Wong Chun Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 202-225). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.ix / Table of Contents --- p.x / List of Abbreviations --- p.xv / List of Figures --- p.xix / List of Tables --- p.xx iii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- "Neurodegeneration, Inflammation and Gliosis" --- p.1 / Chapter 1.2 --- Anatomy of the CNS --- p.5 / Chapter 1.3 --- Astrocytes --- p.6 / Chapter 1.3.1 --- Morphology and Identification of Astrocytes --- p.6 / Chapter 1.3.2 --- Physiological Functions of Astrocytes in the CNS --- p.7 / Chapter 1.3.2.1 --- Induction of Blood-brain Barrier (BBB) --- p.7 / Chapter 1.3.2.2 --- Metabolism of Neurotransmitters --- p.9 / Chapter 1.3.2.3 --- Nursing Role of Astrocytes --- p.9 / Chapter 1.3.2.4 --- Immunological Functions of Astrocytes --- p.10 / Chapter 1.3.3 --- Neonatal Rat Cortical Astrocytes as In Vitro Model --- p.12 / Chapter 1.4 --- Cytokines in Brain Damage --- p.14 / Chapter 1.4.1 --- Lipopolysaccharides (LPS) --- p.16 / Chapter 1.4.2 --- Tumor Necrosis Factor-α (TNF-α) --- p.17 / Chapter 1.4.3 --- Interleukin-1 (IL-1) --- p.19 / Chapter 1.4.4 --- Interleukin-6 (IL-6) --- p.20 / Chapter 1.4.5 --- Interferon-γ (IFN-γ) --- p.21 / Chapter 1.5 --- Cytokines-induced Signaling Cascade --- p.22 / Chapter 1.5.1 --- TNF Receptors --- p.23 / Chapter 1.5.2 --- Ca2+ --- p.25 / Chapter 1.5.3 --- MAPK --- p.26 / Chapter 1.5.4 --- PICA --- p.27 / Chapter 1.5.5 --- NFkB --- p.29 / Chapter 1.6 --- Glucose Metabolism in the Brain and Glucose Transporters --- p.31 / Chapter 1.6.1 --- Glucose Transporters in the Brain --- p.32 / Chapter 1.6.2 --- Glucose Transporters in Brain Damage --- p.34 / Chapter 1.7 --- Ascorbic Acid Metabolism in the Brain --- p.36 / Chapter 1.8 --- Aim and Scope of this Project --- p.39 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Neonatal Sprawley 一Dawley Rats --- p.43 / Chapter 2.1.2 --- Plain Dulbecco Modified Eagle Medium ´ؤ Formula 12 (pDF12) --- p.43 / Chapter 2.1.3 --- Complete DF-12(cDF12) --- p.43 / Chapter 2.1.4 --- Phosphate Buffered Saline (PBS) --- p.44 / Chapter 2.1.5 --- Hank's Buffer (HSB) --- p.44 / Chapter 2.1.6 --- D/L-Homocysteine Buffer --- p.44 / Chapter 2.1.7 --- "LPS, Cytokines and Pentoxifylline" --- p.45 / Chapter 2.1.8 --- Specific TNF Receptor Agonist: TNF antibodies --- p.45 / Chapter 2.1.9 --- Calcium Modulators --- p.45 / Chapter 2.1.10 --- PKA Modulators --- p.46 / Chapter 2.1.11 --- NFkB Inhibitors --- p.47 / Chapter 2.1.12 --- MAPK Inhibitors --- p.47 / Chapter 2.1.13 --- β-Adrenergic Receptor Modulators --- p.47 / Chapter 2.1.14 --- Reagents for RNA and Protein Isolation --- p.48 / Chapter 2.1.15 --- Reagents for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.48 / Chapter 2.1.16 --- Reagents for DNA Electrophoresis --- p.49 / Chapter 2.1.17 --- Reagents for Real-time PCR --- p.51 / Chapter 2.1.18 --- Reagents for Western Blotting --- p.51 / Chapter 2.1.19 --- Reagents for MTT Assay --- p.51 / Chapter 2.1.20 --- Reagents for 3H-Thymidine Incorporation Assay --- p.52 / Chapter 2.1.21 --- Reagents for Glucose Uptake Assay --- p.52 / Chapter 2.1.22 --- Reagents for Ascorbic Acid Accumulation Assay --- p.53 / Chapter 2.1.23 --- Reagents for Immunostammg --- p.53 / Chapter 2.1.24 --- Other Chemicals and Reagents --- p.53 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Preparation of Primary Cultured Rat Astrocytes --- p.55 / Chapter 2.2.2 --- Measuring Cell Viability: MTT Assay --- p.56 / Chapter 2.2.3 --- Measuring Cell Proliferation: 3H Thymidine Incorporation Assay --- p.57 / Chapter 2.2.4 --- Measuring Glucose Uptake: Zero-trans Glucose Uptake Assay --- p.58 / Chapter 2.2.5 --- Measuring Ascorbic Acid Accumulation --- p.60 / Chapter 2.2.6 --- Total Protein Extraction --- p.61 / Chapter 2.2.7 --- Western Blotting --- p.62 / Chapter 2.2.8 --- Immunostaining --- p.64 / Chapter 2.2.9 --- Isolation of RNA --- p.64 / Chapter 2.2.10 --- Measurement of RNA Yield --- p.65 / Chapter 2.2.11 --- RNA Gel Electrophoresis --- p.66 / Chapter 2.2.12 --- Reverse Transcription (RT) --- p.66 / Chapter 2.2.13 --- Polymerase Chain Reaction (PCR) --- p.67 / Chapter 2.2.14 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.67 / Chapter 2.2.15 --- Quantization of PCR Products and Western Blotting --- p.68 / Chapter 2.2.16 --- Real-time PCR --- p.68 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Role of Calcium Ions (Ca2+) in TNF-α-induced Astrocyte Proliferation --- p.70 / Chapter 3.1.1 --- Effects of Changes of Extracellular Ca2+ on Astrocyte Viability --- p.72 / Chapter 3.1.2 --- Effects of Other Divalent Ions on Astrocyte Viability --- p.74 / Chapter 3.1.3 --- Effects of Changes of Intracellular Ca2+ on Astrocyte Viability --- p.78 / Chapter 3.1.4 --- Role of Ca2+ on TNF-α-mduced Proliferation in Astrocytes --- p.85 / Chapter 3.1.5 --- Role of Other Divalent Ions on tnf-α-mduced Proliferation in Astrocytes --- p.90 / Chapter 3.2 --- Effect of Cytokines on Glucose Uptake in Rat Astrocytes --- p.95 / Chapter 3.2.1 --- Basal level of Glucose Uptake in Astrocytes and Effects of Cytokines on Glucose Uptake in Astrocytes --- p.95 / Chapter 3.2.2 --- Signaling Cascade of LPS- and TNF-α-induced Glucose Uptake in Astrocytes --- p.120 / Chapter (A) --- TNFR Subtypes Mediating TNF-a-induced Glucose Uptake --- p.121 / Chapter (B) --- MAPK --- p.125 / Chapter (C) --- PKA --- p.133 / Chapter (D) --- NFkB --- p.139 / Chapter (E) --- Other Mechanisms / Signalling molecules --- p.150 / Chapter (1) --- Interaction with β-Adrenegic Mechanism / Chapter (2) --- Role of cGMP --- p.154 / Chapter (3) --- Effect of Mg2+ on LPS- / TNF-α- induced Glucose Uptake in Astrocytes --- p.156 / Chapter (4) --- Possible Involvement of IGF-1 System --- p.160 / Chapter 3.2.3 --- Summary --- p.163 / Chapter 3.3 --- Effects of LPS and Cytokines on AA Accumulation in Astrocytes --- p.164 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Role of Calcium ions (Ca2+) in TNF-α-induced Astrocyte Proliferation --- p.177 / Chapter 4.1.1 --- Drastic Changes in Extracellular Ca2+ Caused Astrocyte Death --- p.178 / Chapter 4.1.2 --- Extraordinary Role of Ca2+ in Astrocytes Survival --- p.178 / Chapter 4.1.3 --- Elevation of [Ca2+]i Reduced Astrocyte Viability --- p.180 / Chapter 4.1.4 --- Failure of Verapamil to Block TNF-α-induced Astrocyte Proliferation --- p.182 / Chapter 4.2 --- Hypothesis for the Relationship between Cytokines and Energy Metabolism --- p.185 / Chapter 4.2.1 --- Mechanism and Signaling Cascade of the Elevated Glucose Uptake --- p.186 / Chapter 4.2.2 --- Increased Glucose Uptake by Cytokines: Friend or Foe? --- p.191 / Chapter 4.2.3 --- Depletion of AA Pool by LPS --- p.194 / Chapter 4.2.4 --- Possible Bedside Application of the Findings --- p.195 / Chapter 4.3 --- Prospects of This Study and Concluding Remarks --- p.197 / Appendix --- p.201 / References --- p.202

胰腺內的胰島具有極其重要的功能，通過產生并分泌一系列的胰島荷爾蒙，特別是能控制機體葡萄糖利用的胰島素，來調節體內血糖穩態。胰島素的分泌受到多種因素或信號通路的調節。据信，在臨床上表現出來明顯的高血糖症的時候，胰島細胞的分泌功能已經出現典型性的缺陷。由此，大量的研究證據指出，2 型糖尿病表現出來的代謝型缺陷主要為胰島功能紊亂，而并不是周圍組織胰島素抵抗。這表明，胰島素功能缺陷是早於高血糖症的發生的。另一方面，大量證據表明長期性的高血糖會導致胰島　細胞功能紊亂。鑒於此，揭示胰島功能調節的潛在機理并闡明胰島功能与高血糖症之間的關係變得尤為重要。 / 在臨床上表現出能調節胰島功能和血糖控制的相關因子正與日俱增。其中極具研究價值的是一種多肽，稱作胰高血糖素様肽（GLP-1），其作用表現在通過增強胰島素分泌和胰島素敏感性來增強胰島　細胞的功能和增值。GLP-1 在體內的降解能被DPP-4 的抑製劑所延阻。同時，通過對一種名為SGLT2 的葡萄糖轉運蛋白的抑制，機體內的血糖水平能被顯著降低。這一作用是通過阻止腎臟對葡萄糖的重吸收來實現的，並且是不依賴于胰島素的。由於DPP-4 抑制所表現的最終生理作用需要通過胰島素的信號通路來實現，但SGLT2 的抑制卻不依賴於胰島素，由此不難想象，對SGLT2 和DPP-4 的聯合抑制在2 型糖尿病的血糖控制方面具有潛在的協同效應。即通過對SGLT2 的抑制來顯著降低血糖水平，從而促進GLP-1 在體內的作用效應。因此，本研究的第一部分研究SGLT2 和DPP-4 的單一或聯合抑制（利用SGLT2 抑製劑BI-38335 和DPP-4 抑製劑linagliptin）在二型糖尿病動物模型db/db老鼠種對胰島功能和體內葡萄糖穩態的作用。在此研究中，我們比較了SGLT2 和DPP-4 單一抑制或聯合抑制對db/db 老鼠胰島功能的影嚮。研究發現，所有的實驗組都能顯著降低血糖以及糖化血紅蛋白（HbA1c）的水平，而且聯合抑制組表現出更叫顯著的效應。聯合抑制組增強了胰島細胞的胰島素分泌功能，改善葡萄糖耐受并增加胰島素的敏感性。於此一致的是，聯合抑制組降低了β細胞凋亡和胰島免疫細胞標記物，並且抑制了与TLR2 信號通路相關的一系列炎症分子，通過則一系列作用實現對胰島的保護。上述研究表明，對SGLT2 和DPP-4 的聯合抑制在對胰島功能和胰島形態學上的保護至少能夠表現出加性效應，從而更好實現對血糖的調控。 / 在第一部分的工作中，我們利用的動物模型db/db 老鼠是一類較嚴重的糖尿病動物模型，它表現出及其嚴重的高血糖症，糖耐受失調同β細胞缺陷。我們集中于研究SGLT2 和DPP-4 的抑制對這類嚴重糖尿病的胰島功能的調節，具體表現在對胰島β細胞功能的正向調節，包括胰島素分泌功能的增強和β細胞質量的增加。廣為接受的一點是，胰島素抵抗和胰島素分泌功能的缺失最能表徵從正常葡萄糖耐受發展到2 型糖尿病的這一進程。這一進程的早期主要表現為由肥胖或衰老而引起的代償性的胰島素抵抗，此時伴有正常或受損的葡萄糖耐受以及正常的胰島素分泌功能。此時，任何能影響胰島功能的因素都會減緩或加速2 型糖尿病的發生。鑒於此，研究此类因素從而到达阻止2 型糖尿病的发生就显得尤为重要。因此，在本研究的第二部分，我们研究利用高脂飼料诱导的肥胖老鼠模型和老化的老鼠模型来分别研究煙酸（niacin 或 nicotinic acid）对胰岛功能的影響。煙酸是一種臨床上廣汎使用的降血脂藥物，但近年來的研究發現長期或高劑量的使用會導致高血糖症和血糖控制失調的出現，然而這一現象產生的具體機製並不清楚。因此，我們第二部分的研究集中於揭示煙酸引起的高血糖症是否歸因於其對胰島功能的破壞，以及潛在的分子機制。我們的研究發現，在肥胖老鼠和老齡鼠中，煙酸能夠引起高血糖症，破壞葡萄糖體內穩態並且降低胰島素分泌能力；另一方面，煙酸增加饑餓血清胰島素水平並且引起葡萄糖耐受實驗中第一期胰島素分泌缺陷。體內和體外實驗還發現煙酸誘導煙酸受體GPR109a，UCP2 和PPARγ的表達增加以及SIRT1 的表達和NAD,NAD/NADH 降低。通過基因沉默技術降低GPR109a 在β細胞中的表達，我們發現煙酸的上述作用都被極大的減弱，從而揭示了煙酸引起的胰島功能降低是由其受體GPR109a 介導的。 / 總闊來說，我們的研究揭示了DPP-4 同SGLT2 的聯合抑制在增強胰島功能和胰島形態學上的保護以及改善胰島素抵抗等方面能夠表現出加性效應，從而更好實現對血糖的調控。另一方面，我們的研究闡述了煙酸通過它的受體GPR109a 以及其下游信號通路如PPARγ和SIRT1 來損害胰島細胞功能。綜上所述，我們當前的研究證實了一系列因素對胰島功能的調控，從而充實并擴展了我們對胰島功能和血糖控制以及2 型糖尿病之間關係的認識。 / Pancreatic islets are of great importance to govern glucose homeostasis through production and secretion of islet peptide hormones, notably insulin, which functions as a master regulator to control glucose disposal in the body. Insulin secretion is regulated by various factors and signaling pathways. It is well known that islet insulin secretory function is typically lost by the time when signs of hyperglycemia that becomes clinically apparent. Thus, it has been pointed out that islet dysfunction, rather than peripheral insulin resistance, is the primary defect of type 2 diabetes mellitus (T2DM), indicating that deficiencies in islet function are prior to the onset of hyperglycemia. On the other hand, it is also widely accepted that chronic hyperglycemia results in islet β cells dysfunction. In this regard, it is of great importance to unravel the underlying mechanisms by which islet function is regulated, thus elucidating the relationship between hyperglycemia and islet function. / There are ever increasing candidates of clinically relevant factors identified as criticalregulators for islet function and glycemic control. Of great interest is the glucagon-like peptide 1 (GLP-1) that improves β cell function and proliferation and its degradation can be delayed by dipeptidyl peptidase-4 (DPP-4) inhibition. Meanwhile, plasma glucose levels can be remarkably lowered by inhibition of sodium-glucose co-transporter 2 (SGLT2), through blockade of renal glucose reabsorption. In this regard, since the mode of action of SGLT2 inhibition is independent of insulin but the efficacy of DPP-4 inhibition relies on the insulin signalling, it is plausible to hypothesize that sustained lowering of plasma glucose by SGLT2 inhibition can facilitate the actions of GLP-1 from DPP-4 inhibition, thus leading to a potential synergistic effect on islet function and glycemic control. Accordingly, the first part of this study was to investigate the combination effects of SGLT2 and DPP-4 blockade on islet function and glucose homeostasis using an animal model of T2DM, the db/db mice. We compared the effects of either DPP-4 inhibition (by a DPP-4 inhibitor, linagliptin) or SGLT2 inhibition (by an SGLT2 inhibitor, BI-38335) individually and in combination on islet function and glycemic control in db/db mice. Active treatments markedly enhanced islet function, improved glycemic control and reduced islet and peripheral tissue inflammation, with the