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Obesity-induced diabetes affects over 400 million people worldwide. Obesity is a complex metabolic disease and is associated with several co-morbidities, all of which negatively affect the individual’s quality of life. It is commonly considered that obesity is a result of a positive energy misbalance, as increased food intake and lower expenditure eventually lead to the development of this disease. Moreover, the pathology of obesity is attributed to several genetic and epigenetic factors that put an individual at high risk compared to another. Adipose tissue is the main site of the organism’s energy storage. During the time when the nutrients are available in excess, adipocytes acquire triglycerides, which are released during the time of food deprivation in the process of lipolysis (free fatty acids and glycerol released from adipocytes). Uncontrolled lipolysis is the consequent event that contributes to the development of diabetes and paradoxically obesity. To identify the genetic factors aiming for future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the Extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrate that β-adrenergic stimulation stabilizes ERK3 leading to the formation of a complex with the co-factor MAP kinase-activated protein kinase 5 (MK5) thereby driving lipolysis. Mechanistically, we identify a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Moreover, we shed the light on our pharmacological approach in targeting ERK3/MK5 pathways using MK5 specific inhibitor. Already after 1 week of administering the inhibitor, mice showed signs of improvement of their metabolic fitness as showed here by a reduction in induced lipolysis and the elevation in the expression of thermogenic genes. Taken together, our data suggest that targeting the ERK3/MK5 pathway, a previously unrecognized signaling axis in adipose tissue, could be an attractive target for future therapies aiming to combat obesity-induced diabetes. / Adipositas-induzierter Diabetes betrifft weltweit über 400 Millionen Menschen. Adipositas ist eine komplexe Stoffwechselerkrankung und geht mit mehreren Komorbiditäten einher, die sich alle negativ auf die Lebensqualität der Betroffenen auswirken. Es wird generell angenommen, dass Adipositas aus einem positiven Energieungleichgewicht resultiert, da eine erhöhte Nahrungsaufnahme und ein geringerer Verbrauch zu der Ausbildung dieser Krankheit führen. Darüber hinaus ist die Pathologie von Adipositas auf mehrere genetische und epigenetische Faktoren zurückzuführen, wodurch Individuen einem erhöhtem Risiko ausgesetzt sein können. Das Fettgewebe ist der vorwiegende Energiespeicher des Organismus. In Zeiten eines Nährstoffüberschusses speichern Adipozyten Triglyceride, die im Falle eines Nahrungsmangels durch den Prozess der Lipolyse in Form von freien Fettsäuren und Glycerin freigesetzt werden. Unkontrollierte Lipolyse ist ein Folgeereignis, welches zur Entwicklung von Diabetes und paradoxerweise zu Adipositas beiträgt. Um die genetischen Faktoren zu identifizieren, die in Zukunft therapeutische Angriffspunkte darstellen könnten, haben wir ein Hochdurchsatz-Screening durchgeführt und die extrazellulär regulierte Kinase 3 (ERK3) als Treffer identifiziert. Wir zeigen, dass β-adrenerge Stimulation ERK3 stabilisiert, was zur Bildung eines Komplexes mit dem Cofactor MAP-Kinase-aktivierte Proteinkinase 5 (MK5) führt und dadurch die Lipolyse vorantreibt. Mechanistisch identifizieren wir den Transkriptionsfaktor FOXO1, der dem ERK3/MK5-Signalweg nachgeschaltet ist und die Expression des wichtigsten lipolytischen Enzyms ATGL fördert. Darüber hinaus belegen wir, dass die gezielte Deletion von ERK3 in Maus-Adipozyten die Lipolyse hemmt, aber die Energiedissipation erhöht, den mageren Phänotyp fördert und Diabetes lindert. Außerdem nutzen wir einen pharmakologischen Ansatz durch Verwendung eines MK5 spezifischen Inhibitors, um auf den ERK3/MK5-Signalweg abzuzielen. Bereits eine Woche nach Verabreichung des Inhibitors zeigen Mäuse Anzeichen einer verbesserten metabolischen Fitness, die sich durch einer Verringerung der induzierten Lipolyse und eine verstärkte Expression von thermogenen Genen auszeichnet. Zusammenfassend legen unsere Daten nahe, dass der ERK3/MK5-Signalweg, eine zuvor nicht erkannte Signalachse im Fettgewebe, ein attraktiver Ansatzpunkt für zukünftige Therapien zur Bekämpfung von Adipositas-induziertem Diabetes sein könnte.

