.8 Real Time qPCR
RNA (1.0 μg) was reverse-transcribed using iScript RT Supermix (Bio-Rad) according to manufacturer’s instructions. Gene expression was determined using SYBR green or TaqMan Fast Advanced Master Mix real-time qPCR (RT-qPCR) according to the methods specificities. The steady-state mRNA levels of each gene of interest were determined by amplification of cDNA using specific primers or probes against the genes of interest and results were normalized to Gapdh or Rpl13a.
4.9 Statistical Analysis
The variables under study were submitted to the Komogorov-Smirnov test to verify the symmetry. For variables with normal distribution, the Analysis of Variance (ANOVA) was used to compare three independent samples. This test was complemented by Bonferroni multiple comparisons post-test when significant. For the comparison of two independent samples the T-student test was used. For all tests, the significance level was set at 5% (p <0.05).
5.0 RESULTS
5.1 Characterization of the experimental model
Generation of hepatic-specific condition knockout (cKO) (Gnaqfl/fl; Gna11-/-; Alb-Cre) and littermate control (Ctrl) (Gnaqfl/fl; Gna11-/-)mice
To determine the role of Gαq/11 in regulating hepatic glucose production, our goal was to selectively inactive Gnaq in the liver of Gna11 null mice by mating the Alb-Cre and Gnaqfl/fl; Gna11-/- mouse to each other. Both parental lines and the experimental littermate mice bearing the liver-specific conditional deletion of Gnaq are viable and fertile (47, 48). For almost 2 decades the Alb-Cre transgenic mice, having Cre driven by the serum-albumin (alb) gene promoter, have proven useful for studies involving Cre-dependent excision of lox-P flanked (“floxed” or “conditional”) in rodent hepatocytes (58, 59). In our study, qPCR and Western Blot analysis showed that Gαq subunit was not completely abolished but significantly diminished (p<0.05) in the liver of the experimental mice (Fig. 1.A, B, C). The phenomena observed can be related to the liver heterogeneity. Also, since the liver is highly perfused and Gαq/11 proteins are expressed in blood cells, the total recombination was not expected to occur in this tissue (59, 60). Importantly, it was demonstrated by Western Blot that the reduction of Gαq protein expression did not occur in other metabolic tissues but was restricted to the liver (Fig. 1.D). Interestingly, the GPR120 (Ffar4) gene expression is significantly lower in the liver of cKOmice compared to littermate Ctrl, for both chow and HFD treated groups (Fig. 1.E). This decrease in Ffar4 gene expression in the liver of cKO mice can be a consequence of the absence of Gα11 and conditional knockout of Gαq subunits, as the GPR120 signals, mainly, through these G proteins. We also observed that, Ctrl HFD mice showed an increased Gnaq expression (p<0.05) compared to Ctrl chow mice (Fig. 1.A).
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C. D.
E.
Figure 1. Molecular characterization of hepatic-specific conditional knockout (cKO) and littermate control (Ctrl) mice. (A) Quantitative Real-Time PCR (RT-qPCR) of Gnaq in
hepatic tissue of cKO and Ctrl mice fed with chow or HFD. (B, C) Gαq protein expression and
its quantification in the liver. (D) Gαq protein expression in other metabolic tissues:
hypothalamus (Hypot.), pancreas (Panc.), white adipose tissue (WAT) and muscle. (E) Ffar4 (GPR120) gene expression in the liver of cKO and Ctrl mice fed with chow or HFD. Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.
5.2 Behavioral parameters
Food intake and body weight
Food intake and body weight were measured weekly throughout the experimental period of 8 weeks.
cKO and Ctrl mice fed with chow or HFD exhibited similar food intake (Fig. 2. A). However, when compared to chow fed groups, HFD fed animals had lower food intake (Fig. 2.A). This could be explained by the energy content of each diet. Whereas the standard diet (chow) has 3.16 kcal/g, HFD has 5.1 kcal/g and since the HFD has a greater amount of fat (60% of energy) compared to chow (14.92% of
energy), it is expected that HFD fed mice gain more weight although they eat less food than chow fed mice.
No differences were seen in body weight (BW) between chow or HFD cKO mice and their respectively controls (Fig. 2.B, C) suggesting that the conditional knockout of Gαq in the liver does not interfere with weight gain. Meaningful
differences were seen between HFD cKO and chow cKO mice from the 5th week towards the end of the experimental period (Fig. 2.B). Also, the HFD fed groups gained significantly more weight compared to chow fed groups, confirming the development of obesity as a consequence of the HFD consumption (Fig. 2.C).
A. B.
C.
Figure 2. Food intake and body weight. (A) Food intake, (B) body weight and (C) weight
gain of Ctrl and cKO mice fed with chow or HFD for 8 weeks. HFD: 5.1kcal/gram; Chow: 3.16kcal/gram. Error bars represent SEM. *p<0.05; **p<0.01.
