Obesity and diabetes are major public health problems facing the world today. Extending our understanding of adipose tissue biology, and how it changes in obesity, will hopefully better equip our society in dealing with the obesity epidemic. Macrophages and other immune cells accumulate in the adipose tissue in obesity and secrete cytokines that can promote insulin resistance. Adipose tissue macrophages (ATMs) are thought to originate from bone marrow-derived monocytes, which infiltrate the tissue from the circulation. Much work has been done to demonstrate that inhibition of monocyte recruitment to the adipose tissue can ameliorate insulin resistance. While monocytes can enter the adipose tissue, we have shown here that local macrophage proliferation may be the predominant mechanism by which macrophages self-renew in the adipose tissue. We demonstrated that two cell proliferation markers, Ki67 and EdU, can be readily detected in macrophages isolated from adipose tissue of both lean and obese mice. These analyses revealed that 2-4% of ATMs in lean and 10-20% of ATMs in obese mice express the proliferation marker Ki67. Importantly, Ki67+ macrophages were identified within the adipose tissue in crown-like structures. Similarly, a 3-hour in vivo pulse with the thymidine analog EdU showed that nearly 5% of macrophages in epididymal adipose tissue of ob/ob mice were in the S-phase of cell division. Interestingly, obesity increased the rate of macrophage proliferation in adipose tissue but did not affect macrophage proliferation in other tissues. We also used clodronate liposomes to deplete circulating monocytes in obese mice. Surprisingly, monocyte depletion for a total of at least 80 hours did not cause a decrease in ATM content in adipose tissue. Prolonged exposure of mice to EdU in drinking water revealed that approximately half of the ATMs in the epididymal fat pads of ob/ob mice had proliferated locally within 80 hours. Amazingly, these rates were the same with or without monocyte depletion, meaning that the proliferating cells were not freshly recruited monocytes. Overall, these results suggest that local proliferation unexpectedly makes a major contribution to maintaining the large population of macrophages present in the obese adipose tissue in the steady state. This suggests that increased rates of local macrophage proliferation may also be partly responsible for the massive increase in ATM content that occurs in obesity. This information could have implications for future therapeutic strategies in the management of diabetes.

The worldwide prevalence of obesity and metabolic disease is increasing at an exponential rate and current projections provide no indication of relief. This growing burden of obesity-related metabolic disorders, including type 2 diabetes mellitus (T2DM), highlights the importance of identifying how lifestyle choices, genetics and physiology play a role in metabolic disease and place obese individuals at a greater risk for obesity-related complications including insulin resistance (IR). This increased risk of IR, which is characterized by a decreased response to insulin in peripheral tissues including adipose tissue (AT) and liver, is associated with a chronic, low grade inflammatory state; however, the causative connections between obesity and inflammation remains in question. Experimental evidence suggests that adipocytes and macrophages can profoundly influence obesity-induced IR because adipocyte dysfunction leads to ectopic lipid deposition in peripheral insulin sensitive tissues, and obese AT is characterized by increased local inflammation and macrophage and other immune cell populations. Attempts to delineate the individual roles of macrophage-derived pro-inflammatory cytokines, like tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), have demonstrated causative roles in impaired systemic insulin sensitivity, adipocyte function and hepatic glucose and lipid metabolism in obese animal models. Thus, the attenuation of macrophage-derived inflammation is an evolving area of