combined treatment showing the greater effects. These data indicate that combined SGLT2 inhibition with DPP-4 inhibition work additively to exhibit benefits to islet function, inflammation and insulin resistance, thus improving glycemic control. / In the first part, we investigated a positive regulation of islet function in overt diabetic mice, in which there are severe hyperglycemia and β cell failure. It is widely accepted that the progression from normal glucose tolerance to T2DM is characterized by dual defects that include insulin resistance and an insulin secretory defect caused by β cell dysfunction. In the early stage, there is compensated insulin resistance resulting from obesity or aging with normal or even impaired glucose tolerance as well as nearly normal insulin secretory capacity. As such, any factors that affect islet function in this stage may delay or accelerate the onset of diabetes. In this regard, it is noteworthy to study the regulation of such factors in islet function in order to prevent the development of T2DM. Thus, in the second part, we investigated how islet function was regulated by a widely used lipid-lowering drug, niacin (nicotinic acid), in obese mice and aged mice. Niacin has been known to impair euglycemic control during prolonged and high dose treatments but the underlying mechanism(s) whereby the islets are involved remains unclear. As such, we aimed at elucidating whether this hyperglycemic effect is due to the dysfunction of pancreatic islet and, if so, the underlying mechanism(s) involved. We investigated the direct effects of niacin on islet function and insulin resistance in HFD-induced obese (DIO) mice and aged mice. Our results showed that eight-week treatments with niacin impaired glycemic control and islet function in DIO and aged mice. Moreover, niacin treatments significantly induced PPARγ and GPR109a expression but decreased SIRT1 expression in pancreatic islets, while islet morphology remained unchanged. In vitro studies showed that niacin decreased glucose-stimulated insulin secretion (GSIS), cAMP, NAD/NADH ratio, and mitochondrial membrane potential (ΔΨm) but increased reactive oxygen species (ROS) transiently, while upregulated expression levels of UCP2, PPARγ and GPR109a in INS-1E cells. In corroboration, the decrease in GSIS and cAMP levels were abolished by the knockdown of GPR109a. These data indicate that chronic treatment of niacin induces hyperglycemia, which is due, partly, to impaired pancreatic islet function, probably via the mediation of islet niacin receptor GPR109a. / Collectively, our study has revealed that inhibition of DPP-4 or SGLT2 alone can improve islet function, and combined inhibition of DPP-4 and SGLT2 works additively to exhibit benefits to islet cell function/morphology, inflammation and insulin resistance, thus improving glycemic control. On the other hand, we have also elucidated that niacin impairs islet β cell function through GPR109a and downstream signaling pathways such as PPARγ and SIRT1. Taken together, the present study has shown the regulation of is let β cell function by different factors, which has an added advance to our knowledge about the intricate relationship between islet function and hyperglycemia and T2DM. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Lihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 168-195). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / List of Publications --- p.viii / List of Abbreviations / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine pancreas --- p.2 / Chapter 1.1.1 --- Structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Architecture and composition of the islet --- p.3 / Chapter 1.1.3 --- Endocrine cells and their function --- p.5 / Chapter 1.2 --- Disorders of the endocrine pancreas --- p.9 / Chapter 1.3 --- Insulin --- p.10 / Chapter 1.3.1 --- Insulin Structure --- p.10 / Chapter 1.3.2 --- Insulin actions and insulin receptor --- p.11 / Chapter 1.3.3 --- Insulin secretion --- p.12 / Chapter 1.3.3.1 --- Glucose-induced insulin secretion --- p.13 / Chapter 1.3.3.2 --- Phasic insulin secretion --- p.14 / Chapter 1.3.4 --- The regulation of insulin secretion --- p.16 / Chapter 1.3.5 --- Autocrine insulin feedback --- p.20 / Chapter 1.4 --- Diabetes mellitus --- p.21 / Chapter 1.4.1 --- Type 1 diabetes mellitus (T1DM) --- p.22 / Chapter 1.4.2 --- Type 2 diabetes mellitus (T2DM) --- p.23 / Chapter 1.4.3 --- Obesity and T2DM --- p.23 / Chapter 1.4.4 --- Islet dysfunction and T2DM --- p.25 / Chapter 1.5 --- Incretin hormones and DPP-4 inhibition --- p.27 / Chapter 1.5.1 --- Incretin hormones --- p.27 / Chapter 1.5.2 --- Functions of incretin hormones --- p.30 / Chapter 1.5.3 --- Regulation of GLP-1 --- p.34 / Chapter 1.5.4 --- Incretin-based therapy for T2DM --- p.35 / Chapter 1.6 --- Sodium-dependent glucose cotransporter 2 (SGLT2) and its inhibitors --- p.38 / Chapter 1.6.1 --- Sodium-dependent glucose cotransporter 2 (SGLT2) --- p.38 / Chapter 1.6.2 --- Rationale for SGLT2 inhibition --- p.40 / Chapter 1.6.3 --- Consequences of SGLT2 inhibition --- p.41 / Chapter 1.6.4 --- Strategies of SGLT2 inhibition --- p.43 / Chapter 1.6.4.1 --- SGLT2 inhibitors --- p.44 / Chapter 1.6.4.1 --- SGLT2 inhibitors --- p.47 / Chapter 1.7 --- Niacin (nicotinic acid) and its clinical usage --- p.49 / Chapter 1.7.1 --- Niacin general introduction --- p.49 / Chapter 1.7.2 --- General roles of niacin --- p.49 / Chapter 1.7.3 --- Anti-lipolytic effect --- p.50 / Chapter 1.7.4 --- Niacin receptor --- p.51 / Chapter 1.7.5 --- Hyperglycemic effect of niacin --- p.52 / Chapter 1.8 --- General hypothesis --- p.54 / Chapter Chapter 2 --- General Materials