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5.3 Physiological and molecular parameters
Hepatic-specific conditional knockout of Gαq/11 improves gluconeogenesis in
mice fed with HFD for 8 weeks
To evaluate glucose tolerance, Ctrl and cKO mice fed with chow or HFD for 8 weeks were submitted to an intraperitoneal glucose tolerance test (iGTT) (Fig. 3.A). No statistical differences were observed in fasting glucose, neither in the area under the curve (AUC) between Ctrl and cKO groups after 8 weeks of chow or HFD treatment (Fig. 3.B, C). Meaningful differences were seen in fasting glucose between Ctrl mice fed with HFD compared to Ctrl mice fed with chow. Also, HFD Ctrl group exhibited glucose intolerance compared to chow Ctrl group, which was confirmed by the area under the curve (p<0.05) (Fig. 3.A, C). However, this significance was not observed between cKO chow and cKO HFD groups (Fig. 3.A, C).
Next, to access the hepatic glucose production, we performed a pyruvate tolerance test (Fig. 3.D). Regardless of the type of the diet, the conditional deletion of Gαq/11 in the liver did not influence the blood glucose levels after overnight fasting (12h) since no difference was observed in this parameter between Ctrl and cKO animals, but only when we compared HFD to chow fed groups (Fig. 3.E). After 8 weeks of HFD, Ctrl mice exhibited an increased glucose production in relation to Ctrl chow mice, which was demonstrated by the AUC (Fig. 3.F). However, no meaningful differences were seen in the AUC among cKO mice fed with chow and cKO fed with HFD (Fig. 3.F). Besides that, cKO HFD mice produced less glucose upon a pyruvate stimuli compared to HFD Ctrl mice (P<0.05) (Fig.3.F), which indicates that the hepatic-specific conditional knockout ofGαq/11 improves gluconeogenesis in mice fed with HFD for 8 weeks.
B. C.
D.
E. F.
Figure 3. Glucose homeostasis of Ctrl and cKO mice fed with chow or HFD for 8 weeks (A) Blood glucose levels during iGTT, (B) fasting glucose at t=0 of iGTT and (C) AUC of the
iGTT. (D) Blood glucose levels during the PTT, (E) fasting glucose at t=o of PTT and (F) AUC of PTT. Error bars represent SEM. *p<0.05.
Hepatic-specific conditional knockout of Gαq/11 increases FoxO1
phosphorylation (Ser256) in mice fed with HFD for 8 weeks
To investigate whether the liver-specific conditional lack of Gαq was able to improve insulin signaling, thereby diminishing hepatic gluconeogenesis, we analyzed
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the phosphorylation levels of Akt (Ser473) and FoxO1 (Ser256) in the liver of cKO and Ctrl mice fed with chow or HFD for 8 weeks. Western Blot analysis demonstrated that in mice fed with chow, the conditional knockout of Gαq/11 in the liver has no significant effect on Akt or FoxO1 phosphorylation levels (Fig. 4.A, B). However, under a HFD treatment for 8 weeks, though no change was observed on Akt activation, the hepatic conditional knockout of Gαq/11 increased FoxO1 (Ser256) phosphorylation (Fig. 4.C, D).
It´s well established that insulin signaling is required for the suppress of hepatic gluconeogenesis, mainly through the phosphorylation of FoxO1 in serine 256, which results in its extrusion of the cell nucleus followed by degradation by proteasome, thus suppressing its activity in transcribing gluconeogenic genes (27, 29). Therefore, taken together our findings suggest that the hepatic-specific conditional knockout of Gαq/11 positively interferes on glucose homeostasis by promoting a better control of gluconeogenesis, through the increase on FoxO1 (Ser256) phosphorylation levels in the liver of mice fed with HFD for 8 weeks.
A.
C.
D.
Figure 4. Insulin signaling in the liver of mice fed with chow or HFD for 8 weeks (A)
Western blot of Akt (Ser473) and FoxO1 (Ser256) phosphorylation in the liver of Ctrl and cKO mice fed with chow and (B) its respective quantifications. (C) Western blot of Akt (Ser473) and FoxO1 (Ser256) phosphorylation in the liver of Ctrl and cKO mice fed with HFD and (D) its respective quantifications. Error bars represent SEM. *p<0.05.
Hepatic-specific conditional knockout of Gαq/11 decreases
gluconeogenesis but does not restore insulin sensitivity in obese and diabetic mice
To address whether the conditional lack of Gαq/11 signaling in the liver would improve insulin sensitivity and therefore, glucose homeostasis, under obesity and T2DM conditions, cKO and its littermate controls were fed with HFD for 17 weeks. At the end of this period, metabolic tests such as PTT, iGTT and ITT were performed to evaluate hepatic glucose production (HPG) (gluconeogenesis), glucose tolerance and insulin sensitivity respectively.