Protein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in pancreatic cancers (e.g., islet cell carcinoma and pancreatic adenocarcinoma). The progression of pancreatic ductal adenocarcinoma (PDAC), the most lethal common cancer, is initiated by activation mutations of the KRas oncogene. This leads to additional molecular changes, such as activation of the AKT1 oncogene, which drives PDAC progression and tumor formation. By mating transgenic mice with activation of KRas (Pdx- Cre;LSL-KRasG12D) and mice with activation of AKT1 (Pdx- Tta;TetO-MyrAKT1) we were able to produce mice with two activated oncogenes (AKT1Myr/KRasG12D) for comparative studies. Kaplan-Meier survival curves, histology, and genomic/proteomic analysis were used to characterize the incidence and frequency of histological (e.g. presence of mucin-4 in pancreatic intraepithelial neoplasms) and genetic (e.g. loss of tumor suppressors p16Ink4a and p19Arf) alterations known to commonly occur in human pancreatic cancer, as well as delineate the role of AKT1 in accelerating pancreatic tumor progression and metastasis. We determined that AKT1Myr/KRasG12D mice, unlike other PDAC mouse models, accurately mimic the human PDAC progression molecularly, structurally, and temporally. Interestingly, the AKT1Myr and AKT1Myr/KRasG12D models both exhibit a pre-tumor, diabetic phenotype. While, AKT1 hyperactivation in various cancers has been thoroughly studied, its role in glucose metabolism has been noted, but comparatively overlooked. As early as the 1900s a relationship between diabetes and pancreatic cancer has been proposed. With 80% of PDAC patients suffering from hyperglycemia or diabetes prior to diagnosis, one prevailing theory is that new onset diabetes is an early marker for pancreatic cancer. This is also supported by experimental and clinical studies, such as the resolution of diabetes with tumor removal and the induction of hyperglycemia with the implantation of cancer cell lines. To better understand the role of AKT1 and its hyperactivation in glucose metabolism, AKT1Myr mice were characterized via metabolic (e.g. glucose/insulin tolerance test) and histological (e.g. immunohistochemistry) studies. Beginning at weaning, 3 weeks of age, the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. Additionally, AKT1Myr/KRasG12D mice currently being studied, appear to display a more severe diabetic phenotype, with fasted hyperglycemia noticeable at an earlier age, fasted hyperglucagonemia, polyuria, muscle wasting, and bloating. Treatment of both models with doxycycline diet, to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. These newly revealed roles of AKT1, along with future studies of these mouse models, will better delineate the molecular mechanisms responsible for the individual and joint roles of AKT1 and KRas in pancreatic cancer oncogenesis, the initiation of cancer associated diabetes, and the association of these two diseases.

Excess body fat, diabetes, and metabolic syndrome are associated with certain types of cancers. Although obesity is strongly associated with metabolic dysfunction (MetDys), some overweight individuals do not develop MetDys, although certain lean individuals do. The objective of this dissertation was to determine whether MetDys modifies the effect of excess body fat on cancer risk. The independent and combined effects of body mass index (BMI), waist circumference (WC), and MetDys on obesity-related cancer (ObCa) risk was examined among 3,818 45–69 year-olds in the Framingham Offspring Study using Cox proportional hazard’s analysis. Primary ObCa types included postmenopausal breast, endometrial, and colon cancer. Markers of MetDys included fasting glucose, triglycerides, high density lipoprotein-cholesterol, and hypertension. Subjects with ≥2 metabolic abnormalities were considered to have prevalent MetDys. Obesity alone (BMI≥30) in both men and women led to about a two-fold increased risk of ObCa. WC and waist-to-height ratio (WHtR) were somewhat stronger risk predictors in men than in women. There was little evidence of effect modification of WC or WHtR by MetDys in either men or women. In obese men, however, prevalent MetDys was associated with a 2.5-fold increased risk (95% CI: 1.36–1.46) of ObCa while obese men without MetDys had only a non-statistically significant 1.5-fold increased risk (0.70–3.03). There was no effect modification in women. To separate the effects of overall body fat from fat distribution, hip circumference was added to the multivariable models. Here, the effect of a WC >40 inches in men was strengthened (HR=2.59; 95% CI: 1.53–4.36) by controlling for HC. Conversely, when controlling for WC, the smallest HC in men conveyed a higher cancer risk (HR=2.68; 95%CI: 1.46–4.89). Subjects gaining ≥1 lb/year over approximately 14 years and who developed MetDys during that period had a 77% (95%CI: 1.21–2.59) increased ObCa risk while weight gainers not developing MetDys had a 31% (95%CI: 1.00–1.71) increased risk. Finally, weight gainers who became overweight/obese had a higher cancer risk than those with long-term overweight/obesity. This study suggests that those with excess body fat and long-term weight gain have higher ObCa risk, with little evidence that metabolic abnormalities modified the effect of obesity.