interest to provide insight into the underlying mechanism(s) leading to obesity-induced IR. Thus, in the first chapter of this thesis, I describe experiments to refine the current paradigm of obesity-induced AT inflammation by combining gene expression profiling with computational analysis of two anatomically distinct AT depots, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) to address whether the inflammatory signature of AT is influenced by diet-induced obesity (DIO). Microarray and qRT-PCR analysis data revealed that DIO mouse SAT is resistant to high fat diet (HFD)-induced inflammation and macrophage infiltration, and our data support the current model of obesity-induced visceral adipose tissue macrophage (VATM) enrichment. Our data demonstrated robust increases in VAT pro-inflammatory cytokine expression, which are consistent with the significant increases in macrophage-specific gene expression and consistent with previous reports in which VAT inflammation is enhanced and attributed to classically activated (M1) macrophage infiltration. However, these data are only observed relative to the expression of invariant housekeeping gene expression. When M1-specific genes are expressed relative to macrophage-specific standards like F4/80 expression, these inflammatory makers are unchanged. These data indicate that the changes in the overall inflammatory profile of DIO mouse VAT is because of quantitative changes in adipose tissue macrophage (ATM) number and not qualitative changes in activation state. These observations are consistent with the idea that infiltrating ATMs may have roles other than the previously described role in mediating inflammation in obese adipose tissue. Hepatic IR occurs partly as a consequence of adipocyte dysfunction because the liver becomes a reservoir for AT-derived fatty acids (FAs), which leads to obesity-related non-alcoholic fatty liver disease (NAFLD). In the second part of my thesis, I used clodronate liposome-mediated macrophage depletion to define the role of macrophages in hepatic lipid metabolism regulation. We discovered that i.p. administration of clodronate liposomes depletes Kupffer cells (KCs) in ob/ob mice without affecting VATM content, whereas clodronate liposomes depletes both KCs and VATMs in DIO mice. To this end, we established that clodronate liposome-mediated KC depletion, regardless of VATM content in obese mice, abrogated hepatic steatosis by reducing hepatic de novo lipogenic gene expression. The observed reductions in hepatic inflammation in macrophage-depleted obese mice led to the hypothesis that IL-1β may be responsible for obesity-induced increased hepatic triglyceride (TG) accumulation. We determined that IL-1β treatment increases fatty acid synthase (Fas) protein expression and TG accumulation in primary mouse hepatocytes. Pharmacological inhibition of interleukin-1 (IL-1) signaling by interleukin-1 receptor antagonist (IL-1Ra) administration recapitulated these results by reducing hepatic TG accumulation and lipogenic gene expression in DIO mice. Thus, these data highlight the importance of the inflammatory cytokine IL-1β in obesity-driven hepatic steatosis and suggests that liver inflammation controls hepatic lipogenesis in obesity. To this end, the studies described herein provide new insight and appreciation to the multi-functional nature of macrophages and clinical implications for anti-inflammatory therapy in obesity and NAFLD treatment. We demonstrate the complexities of macrophage-mediated functions in insulin sensitive tissues and a role for obesity-induced inflammatory cytokine IL-1β in hepatic lipid metabolism modulation, which is reversed via IL-1Ra intervention. The use of anti-inflammatory therapy to ameliorate obesity-associated NAFLD was perhaps the most important contribution to this body of work and is full of promise for future clinical application. It is likely that the future of therapeutics will be multi-faceted and combine therapeutic approaches to enhance glucose tolerance and overall health in obese, IR and T2DM patients.