and Methods --- p.56 / Chapter 2.1 --- Experimental animal models --- p.57 / Chapter 2.1.1 --- Animal model of type 2 diabetes --- p.57 / Chapter 2.1.2 --- High-fat diet-induced obese mice --- p.58 / Chapter 2.1.3 --- Aged mice --- p.59 / Chapter 2.2 --- INS-1E cell culture and treatment --- p.59 / Chapter 2.2.1 --- Mouse pancreatic islet isolation --- p.59 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets --- p.60 / Chapter 2.3 --- Pancreatic islet isolation and culture --- p.60 / Chapter 2.4 --- Glucose-stimulated insulin secretion (GSIS) assay --- p.61 / Chapter 2.5 --- Assessment of glucose homeostasis --- p.61 / Chapter 2.6 --- Determination of mRNA expression --- p.62 / Chapter 2.6.1 --- Design of specific primers --- p.63 / Chapter 2.6.2 --- Total RNA extraction and cDNA synthesis --- p.63 / Chapter 2.6.3 --- Real-time PCR analysis --- p.64 / Chapter 2.7 --- Detection of protein expression --- p.64 / Chapter 2.7.1 --- Western blotting analysis --- p.64 / Chapter 2.7.2 --- Immunofluorescent staining --- p.65 / Chapter 2.8 --- Biochemical analyses --- p.65 / Chapter 2.8.1 --- Plasma insulin and blood HbA1c levels --- p.65 / Chapter 2.8.2 --- Detection of cAMP --- p.66 / Chapter 2.8.3 --- NAD and NADH determination --- p.66 / Chapter 2.9 --- Detection of intracellular ROS --- p.67 / Chapter 2.10 --- Detection of mitochondrial membrane potential --- p.67 / Chapter 2.11 --- Statistical analysis --- p.67 / Chapter Chapter 3 --- Effects of Combining Linagliptin Treatment with BI-38335, A Novel SGLT2 Inhibitor, on Pancreatic Islet Function and Inflammation in db/db Mice --- p.70 / Chapter 3.1 --- Abstract --- p.71 / Chapter 3.2 --- Introduction --- p.72 / Chapter 3.3 --- Materials and Methods --- p.74 / Chapter 3.3.1 --- Animal model and experimental design --- p.74 / Chapter 3.3.2 --- In vivo glucose homeostasis --- p.75 / Chapter 3.3.3 --- Pancreas and islet studies --- p.76 / Chapter 3.3.4 --- Biochemical analyses --- p.77 / Chapter 3.3.5 --- Real-time PCR analyses --- p.77 / Chapter 3.3.6 --- Statistical analysis. --- p.78 / Chapter 3.4 --- Results --- p.78 / Chapter 3.4.1 --- Treatments with DPP-4 and SGLT2 inhibitors lower plasma glucose --- p.78 / Chapter 3.4.2 --- Treatments with DPP-4 and SGLT2 inhibitors improve glycemic --- p.80 / Chapter 3.4.3 --- Pancreatic islet function in db/db mice --- p.83 / Chapter 3.4.4 --- Pancreatic islet and peripheral tissue inflammation --- p.86 / Chapter 3.4.5 --- Islet morphology and preserved beta cells --- p.89 / Chapter 3.5 --- Discussion --- p.93 / Chapter Chapter 4 --- Niacin-Induced Hyperglycemia Is Mediated via Niacin Receptor GPR109a in Pancreatic Islets --- p.98 / Chapter 4.1 --- Abstract --- p.99 / Chapter 4.2 --- Introduction --- p.100 / Chapter 4.3 --- Research design and methods --- p.102 / Chapter 4.3.1 --- Animal model and experimental design --- p.102 / Chapter 4.3.2 --- In vivo glucose homeostasis --- p.102 / Chapter 4.3.3 --- Pancreas and islet studies --- p.103 / Chapter 4.3.4 --- INS-1E cell culture and treatment --- p.103 / Chapter 4.3.5 --- Construction of small interfering RNA for GPR109a --- p.103 / Chapter 4.3.6 --- Real-time PCR analyses --- p.104 / Chapter 4.3.7 --- Western blotting assay --- p.104 / Chapter 4.3.8 --- Detection of intracellular and mitochondrial ROS --- p.105 / Chapter 4.3.9 --- Detection of mitochondrial membrane potential (ΔΨm) --- p.105 / Chapter 4.3.10 --- Measurement of cAMP levels --- p.105 / Chapter 4.3.11 --- Determination of NAD and NADH levels --- p.106 / Chapter 4.3.12 --- Measurement of cell viability --- p.106 / Chapter 4.3.13 --- Statistical analysis --- p.106 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Glycemic control in HFD-induced obese mice --- p.106 / Chapter 4.4.2 --- Pancreatic islet function in HFD-induced obese mice --- p.110 / Chapter 4.4.3 --- Pancreatic islet morphology and gene expression --- p.112 / Chapter 4.4.4 --- INS-1E function and intracellular levels of cAMP, NAD, and NADH --- p.114 / Chapter 4.4.5 --- Gene expression in INS-1E cells --- p.117 / Chapter 4.4.6 --- Status of ROS and ΔΨm in INS-1E cells --- p.119 / Chapter 4.4.7 --- GPR109a knockdown in INS-1E cells --- p.122 / Chapter 4.5 --- Discussion --- p.129 / Chapter Chapter 5 --- Niacin Impairs Pancreatic Islet Glucose-Stimulated Insulin Secretion in Aged Mice through The Suppression of SIRT1 Signaling --- p.134 / Chapter 5.1 --- Abstract --- p.135 / Chapter 5.2 --- Introduction --- p.136 / Chapter 5.3 --- Research design and methods --- p.139 / Chapter 5.3.1 --- Animal model and experimental design --- p.139 / Chapter 5.3.2 --- In vivo glucose homeostasis --- p.139 / Chapter 5.3.3 --- Pancreas and islet studies --- p.140 / Chapter 5.3.4 --- Real-time PCR analyses --- p.140 / Chapter 5.3.5 --- Western blotting assay --- p.140 / Chapter 5.3.6 --- NAD and NADH determination --- p.141 / Chapter 5.3.7 --- NEFA determination --- p.141 / Chapter 5.3.8 --- Statistical analysis --- p.141 / Chapter 5.4 --- Results --- p.142 / Chapter 5.4.1 --- Glycemic control in middle aged mice --- p.142 / Chapter 5.4.2 --- Pancreatic islet function in HFD-induced obese mice --- p.147 / Chapter 5.4.3 --- NAD, NADH levels in pancreatic islet --- p.149 / Chapter 5.4.4 --- Genes expression in pancreatic islet --- p.151 / Chapter 5.5 --- Discussion --- p.150 / Chapter Chapter 6 --- General discussion --- p.156 / Chapter 6.1 --- Combined inhibition of DPP-4 with SGLT2 on islet function, inflammation and insulin resistance in T2DM --- p.158 / Chapter 6.2 --- Niacin impairs islet function in high-fat diet-induced obese mice and aged mice --- p.161 / Chapter 6.3 --- General conclusion --- p.164 / Chapter 6.4 --- Future directions --- p.166 / Chapter Chapter 7 --- Bibliography --- p.167