Patients with Type 2 Diabetes (T2D) have been found to have increased mortality and morbidity for COVID-19 and are at higher risk for severe disease once infected with COVID-19. Clearly, there exists a relationship between T2D and COVID-19 that requires more attention. In order to understand the mechanisms by which T2D contributes to more severe COVID-19 disease, attention was turned to extracellular vesicles (EVs). It was speculated that viral RNA components of the COVID-19 virus may have originated from circulating EVs that came from infected cells and use a Trojan Exosome method to infect host cells. It is necessary to characterize the EVs and viral RNA components of COVID-19 patients to understand the infection mechanisms. EV purification, liquid chromatography-tandem mass spectrophotometry, nanoparticle tracking analysis, Real Time-Polymerase Chain Reaction (qPCR), and Bioanalyzer analysis were performed for this using patient samples from Advent Health Orlando. Results found that qPCR was unable to detect COVID-19 viral RNA in the EVs of these patients, most likely a result of poor sensitivity. This study contributes towards defining the proteomic landscape of circulating EVs in people with COVID-19 and T2D and identifying biological mechanisms driving the interaction between the two diseases. Future directions include profiling the small noncoding RNAs found in COVID-19 patients and utilizing different methods to analyze isolated RNA to identify COVID-19 viral materials in EVs.

Epilepsy is one of the most common neurological disorders in the world, involving recurrent and spontaneous seizures which can have devastating effects on individual health and quality of life, as well as on the economic health of nations. While currently available anti-seizure drugs can control the symptoms of epilepsy, these drugs do not work in one-third of patients. Drug-resistant epilepsy is a serious concern. Even in patients responding to anti-seizure drugs, these treatments are not curative – they must be taken chronically and have limited to no ability to reverse or prevent the course of epilepsy development, called epileptogenesis. Epileptogenesis is a dynamic process involving underlying changes in the development of epilepsy, including changes in network excitability and organization, cellular plasticity, and inflammation. As epilepsy research has refocused on finding anti-epileptogenic agents to meet the unmet need of patients with drug-resistant epilepsy, dysregulated metabolism has come to light as a new area of research – along with a revisiting of previously tried metabolic treatments for epilepsy such as the ketogenic diet.

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Carboxylesterases (CEs) metabolize a wide range of endogenous compounds and xenobiotics containing ester bonds. Crystal structures of mammalian CEs indicate a ?side door? located adjacent to the catalytic gorge that may act as an alternative pore for the trafficking of substrates and products. This study investigated the role of the ?gate? residue of the side door during para-Nitrobenzyl esterase (pnb CE)-catalyzed hydrolysis of esters. Purified recombinant pnb CE proteins were examined for their hydrolytic activity toward several esters. Mutation of the gate residue altered the kinetic parameters of pnb CE toward these substrates, demonstrated by increased Km values and decreased Vmax values. Site-specific mutations of the ?gate? residue also affected the sensitivity of the enzyme toward inhibiting organophosphate compounds. A distinct possibility is that the side door mutants affect substrate hydrolysis by increasing the steric hindrance and/or electrostatic repulsion between the substrate and the active site catalytic residues.

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