<p>Uvod: Febrilna neutropenija, kao prvi znak infekcije, je česta komplikacija u fazi postterapijske aplazije kostne srži u obolelih od akutne nelimfoblastne leukemije. Klinička slika febrilne neutropenije može biti suptilna, a progresija u stanje septičnog &scaron;oka znatno brža nego kod imunokompetentnih bolesnika. Rana predikcija rizika od komplikacija febrilne neutropenije i uvođenje empirijske antibiotske terapije može da pobolj&scaron;a prognozu bolesnika. Insulinska rezistencija, dislipidemija i inflamacija masnog tkiva se javljaju u sklopu sistemske inflamacije. Njihova uloga i značaj kao potencijalnih faktora predikcije toka i ishoda febrilne neutropenije nisu ispitani. Ciljevi istraživanja: Ustanoviti promene pokazatelja stepena insulinske senzitivnosti, ukupnog holesterola, triglicerida, HDL - holesterola, LDL - holesterola, apolipoproteina A-I, lipoproteina (a) i adiponektina pre i u fazi febrilne neutropenije kod bolesnika sa akutnom nelimfoblastnom leukemijom. Uporediti vrednosti pokazatelja stepena insulinske senzitivnosti, ukupnog holesterola, triglicerida, HDL - holesterola, LDL - holesterola, apolipoproteina A-I, lipoproteina (a) i adiponektina bolesnika sa akutnom nelimfoblastnom leukemijom pre početka febrilne neutropenije i kontrolne grupe gojaznih. Uporediti vrednosti pokazatelja stepena insulinske senzitivnosti, ukupnog holesterola, triglicerida, HDL - holesterola, LDL - holesterola, apolipoproteina A-I, lipoproteina (a) i adiponektina bolesnika sa akutnom nelimfoblastnom leukemijom u fazi febrilne neutropenije i kontrolne grupe gojaznih. Utvrditi da li su pokazatelj stepena insulinske senzitivnosti, ukupni serumski holesterol, trigliceridi, HDL - holesterol, LDL - holesterol, apolipoprotein A-I, lipoprotein (a) i adiponektin bolesnika sa akutnom nelimfoblastnom leukemijom u fazi febrilne neutropenije u korelaciji sa vrednostima parametara inflamacije, njenim tokom i ishodom. Materijal i metode: Istraživanje je sprovedeno u Klinici za hematologiju i Klinici za endokrinologiju, dijabetes i bolesti metabolizma. Obuhvatilo je 60 ispitanika, od kojih je 30 ispitanika obolelo od akutne nelimfoblastne leukemije, a 30 ispitanika je činilo kontrolnu grupu gojaznih. Nakon uključivanja u istraživanje, ispitanicima su urađeni predviđeni pregledi i laboratorijske analize u cilju procene insulinske senzitivnosti, metaboličkog statusa i serumskog adiponektina. Navedena merenja su urađena pre hemioterapije i u febrilnoj neutropeniji. Zdravstveno stanje ispitanika je praćeno do kraja prve hospitalizacije. Statistička obrada je izvr&scaron;ena uz pomoć statističkog paketa Statistica. Podaci su predstavljeni tabelarno i grafički, a statistička značajnost je odreĎivana na nivou p &lt; 0.05. Rezultati: U febrilnoj neutropeniji bolesnika sa akutnom leukemijom je do&scaron;lo do razvoja insulinske rezistencije (t = - 2.43, p = 0.021), dislipidemije sa značajnim sniţenjem ukupnog holesterola (t = 3.59, p = 0.0012), LDL &ndash; holesterola (t = 3.56, p = 0.0013) i apoA &ndash; I (t = 2.27, p = 0.03). Oboleli od akutne nelimfoblastne leukemije u febrilnoj neutropeniji su razvili metaboličke promene viđene kod gojaznih osoba sa insulinskom rezistencijom. Nastanak i progresija insulinske rezistencije je bila u pozitivnoj korelaciji sa fibrinogenom kao pokazateljem težine inflamacije (r = 0.59, p &lt; 0.05) dok je apoA - I negativno korelirao sa CRP (r = - 0.37, p &lt; 0.05). Ispitanici sa nižom insulinemijom i vrednostima HDL - holesterola pre hemoterapije su imali značajno bolji tok febrilne neutropenije (t = -2.38, p = 0.024 vs. t = - 2.87, p = 0.007). Ispitanici sa većim indeksom telesne mase (BMI) i obimom struka imali su povoljniji ishod febrilne neutropenije (r = - 0.47, p &lt; 0.05 vs. r = - 0.40, p &lt; 0.05). Drugi pokazatelji insulinske senzitivnosti, metaboličkog statusa i adiponektin nisu značajno uticali na tok i ishod febrilne neutropenije. Normalna telesna masa pre hemioterapije, a u febrilnoj neutropeniji temperatura u trajanju dužem od 7 dana, niže vrednosti MASCC indeksa rizika, vi&scaron;e vrednosti CRP, vi&scaron;e vrednosti adiponektina, niže vrednosti Lp(a) i komplikovan tok febrilne neutropenije su bili prediktori lo&scaron;ije prognoze febrilne neutropenije. Zaključak: Pored klasičnih hematolo&scaron;kih parametara potrebno je uzeti u obzir antropometrijske karakteristike, redistribuciju masne mase, disfunkcionalnost masne mase, insulinsku rezistenciju i metaboličke parametre u cilju praćenja i predviđanja mogućih komplikacija i komorbiditeta.