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell membrane. It also promotes glucose utilisation in type 2 diabetic patients via increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13- kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection exists between these two pathways upstream of GLUT4 translocation. Understanding this connection is important in the development of treatment strategies for type 2 diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK) activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4- carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'- aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue. Aim: To investigate the effect of two pharmacological AMPK-activating compounds, ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt as well as possible feedback on insulin-stimulated glucose uptake and GLUT4 translocation. Materials and Methods: Adult ventricular cardiomyocytes were isolated from male Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13- kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation. PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain results were confinned or refuted by repeating experiments using the isolated rat heart model. Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but in fact inhibited basal glucose uptake, although GLUT4 translocation from cytosol to membrane occurred. Both compounds also attenuated insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin, AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and ZMP increased glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. However both compounds increased GLUT4 translocation, clearly demonstrating that translocation and activation of GLUT4 are separate processes. 8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP indicating that there was no involvement of the purinergic receptors. Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. Conversely, both compounds inhibited both basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation. Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto unknown effects of AICAR and ZMP on PKB phosphorylation. / AFRIKAANSE OPSOMMING: Agtergrond: Oefening verhoog skeletspier glukose opname via AMP-geaktiveerde protein kinase (AMPK) aktivering en GLUT4 translokering vanaf die sitosol na die selmembraan. Dit verbeter ook glukose verbruik in tipe 2 diabetes pasiënte via verhoogde insulien sensitiwiteit. Insulien stimuleer GLUT4 translokering deur P13- kinase en protein kinase B (PKB/Akt) te aktiveer. Dit word dus gepostuleer dat daar 'n verbinding tussen hierdie twee paaie, wat beide betrokke is by GLUT4 translokering, bestaan. Dit is belangrik om hierdie verbinding te verstaan aangesien dit in behandelingstrategieë van tipe 2 diabetes geteiken kan word. Die oefening geïnduseerde verhoging in AMPK aktivering, kan deur 'n farmakologiese middel 5'- aminoimidasool-4-karboksamied ribonukleosied (AICAR), wat intrasellulêr omgesit word na 5'-aminoimidasool-4-karboksamied-ribonukleosiedfosfaat (ZMP), 'n AMP analoog, nageboots word. Doel: Om die effek van twee farmakologiese AMPK-aktiveringsmiddels, AICAR en ZMP, op die fosforilering van AMPK en PKB/Akt, sowel as moontlike effekte daarvan op insulien-gestimuleerde glukose opname en GLUT4 translokering, te ondersoek. Materiale en Metodes: Volwasse ventrikulêre kardiomiosiete is uit manlike Wistar rotharte geïsoleer d.m.v kollagenase perfusies en behandel met 1 mM AICAR of 1 mM ZMP in die teenwoordigheid of afwesigheid van 100 nM insulien of 100 nM wortmannin. Glukose opname is gemeet via intrasellulêre [3H]-2-deoksiglukose akkumulasie; PKB/Akt en AMPK fosforilering sowel as GLUT4 translokering is bepaal deur Western blot analises. Purinergiese reseptore is geblokkeer met 8-siklopentiel- 1,3-dipropielxanthien (8CPT) en die effek daarvan op AMPK fosforilering genoteer. Ten einde resultate wat in die geïsoleerde kardiomiosiet-model verkry is, te bevestig, is sekere eksperimente in die geïsoleerde rothart herhaal. Resultate: Beide AIGAR en ZMP stimuleer AMPK fosforilering. Die middels kan nie glukose opname verhoog nie, inteendeel, basale glukose opname is onderdruk alhoewel GLUT4 translokering vanaf die sitosol na die selmembraan wel plaasgevind het. Wortmannin kon insulien gemedieerde glukose opname en PKB/Akt fosforilering onderdruk. In die teenwoordigheid van wortmannin het beide AIGAR en ZMP PKB/Akt fosforilering verhoog. Alhoewel beide AIGAR en ZMP glukose opname in skeletspier verhoog, was dit nie die geval in kardiomiosiete nie. Beide middels het wel GLUT 4 translokering verhoog, wat duidelik demonstreer dat die translokering en aktivering van GLUT4, verskillende prosesse is. 8GPT het geen effek gehad op die fosforilering van AMPK deur AIGAR of ZMP nie, wat bewys dat daar geen betrokkenheid van die purinergiese reseptore was nie. Gevolgtrekking: Alhoewel AIGAR en ZMP glukose opname in skeletspier verhoog is dit nie die geval in kardiomiosiete nie. Beide middels inhibeer basale en insuliengestimuleerde glukose opname maar stimuleer GLUT4 translokeering. Inhibisie van PI3-kinase in die teenwoordigheid of afwesigheid van insulien, ontmasker voorheen onbekende effekte van AIGAR en ZMP op PKB/Akt fosforilering.