We accessed gluconeogenesis by performing the PTT and we observed that after 12h overnight fasting, cKO mice exhibited significantly lower blood glucose level compared to Ctrl mice (Fig. 5.A, B). Additionally, cKO mice showed lower blood glucose levels throughout the PTT (Fig. 5.A), as well as smaller AUC (p<0.05) (Fig.
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5.C), which indicates that cKO mice produced less glucose upon a pyruvate stimuli, compared to Ctrl mice.
Then, to verify whether the conditional knockout of Gαq/11 in the liver could impact the systemic glucose homeostasis, we performed the iGTT and ITT. After 6h fasting, no differences were seen on blood glucose levels neither on the clearance of glucose throughout the iGTT, between Ctrl and cKO animals (Fig. 5.D, E). However, cKO mice showed diminished fasting glucose (p<0.05) previously the insulin stimuli (Fig. 5.F). Nevertheless, when we normalized the starting point, no difference was observed in the AUC among Ctrl and cKO mice (Fig. 5.G, H).
Overall, these results suggest that the conditional knockout of Gαq/11 in the liver promotes a better control of gluconeogenesis (HPG), but it does not restore peripheral insulin sensitivity in obese and diabetic mice, fed with HFD for 17 weeks.
A.
D. E.
F.
G. H.
Figure 5. Hepatic glucose production and glucose homeostasis of obese and diabetic Ctrl and cKO mice. (A) Overnight fasting (12h) glucose levels, (B) blood glucose levels
during the PTT and (C) AUC of PTT. (D) Fasting (6h) glucose levels at t=0 of iGTT and (F) of ITT (*p<0.05). (E) Blood glucose levels during iGTT and (G) ITT with normalized starting (t=0) point. (H) AUC of normalized ITT. Mice were fed with HFD for 17 weeks. Error bars represent SEM. *p<0.05.
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Omega-3 treatment has a transient beneficial effect on glycemic homeostasis in obese and diabetic cKO mice
Since it´s well know that ω-3 fatty-acids can exert beneficial effects on glucose homeostasis (31–35), we decided to investigate whether these nutrients could reverse the insulin resistance in Ctrl mice but not in cKO mice, due to the impairment of Gαq/11 coupled-signalingin the liver.
At the end of 17 weeks in HFD when the knockdown of hepatic Gαq/11 showed no effect on restoring glucose tolerance and insulin sensitivity, we decided to start the DHA treatment. Thus, Ctrl and cKO mice were daily supplemented via orogastric route (gavage) with oil rich in DHA at 2 g/kg of body weight, which corresponded to 70 mg/day of DHA for 3 weeks. After 1 week of DHA supplementation, animals were submitted to the iGTT, and it was demonstrated that the DHA treatment significantly improved the glucose tolerance in cKO mice (Fig. 6.A). No difference was observed in the fasting glucose level, however, cKO mice showed significantly lower AUC compared to Ctrl mice, which suggests that the cKO mice had a better clearance of glucose throughout the test (Fig. 6.B, C).
In subsequent studies, to understand whether the DHA treatment would improve glucose homeostasis within each genotype, we included water control groups in our experiments. Thus, Ctrl and cKO groups were splitted into 2 other groups based on the orogastric treatments: Ctrl and cKO groups that received water via gavage (referred to as “Ctrl water” and “cKO water”) and Ctrl and cKO groups that received DHA via gavage (referred to as “Ctrl DHA” and “cKO DHA”).
After 2 weeks of water / DHA treatment, Ctrl and cKO mice were submitted to iGTT (Fig. 6.D), and we saw that, the daily DHA supplementation for 3 weeks at 2 g/kg of BW, did not improve glucose tolerance in obese and diabetic Ctrl mice compared to Ctrl water mice (Fig. 6.D, F). Also, and surprisingly, it seemed that, despite the significant lower fasting glucose levels observed in cKO DHA mice (Fig. 6.E), the DHA treatment for a prolonged period of time, had no longer effect in improving glucose tolerance in cKO animals, since the cKO DHA group showed similar AUC as the cKO water group (Fig. 6.D, F). Additionally, both cKO water and cKO DHA groups exhibited similar glucose intolerance as the Ctrl water and Ctrl DHA groups, as it was demonstrated by the AUC of the iGTT (Fig. 6.F).
Besides the iGTT, we also conducted the PTT to investigate if the extended period of DHA treatment could impact the hepatic glucose production
(gluconeogenesis) (Fig. 6.G). Although the cKO DHA mice displayed lower blood glucose levels after 12 h fasting compared to cKO water mice (Fig. 6.H), no differences were observed in the AUC between Ctrl and cKO groups treated with water or DHA (Fig. 6.I).