</p> / <p>Introduction: Febrile neutropenia is a common complication in posttreatment aplasia in patients with acute nonlymphoblastic leukemia. Its clinical manifestation can be subtle. However, it can progress to septic shock more quickly than in immunocompetent patients. Early prediction of complications and recognition of risk factors can improve outcome. Systemic inflammation is characterized by insulin resistance, dyslipidemia and adipocyte dysfunction. However, their importance in predicting complications and outcome of febrile neutropenia is not entirely known.<br />Aims: To determine changes in HOMA-IR, total cholesterol, triglycerides, HDL - cholesterol, LDL - cholesterol, apolipoprotein A-I, lipoprotein (a) and adiponectin in patients before chemotherapy and during febrile neutropenia. To compare HOMA-IR, total cholesterol, triglycerides, HDL - cholesterol, LDL - cholesterol, apolipoprotein A-I, lipoprotein (a) and adiponectin in patients before chemotherapy and the obese. To compare HOMA-IR, total cholesterol, triglycerides, HDL - cholesterol, LDL - cholesterol, apolipoprotein A-I, lipoprotein (a) and adiponectin in patients during febrile neutropenia and the obese. To determine whether HOMA-IR, total cholesterol, triglycerides, HDL - cholesterol, LDL - cholesterol, apolipoprotein A-I, lipoprotein (a) and adiponectin in febrile neutropenia are in correlation with the severity of the infection, appearance of complications and outcome. Materials and methods: The study was conducted at the Clinic for hematology and Clinic for endocrinology, diabetes, and metabolic disorders. 60 patients who fulfilled the inclusion criteria were included in the study. 30 patients had acute leukemia, and 30 were obese. Clinical and laboratory examination to assess insulin sensitivity, metabolic disorders and adiponectin was done before chemotherapy and during febrile neutropenia. Patients were followed up until the end of the first hospitalization. Data were analyzed with Statistica software and presented in tables and graphs. Statistical significance was set at p&lt;0.05. Results: During febrile neutropenia, patients with acute leukemia developed insulin resistance (t = - 2.43, p = 0.021), alongside significant decline of total cholesterol (t = 3.59, p = 0.0012), LDL &ndash; cholesterol (t = 3.56, p = 0.0013) and apoA &ndash; I (t = 2.27, p = 0.03). In acute inflammation, metabolic changes in patients with acute leukemia resembled those in the obese with insulin resistance. HOMA-IR values were in positive correlation with fibrinogen (r = 0.59, p &lt; 0.05) whereas apoA-I was in negative correlation to CRP (r = - 0.37, p &lt; 0.05). Patients with higher body mass index and waist circumference had better course and outcome of febrile neutropenia (r = - 0.47, p &lt; 0.05 vs. r = - 0.40, p &lt; 0.05). Patients with lower insulin levels and HDL - cholesterol prior to chemotherapy had a significantly better course of febrile neutropenia (t = -2.38, p = 0.024 vs. t = - 2.87, p = 0.007). Other parameters of insulin sensitivity, metabolic status, and adiponectin did not influence the course and outcome of inflammation significantly. Normal body weight, duration of febrile neutropenia for longer than 7 days, lower MASCC risk index, higher CRP and adiponectin, low Lp(a) in febrile neutropenia and a complicated course od febrile neutropenia were predictors of a worse outcome. Conclusion: Besides known hematological risk factors for complications in febrile neutropenia, anthropometric characteristics, fat mass distribution and disfunction, insulin resistance and metabolic parameters are useful predictors of the course and outcome of febrile neutropenia.</p>