Given the high and increasing social, health and economic costs of type 2 diabetes, early diagnosis and prevention are critical. Insulin sensitivity and insulin secretion are important etiological factors of type 2 diabetes and are used to define an individual’s risk or progression to the disease state. The dynamic insulin sensitivity and secretion test (DISST) concurrently measures insulin sensitivity and insulin secretion. The protocol uses glucose and insulin boluses as stimulus, and the participant response is observed during a relatively short protocol via glucose, insulin and C-peptide assays. In this research, the DISST insulin sensitivity value was successfully validated against the gold standard euglycaemic clamp with a high correlation (R=0.82), a high insulin resistance diagnostic equivalence (ROC c-unit=0.96), and low bias (-10.6%). Endogenous insulin secretion metrics obtained via the DISST were able to describe clinically important distinctions in participant physiology that were not observed with euglycaemic clamp, and are not available via most established insulin sensitivity tests. The quick dynamic insulin sensitivity test (DISTq) is a major extension of the DISST that uses the same protocol but uses only glucose assays. As glucose assays are usually available immediately, the DISTq is capable of providing insulin sensitivity results immediately after the final blood sample, creating a real-time clinical diagnostic. The DISTq correlated well with the euglycaemic clamp (R=0.76), had a high insulin resistance diagnostic equivalence (ROC c-unit=0.89), and limited bias (0.7%). These DISTq results meet or exceed the outcomes of most validation studies from established insulin sensitivity tests such as the IVGTT, HOMA and OGTT metrics. Furthermore, none of the established insulin sensitivity tests are capable of providing immediate or real-time results. Finally, and most of the established tests require considerably more intense clinical protocols than the DISTq. A range of DISST-based tests that used the DISST protocol and varying assay regimens were generated to provide optimum compromises for any given clinical or screening application. Eight DISST-based variants were postulated and assessed via their ability to replicate the fully sampled DISST results. The variants that utilised insulin assays correlated well to the fully sampled DISST insulin sensitivity values R~0.90 and the variants that assayed C-peptide produced endogenous insulin secretion metrics that correlated well to the fully-sampled DISST values (R~0.90 to 1). By taking advantage of the common clinical protocol, tests in the spectrum could be used in a hierarchical system. For example, if a DISTq result is close to a diagnostic threshold, stored samples could be re-assayed for insulin, and the insulin sensitivity value could be ‘upgraded’ without an additional protocol. Equally, adding C-peptide assays would provide additional insulin secretion information. Importantly, one clinical procedure thus yields potentially several test results. In-silico investigations were undertaken to evaluate the efficacy of two additional, specific DISTq protocol variations and to observe the pharmacokinetics of anti-diabetic drugs. The first variation combined the boluses used in the DISTq and reduced the overall test time to 20 minutes with only two glucose assays. The results of this investigation implied no significant degradation of insulin sensitivity values is caused by the change in protocol and suggested that clinical trials of this protocol are warranted. The second protocol variant added glucose content to the insulin bolus to enable observation of first phase insulin secretion concurrently with insulin sensitivity from glucose data alone. Although concurrent observation was possible without simulated assay noise, when clinically realistic noise was added, model identifiability was lost. Hence, this protocol is not recommended for clinical investigation. Similar analyses are used to apply the overall dynamic, model-based clinical test approach to other therapeutics. In-silico analysis showed that although the pharmacokinetics of insulin sensitizers drugs were described well by the dynamic protocol. However, the pharmacokinetics of insulin secretion enhancement drugs were less observable. The overall thesis is supported by a common model parameter identification method. The iterative integral parameter identification method is a development of a single, simple integral method. The iterative method was compared to the established non-linear Levenberg-Marquardt parameter identification method. Although the iterative integral method is limited in the type of models it can be used with, it is more robust, accurate and less computationally intense than the Levenberg-Marquardt method. Finally, a novel, integral-based method for the evaluation of a-priori structural model identifiability is also presented. This method differs significantly from established, derivative based approaches as it accounts for sample placement, measurement error, and probable system responses. Hence, it is capable of defining the true nature of identifiability, which is analogous, not binary as assumed by the established methods. The investigations described in this thesis were centred on model-based insulin sensitivity and secretion identification from dynamic insulin sensitivity tests with a strong focus on maximising clinical efficacy. The low intensity and informative DISST was successfully validated against the euglycaemic clamp. DISTq further reduces the clinical cost and burden, and was also validated against the euglycaemic clamp. DISTq represents a new paradigm in the field of low-cost insulin sensitivity testing as it does not require insulin assays. A number of in-silico investigations were undertaken and provided insight regarding the suitability of the methods for clinical trials. Finally, two novel mathematical methods were developed to identify model parameters and asses their identifiability, respectively.