This led us to conclude that, with the aggravation of obesity and T2DM, the hepatic conditional knockout of Gαq/11 has no longer positive impact on gluconeogenesis, as it was demonstrated when mice were fed with HFD for 8 weeks. In addition to that, the results from metabolic tests suggest that the DHA supplementation might have a transient beneficial effect on glucose homeostasis in cKO animals under obesity and T2DM conditions.
A.
38 D. E. F. G. H. I.
Figure 6. Glucose tolerance and gluconeogenesis in obese and diabetic Ctrl and cKO mice supplemented with DHA. (A) Blood glucose levels, (B) fasting glucose and (C) AUC
of iGTT in Ctrl and cKO mice treated with DHA (2 g/kg of body weight) via orogastric route (gavage) for 1 week. (D) Blood glucose levels, (E) fasting glucose and (F) AUC of iGTT in Ctrl and cKO mice treated with water or DHA via gavages for 3 weeks. (G) Blood glucose levels upon pyruvate bolus, (H) overnight fasting glucose and (I) AUC of PTT in Ctrl and cKO mice treated with water or DHA via gavages for 3 weeks. Error bars represent SEM. *p<0.05.
Next, we aimed to investigate the impact of DHA supplementation on the hepatic expression of the ω-3 receptor, GPR120 (Ffar4) and its subsequent protein that also mediates the ω-3 effects, β-arrestin2 (Arr2b). Additionally, we investigated the impact of DHA treatment on the activation of insulin signaling proteins (Akt and FoxO1) as well as on gluconeogenesis genes (Pck1, G6pc). Before we start our analysis on these genes/proteins, we firstly confirmed that the Gαq protein expression was successfully diminished in the liver of cKO compared to Ctrl mice, regardless the orogastric treatments (Fig. 7.A, B). Interestingly, the ω-3 treatment significantly increased the Ffar4 and the Arrb2 gene expression in the liver of Ctrl mice (Fig. 7.C, D). No differences were observed for these genes between cKO groups (Fig. 7.C, D). On the other hand, the Ffar4 hepatic expression was down-regulated in cKO DHA compared to Ctrl DHA animals while no differences were observed between cKO and Ctrl water treated animals (Fig. 7.C).
40
C. D.
Figure 7. Gnaq (Gαq) and Ffar4 (GPR120) expression in the liver of obese and diabetic Ctrl and cKO mice treated with water or DHA. (A - B) Western blot of Gαq and its
respective quantificationsin the liver of Ctrl and cKO mice treated with water or DHA via gavages for 3 weeks. (C) RT-qPCR of Ffar4 and of (D) Arrb2 in the liver of Ctrl and cKO mice treated with water or DHA via gavages for 3 weeks. Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.
We next evaluated the phosphorylation levels of Akt (Ser473) and FoxO1 (Ser256) and the gene expression of gluconeogenic enzymes, Pck1 and G6pc. For this end, in the day of the experiment, after 6 h fasting, obese and diabetic Ctrl and cKO mice treated with water or DHA for 3 weeks, were injected (intraperitoneally – ip) with PBS (phosphate-buffered saline) or insulin (1 U/kg/BW) and fragments of liver were collected, 10 minutes upon the stimuli, for subsequent analysis of Western blot and RT-qPCR.
When we compared Ctrl and cKO groups, submitted to the same orogastric treatment (water or DHA), we only observed an increase on Akt phosphorylation (Ser473) in the liver of mice under insulin stimuli in relation to Ctrl and cKO mice that received PBS ip (Fig. 8.A – D). Also, independently of the orogastric treatment, no differences were observed on FoxO1 (Ser256) phosphorylation levels among Ctrl and cKO, even in the liver of mice that were stimulated with insulin via ip (Fig. 8.A - B; E - F).
A. B.
C. D.
E. F.
Figure 8. Phosphorylation levels of insulin signaling proteins in the liver of obese and diabetic Ctrl and cKO mice submitted to orogastric treatments. (A) Western blot of p-Akt
(Ser473) and p-FoxO1 (Ser256) in the liver of Ctrl and cKO mice that were orally treated with water and (B) DHA for 3 weeks and its respective quantifications: (C – D) p-Akt / Akt and (E
– F) p-FoxO1 / FoxO1. Ctrl and cKO animals were stimulated with PBS or insulin (1
U/kg/BW) via intraperitoneal and fragments of liver were collected 10 minutes upon the stimuli. Error bars represent SEM. *p<0.05.
It was also important to determine whether the DHA supplementation could impact on the activation of Akt and FoxO1, as well as on the expression of gluconeogenic enzymes such as Pck1 and G6pc, within the genotypes.
No differences were seen on p-Akt (Ser473) within the genotypes when we compared water to DHA treated animals. We only saw enhanced (p<0.05) p-Akt