In the late 90’s, emerging evidence revealed that the brain is insulin-sensitive, highlighted by broad expression of brain-specific insulin receptors and reports of circulating brain insulin. Contemporary literature robustly supports the role of insulin signaling in normal brain function and suggests that insulin-related processes diminish with aging, evidenced by decreased signaling markers, reduced insulin receptor density, and lower levels of insulin transport across the blood-brain barrier. In the context of pathological cognitive decline, clinical trials using intranasal insulin delivery have reported positive outcomes on memory and learning in patients with mild cognitive decline or early-stage Alzheimer’s disease. However, while the importance of insulin and its related actions in the brain are robustly supported, the distinct mechanisms and pathways that mediate these effects remain unclear. To address this, I conducted a series of experiments exploring the impact of insulin on memory and learning in two models: primary hippocampal cell cultures and the Fisher 344 animal model of aging. These studies attempted to identify relationships between insulin receptor signaling, neuronal gene expression, glucose metabolism, and calcium homeostasis in the hippocampus using either expression of a constitutively active human insulin receptor or administration of intranasal insulin. The following dissertation summarizes this work and provides valuable insights into the potential pathways mediating these relationships. Of note, intranasal studies reported that insulin is able to significantly alter gene expression patterns in the hippocampus of both young and aged rats following chronic, repeated exposure to the ligand. In cell culture, constitutive insulin signaling correlated with significantly elevated neuronal glucose uptake and utilization, as well as with significant alterations in the overall expression and localization of the neuron-specific glucose transporter 3. Interestingly, continued activity of the insulin receptor did not appear to alter voltage-gated calcium channels in hippocampal neurons despite prior evidence of the ligand’s role in other calcium-related processes. The results reported in this manuscript suggest that in the brain, insulin may be involved in a myriad of complex and dynamic events dependent on numerous variables, such as age, length of the exposure, and/or the insulin formulation used. Nevertheless, this work highlights the validity of using insulin to ameliorate age-related cognitive decline and supports the need for further studies exploring alternative approaches to enhance insulin receptor signaling in the brain.

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Les maladies métaboliques ont pris une ampleur considérable dans le monde ces dernières décennies, telle que certains parlent à ce jour de pandémie. Le diabète de type 2 est l’une de celles qui progressent avec la plus importante prévalence. L'un des facteurs à l’origine du développement de cette physiopathologie est l’insulino-résistance. Il s'agit d'une altération de la réponse à l’insuline des tissus cibles tels que le muscle squelettique, le tissu adipeux et le foie, induisant une dérégulation de l'homéostasie du glucose. Les tissus sensibles deviennent incapables, entre autres, d'absorber adéquatement le glucose sanguin conduisant ainsi à l’établissement d’une hyperglycémie chronique. Les travaux présentés dans cette thèse ont pour objectif de caractériser le rôle de la protéine Growth-arrest specific 6 (GAS6) dans la dérégulation de l’équilibre glycémique et le développement de la résistance à l’insuline. GAS6 est une protéine γ-carboxylée sécrétée qui agit comme ligand pour la famille des récepteurs tyrosines kinases TAM comprenant : TYRO3, AXL et MERTK. GAS6 et ses récepteurs jouent un rôle essentiel dans le système immunitaire, la progression tumorale et les métastases cancéreuses. Cependant, des études récentes menées chez l’humain ont montré que les niveaux circulants de GAS6 ou des variations dans le gène GAS6 sont associés à l’hyperglycémie, la résistance à l'insuline et le risque de développer le diabète de type 2. Cependant, le mécanisme par lequel GAS6 influence ces désordres métaboliques reste méconnu. Dans une première étude, nous avons évalué, pour la première fois dans une cohorte de femmes canadiennes, la corrélation éventuelle entre les niveaux circulants de