No description available.

Background: The NKG2D ligands are a set of cell surface proteins, the expression of which can make cells susceptible to immunity mediated by NKG2D receptor expressing cells, which include NK cells, CD8⁺ αβ T cells and γδ T cells. The NKG2D ligands are known to be expressed in distinct settings, including viral infection, cancer, T cell activation, and cellular proliferation, settings also tightly associated with Warburg metabolism. The molecular events which determine NKG2D ligand expression status are unknown. Aims: We aim to enhance understanding of the deterministic molecular events that control NKG2D ligand expression. Specifically, to explore the relationship between Warburg metabolism and NKG2D ligand expression in cell line and physiological models, and second, to identify open chromatin elements at NKG2D ligand loci, and develop computational methods to analyse this data. Methods: We use a range of molecular biology techniques to delineate the role of glucose metabolism in NKG2D ligand expression in a HEK293T cell model. We develop a physiological CMV-primary fibroblast model of NKG2D ligand induction to validate our key findings. We adapt, optimise and validate a DNaseI-seq protocol, to define open chromatin sites at the NKG2D ligand loci. We develop a data analysis `pipeline', including our own peak-finding software (“PeakHunter"), to identify open chromatin sites in the data. Key results: Glucose drives NKG2D ligand expression. This effect requires cellular uptake and metabolism of glucose. Purine nucleotides are a key glucose metabolite for this effect, and purine nucleosides are sufficient to induce NKG2D ligand expression in our HEK293T model. We have identified the open chromatin sites at the NKG2D loci in MCF7 breast cancer cells, and optimised and validated this protocol. Finally we have developed “PeakHunter" a multifunctional software tool for mapped DNaseI-seq data analysis. Conclusions: Glucose and its contribution to purine metabolism play a central role in the induction of NKG2D ligand expression in physiological settings. The influence of glucose leads to significant alterations in cellular NKG2D-dependent immunogenicity. PeakHunter is a useful tool for analysis of mapped DNaseI-seq data.

Obesity is widely considered a primary risk factor for type 2 diabetes (T2D). However, less is known about the early adaptive responses to short-term periods of high-fat energy excess (HFEE). Previous reports detailing whole-body adaptation to fat and energy oversupply are equivocal, perhaps, in part, owing to use of different experimental protocols, varying durations of dietary manipulation and participant cohorts with individuals of varying characteristics. In addition to use of different dietary protocols between studies, alterations in functional end-point measures due to the type of dietary fat consumed warrants consideration. Daily n-3 PUFA intake, commonly obtained from pelagic fish oil (FO) consumption, has been shown to positively associate with insulin sensitivity in epidemiological studies and thus may be a useful dietary strategy for slowing insulin resistance development. Chapter 2 of this thesis extends previous literature by demonstrating that 6 d HFEE (150 % habitual energy intake; 60 % of energy from fat) does not clearly alter whole- body insulin sensitivity, irrespective of FO consumption. However, investigation of metabolism at the tissue level, as presented in Chapter 3 of this thesis, offers insight into a potential tissue-specific level of regulation that precedes whole-body regulation. Skeletal muscle insulin signalling protein (e.g. protein kinase B (PKB)) activity, levels of certain ceramide species, and AMPK α2 activity were altered following HFEE and may explain the early maladaptive responses to short-term HFEE. Moreover, FO intake as 10 % of total fats mediated some of these molecular responses, including PKB and AMPK α2 activity, reflecting possible functional effects of FO at the subcellular level. Regulation of these metabolic / molecular responses at both the tissue and whole- body level can be explained, in part, by genetic predisposition, environmental influence and more recently epigenetics, including microRNAs (miRNAs). In Chapter 4, we characterised the plasma and skeletal muscle miRNA responses to HFEE and oral glucose ingestion. We demonstrate transient changes in levels of certain miRNAs following oral glucose ingestion in both tissue types and in response to HFEE in skeletal muscle. However, no significant correlations between basal plasma and skeletal muscle miRNA levels were observed, suggesting that our candidate plasma miRNAs may be co-ordinating functional changes in other tissue types. Plasma miR- 145-5p and skeletal muscle miR-204-5p predicted a significant proportion of the variance in mean whole-body insulin sensitivity change in response to HFEE. These data indicate that these miRNAs may be useful biomarkers of insulin resistance development following HFEE. A constraint of this thesis is that all conclusions are made within the context of statistically unaltered insulin sensitivity. Therefore, future investigations of diet-induced maladaptation should consider establishing a time course of insulin resistance development in response to HFEE, or use different study populations. Populations that are more susceptible to T2D development, e.g., overweight, sedentary individuals would be of particular interest. These data would aid development of a working model of diet-induced insulin resistance that has more direct application to T2D progression and extends the data presented herein.