GAS6 et des facteurs de risque liés au diabète. Cette cohorte nommée MONET (Montréal and Ottawa New Emerging Team) est constituée de 126 femmes post-ménopausées, en surpoids ou obèses. Ces femmes ne sont pas diabétiques, mais présentent un risque plus élevé de développer la maladie à cause de leur poids et de leur statut sédentaire. Nous avons constaté que les femmes ayant des taux élevés de GAS6 dans le sang ont une tolérance au glucose significativement plus faible que celles avec des niveaux plus faibles de GAS6. Par ailleurs, certains paramètres de dysfonctionnements hépatiques (AST, ALT) et des marqueurs d’inflammation (IL-6) concordent positivement avec des taux élevés de GAS6. Nos résultats suggèrent que GAS6 pourrait être un biomarqueur de l’intolérance au glucose chez des patientes obèses et qu’il pourrait être associé à l’inflammation et à certains problèmes hépatiques, qui sont des facteurs impliqués dans le développement du diabète. Dans une seconde étude, à l’aide de modèles murins modifiés génétiquement, nous avons pu démontrer que la délétion du gène Gas6 est suffisante pour améliorer la sensibilité à l’insuline et la tolérance au glucose, sans affecter la sécrétion d’insuline. Par ailleurs, les souris déficientes pour GAS6 sont protégées contre la résistance à l'insuline induite par un régime alimentaire riche en graisses et en sucres. À l’inverse, l'augmentation in vivo des taux circulants de GAS6 est suffisante pour réduire la sensibilité à l'insuline. L'analyse de l'expression génique des récepteurs TAM dans les tissus sensibles à l’insuline a révélé qu’Axl est fortement exprimé dans le muscle squelettique. Dans une lignée de cellules musculaires, nous avons démontré que la voie de signalisation de GAS6-AXL affecte la réponse à l'insuline en inhibant la phosphorylation du récepteur de l'insuline (RI) et de son effecteur en aval AKT. Mécaniquement, AXL s'hétérodimérise avec le RI et GAS6 reprogramme les voies de signalisation en aval du RI dans les cellules musculaires. Il en résulte une activation accrue de la voie des Rab, notamment Rab7 induisant une internalisation de RI. Ensemble, ces résultats décrivent le mécanisme cellulaire par lequel GAS6 et AXL influencent la sensibilité à l'insuline. Finalement, nos derniers résultats soulignent un autre mécanisme d’action de GAS6 sur le métabolisme des cellules musculaires. Nous avons démontré, par protéomique, que GAS6 augmente significativement les niveaux protéiques de plusieurs enzymes impliquées dans la glycolyse et la production de lactate. Le profil métabolique des cellules musculaires traitées avec GAS6 démontre une augmentation du niveau de la glycolyse anaérobique et de la production de lactate. Par ailleurs, nos résultats suggèrent que le lactate lui-même induit une inhibition de la phosphorylation du RI en réponse à l’insuline. Ainsi, GAS6, en reprogrammant les voies métaboliques et l’utilisation du glucose des cellules musculaires, favoriserait la production de lactate induisant une diminution de la sensibilité à l'insuline. / Metabolic diseases have taken on a considerable scale in the world in recent decades, such that some speak of a pandemic. Type 2 diabetes is one of those diseases that progress with the highest prevalence. One of the factors behind the development of this pathophysiology is insulin resistance. It is an alteration of the insulin response of targeted tissues such as skeletal muscle, adipose tissue and liver, inducing dysregulation of glucose homeostasis. Sensitive tissues become incapable, among other things, of adequately absorbing blood glucose, thus leading to the establishment of chronic hyperglycemia. The work presented in this thesis focuses on characterizing the role of Growth-arrest specific protein 6 (GAS6) in the dysregulation of glycemic balance and the development of insulin resistance. GAS6 is a secreted γ-carboxylated protein that acts as a ligand for the TAM family of receptor tyrosine kinases including: TYRO3, AXL and MERTK. GAS6 and its receptors play an essential role in the immune system, tumor progression and cancer metastasis. However, recent studies in humans have shown that circulating GAS6 levels or variations in GAS6 gene are associated with hyperglycemia, insulin resistance and the risk of developing type 2 diabetes. However, the mechanism by which GAS6 influences these metabolic disorders remains unknown. In a first study, carried out for the first time in a cohort of Canadian women, we evaluated the potential correlation between circulating GAS6 levels and risk factors linked to diabetes. This cohort, named MONET (Montreal and Ottawa New Emerging Team), is composed of 126 post-menopausal, overweight or obese women. These women are not diabetic but have high risks of developing the disease because of their weight and sedentary status. We found that women with high levels of GAS6 in the blood have significantly lower glucose tolerance than those with lower levels of GAS6. In addition, certain liver dysfunction parameters (AST, ALT) and inflammation markers (IL-6) positively correlated with high levels of GAS6. Our results suggest that GAS6 could be a biomarker of glucose intolerance in obese patients and be associated with inflammation and certain liver problems, which are factors involved in the development of diabetes.  In a second study, using genetically modified mouse models, we were able to demonstrate that deletion of the Gas6 gene was sufficient to improve insulin sensitivity and glucose tolerance, without affecting insulin secretion. Furthermore, GAS6-deficient mice were protected against insulin resistance induced by a diet high in fats and sugars. Conversely, in vivo, increase of GAS6 circulating levels is sufficient to reduce insulin sensitivity. Analysis of TAM receptors gene expression in insulin-responsive tissues revealed that Axl is highly expressed in skeletal muscle. In a muscle cell line, we demonstrated that the GAS6-AXL signaling pathway affects the insulin response by inhibiting the phosphorylation of the insulin receptor (IR) and its downstream effector AKT. Mechanistically, AXL heterodimerizes with IR and GAS6 reprograms signaling pathways downstream of IR in muscle cells. This results in an increased activation of the Rab pathway, in particular Rab7, inducing an internalization of IR. Together, these results describe the cellular mechanism by which GAS6 and AXL influence insulin sensitivity. Finally, our latest results highlight another mechanism of action of GAS6 on muscle cell metabolism. We demonstrated by proteomics that GAS6 significantly increases the protein levels of several enzymes involved in glycolysis and lactate production. The metabolic profile of muscle cells treated with GAS6 demonstrates an increase in the level of anaerobic glycolysis and lactate production. Furthermore, our results suggest that lactate itself induces an inhibition of IR phosphorylation in response to insulin. Thus, GAS6, by reprogramming the metabolic pathways and the use of glucose in muscle cells, would promote lactate production inducing a decrease in insulin sensitivity.

Genome-wide association studies have identified adenylyl cyclase type 5 (ADCY5) as candidate gene for diabetes-related quantitative traits and an increased risk of type 2 diabetes. Mice with a whole-body deletion of Adcy5 (Adcy5–/–) do not develop obesity, glucose intolerance and insulin resistance, have improved cardiac function and increased longevity. Here, we investigated Adcy5 knockout mice (Adcy5–/–) to test the hypothesis that changes in adipose tissue (AT) may contribute to the reported healthier phenotype. In contrast to previous reports, we found that deletion of Adcy5 did not confer any physiological or biochemical benefits. However, this unexpected finding allowed us to investigate the effects of Adcy5 depletion on AT independently of lower body weight and a metabolically healthier phenotype. Adcy5–/– mice exhibited an increased number of smaller adipocytes, lower mean adipocyte size and a distinct AT gene expression pattern with midline 1 (Mid1) as the most significantly downregulated gene compared to control mice. Our Adcy5–/– model challenges previously described beneficial effects of Adcy5 deficiency and suggests that targeting Adcy5 does not improve insulin sensitivity and may therefore limit the relevance of ADCY5 as potential drug target.

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