A oligopeptidase neurolina (E.C.3.4.24.16; nln ) foi identificado pela primeira vez em membranas sinápticas de cérebro de ratos como sendo capaz de participar no metabolismo de peptídeos bioativos, como neurotensina e bradicinina. Recentemente, foi sugerido que a ausência de Nln pode melhorar a sensibilidade a insulina. Aqui, nós mostrado que camundongos knockout para Nln (KO) são mais tolrerantes à glicose, sensíveis à insulina e apresentam maior gliconeogênese. Os animais KO apresentou um aumento na expressão de mRNA de vários genes relacionados com a gliconeogênese no fígado. A semiquantificação de peptídeos intracelulares revelou um aumento em peptídeos intracelulares específicos no gastrocnêmio e tecido adiposo epididimal, que estão envolvidos com o aumento da tolerância a glicose e maior sensibilidade à insulina nos animais KO. Esses resultados sugerem fortemente a nova possibilidade de que Nln é uma enzima chave no metabolismo energético e pode ser um novo alvo terapêutico para melhorar a captação de glicose e sensibilidade a insulina. / The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

Glucose isomerase (GI) catalyzes the isomerization of glucose to fructose and consequently is one of the bulkiest industrial enzyme for the manufacture of high fructose corn syrup and crystalline fructose. The GI is used in industry mainly in the form of immobilized enzyme. / In this work, the immobilization of GI had been studied by several methods: ion exchange adsorption, covalent binding, alginate cells entrapment and cells cross-linking. Three kinds of carrier support (ion exchange resin, epoxy resin and amino resin) have been used in the immobilization of cells-free enzyme; the whole cells immobilization of GI by cross-linking agents polyethyleneimid and glutaraldehyde were critically examined. The results show that the cells cross-linking is the best method to prepare the immobilized GI products, as it is high in specific activity and thermostability, and low the cost. The method is likely to make significant contribution to the field of immobilization, its application has expanding rapidly in many walks of the society, including environment protection, food and pharmaceutical industries. / Jin, Caike. / "August 2005." / Adviser: Jun Wang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 125-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Nós investigamos se o ácido palmitoléico modula o metabolismo de glicose e triacilglicerol (TAG) em adipócitos. Assim, células 3T3-L1 tratadas com ácido palmitoleico (16:1n7 , 200 mM) ou palmítico (16:0, 200 mM) por 24h e adipócitos epididimais de camundongos selvagem ou deficientes para PPARa tratados com 16:1n7 o ácido oleico (18:1n9, 300 mg / kg / dia), via gavagem durante 10 dias, foram avaliados. O tratamento com palmitoleico aumenta a captação de glicose e o conteúdo de GLUT4 e pThr172AMPKa. O aumento de GLUT4 foi abolido pela inibição da AMPK. Palmitoleico aumenta a conversão de glicose em lactato e CO2 e diminui a síntese de novo de ácidos graxos. O tratamento de células 3T3-L1 com ácido palmitoleico aumentou a lipólise o mRNA da ATGL e HSL, além do conteúdo proteico da ATGL e pSer660HSL. O aumento na lipólise foi abolido pela inibição de PPARa. Também, o tratamento de camundongos selvagens, mas não os deficientes para PPARa, com palmitoleico aumentou a lipólise e o mRNA da ATGL e HSL em adipócitos. Em resumo, o ácido palmitoleico aumenta a captação de glicose e sua utilização pelos adipócitos, um efeito que está associado com a expressão de GLUT4 e AMPK. Além disso, este ácido aumenta a lipólise e lipases em adipócitos viA PPARa. / We investigated whether palmitoleic acid modulates glucose and triacylglycerol (TAG) metabolism in white adipocytes. For this, 3T3-L1 cells treated with palmitoleic (16:1n7, 200 µM) or palmitic acid (16:0, 200 mM) for 24h and epididimal adipocytes from wild type or PPARa deficient mice treated with 16:1n7 or oleic acid (18:1n9, 300 mg/kg/day) by gavage for 10 days were evaluated. Thus, treatment with palmitoleic increases glucose uptake and the content of GLUT4 and pThr172AMPKa. The increase in GLUT4 was prevented by AMPK inhibition. Also, palmitoleic increases glucose conversion into lactate and CO2, and decreases de novo fatty acids synthesis. Furthermore, treatment of 3T3-L1 cells with palmitoleic increased lipolysis, mRNA levels of ATGL and HSL and protein content of ATGL and pSer660-HSL. Such increase in lipolysis can be prevented by PPARa inhibition. Treatment of wild type, but not PPARa deficient mice, with palmitoleic increased adipocytes lipolysis and ATGL and HSL mRNA levels. In conclusion, palmitoleic acid increases glucose uptake and utilization by adipocytes, associated with GLUT4 expression and AMPK activation. Furthermore, palmitoleic acid increases adipocyte lipolysis and lipases via PPARa.