目前，骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展，採用核酸干擾策略進行治療的很多問題已被解決。然而，小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大，其系統給藥後可能對其他非骨組織產生副作用。所以，亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統，以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞，使其具有高度的細胞選擇性。在該研究中，我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統：首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備（天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑，ζ電位，包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右，其包封率可高達80%。該遞送系統較穩定，可使攜載的小干擾核酸具有較高的基因沉默效率，而且沒有明顯的細胞毒性。體內試驗表明，該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因，且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞，避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp，Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統：將針對大鼠成骨細胞（ROS 17/2.8細胞系）進行正向篩選，大鼠肝細胞（BRL-3A細胞系）和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑，ζ電位，包封率和形態學進行考察。在體外評價實驗中，考察了該遞送系統的選擇性，細胞毒性，基因沉默效率以及細胞攝取機制。在體內實驗中，對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm，其電勢為-23 ± 2 mV，包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系（靶向細胞）中的攝取， 然而在BRL-3A 細胞系（非靶向細胞）中攝入很少。該遞送系統沒有明顯細胞毒性，在10 nM小干擾核酸的低濃度下，體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈，降低其被肝組織的攝取。在肝组织冰凍切片中，肝血竇和肝細胞中沒有明顯的小干擾核酸分佈，進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞，避免被前破骨細胞/破骨細胞吞噬。 / 重要意義：本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑，實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下，首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具，為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130

Folate deficiency, a prevalent vitamin deficiency in America, can stem from environmental and/or genetic causes. The most common inborn error of folate metabolism is deficiency of methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Severe MTHFR deficiency results in hyperhomocysteinemia and homocystinuria; patients present with developmental delay, and various neurological and vascular disorders. This thesis describes three mutations identified in the MTHFR locus in patients with severe deficiency: 1025T→C (M→T), 1027T→G (W→G), and 1768G→A (E→K). Genotype-phenotype correlations are described, along with biochemical characterization of three mutations (983A→G (N→S), 1025T→C, 1027T→G). All three mutations exert their effect by decreasing Vmax without changing the enzyme's affinity for its substrate, 5-methyltetrahydrofolate. The 983A→G variant also conferred decreased affinity for FAD, a cofactor. / The more common and mild deficiency observed in the general healthy population is probably due in part to insufficient dietary intake of folate. Folate deficiency has been associated with increased risk for colon cancer. In a pilot study presented here, the impact of altered folate intake on tumor multiplicity in the Min mouse, a model for multiple intestinal neoplasia, was assessed. Folate deficient diets did not produce a consistent change in tumor numbers. However, a linear correlation between S-adenosylmethionine and S-adenosylhomocysteine content of preneoplastic tissue and tumor multiplicity was identified. / This thesis contributes to our understanding of the impact of genetic- and/or dietary-induced folate deficiency on cellular and organismal functions.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 137-143).

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Iron metabolism disorders comprise the most common disorders in humans. Hereditary haemochromatosis (HH) is a common condition resulting from inappropriate iron absorption. The most common form of the disease (Type 1) is associated with mutations in the HFE gene. The C282Y homozygous genotype accounts for approximately 80% of all reported cases of HH within the Caucasian population. A second HFE mutation, H63D, is associated with less severe disease expression. The C282Y mutation is extremely rare in Asian and African populations. The H63D mutation is more prevalent and has been observed in almost all populations. Iron overload resulting from haemochromatosis is predicted to be rare in Asian Indian populations and is not associated with common HFE mutations that are responsible for HH in the Caucasian population. The aberrant genes associated with HH in India have not yet been identified. The present study attempted to identify variants in six iron regulatory genes that were resulting in the Type 1 HH phenotype observed in two Asian Indian probands from a highly consanguineous family. The promoter and coding regions of the HMOX1, HFE, HAMP, SLC40A1, CYBRD1 and HJV genes were subjected to mutation analysis. Gene fragments were amplified employing the polymerase chain reaction (PCR) and subsequently subjected to heteroduplex single-strand conformational polymorphism (HEX-SSCP) analysis. Samples displaying aberrations were then analysed using bi-directional semi-automated DNA sequencing analysis to identify any known or novel variants within the six genes. Variants disrupting restriction enzyme recognition sites were genotyped employing restriction fragment length polymorphism (RFLP) analysis. Mutation analysis of the six genes revealed 24 previously identified variants, five novel variants (HFE: 5’UTR-840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR- 1272T→C; HJV: 5’UTR-534G→T, 5’UTR-530G→T), one previously described microsatellite and two novel repeats. Variants identified within the SLC40A1, CYBRD1 and HJV genes do not seem to be associated with the iron overload phenotype. A previously described HAMP variant (5’UTR-335G→T) was observed in the homozygous state in both probands. This variant seems to be the genetic aberration responsible for iron overload in this Indian family. The severe juvenile haemochromatosis phenotype usually associated with HAMP mutations, was not exhibited by the two Indian probands. Their symptoms resembled those observed in classic Type 1 HH. It is suggested that variants identified in the HMOX1 and HFE genes are modifying the effect of the HAMP variant and resulting in the less severe disease phenotype. Although this variant has only been identified in one Indian family, it could shed some light in the hunt for the iron-loading gene in India. / AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is ‘n algemene siektetoestand wat ontstaan as gevolg van oneffektiewe opname van yster in die liggaam. Die mees algemene vorm van die siekte (Tipe 1) word geassosieer met mutasies in die HFE-geen. Die C282Y homosigotiese genotipe is verantwoordelik vir ongeveer 80% van alle gerapporteerde gevalle van OH binne die Kaukasiese bevolking. ‘n Tweede HFE mutasie, H63D, word geassosieer met minder ernstige siekte simptome. Die C282Y mutasie is besonder skaars in Asiese en Afrika bevolkings. Daar word bespiegel dat oorerflike ysteroorlading as gevolg van hemochromatose skaars is in Asiese Indiër bevolkings en word nie geassosieer met algemene HFE mutasies wat verantwoordelik is vir OH in Kaukasiese bevolkings nie. Die abnormale gene wat wél geassosieer word met OH in Indië is tot dusver nog nie identifiseer nie. Die doel van hierdie studie was om die variante in ses yster-regulerende gene te identifiseer wat die Tipe 1 OH fenotipe in hierdie familie veroorsaak. Hierdie fenotipe is waargeneem in twee Asies Indiese familielede afkomstig van ‘n bloedverwante familie. Die promotor en koderingsareas van die HMOX1, HFE, HAMP, SLC40A1, CYBRD1 en HJV gene is gesif vir mutasies. Geen fragmente is geamplifiseer met behulp van die polimerase kettingsreaksie (PKR) en daarna aan heterodupleks enkelstring konformasie polimorfisme (HEX-SSCP) analise blootgestel. PKR produkte wat variasies getoon het, is daarna geanaliseer deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise om enige bekende of nuwe variante binne die ses gene te identifiseer. Variante waar restriksie ensiem herkenningsetels teenwoordig is, is verder analiseer met behulp van die restriksie fragment lengte polimorfisme (RFLP) analise sisteem. Mutasie analise van die ses gene het 24 bekende variante, vyf nuwe variante (HFE: 5’UTR- 840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-1272T→C, HJV: 5’UTR-534G→T, 5’UTR-530G→T), een bekende herhaling en twee nuwe herhalings gewys. Variante wat binne die SLC4041, CYBRD1 en HJV gene geïdentifiseer is, blyk nie om by te dra tot die ysteroorladings-fenotipe nie. Die bekende HAMP variant (5’UTR-335G→T) is waargeneem in die homosigotiese toestand in beide van die aangetaste individue. Hierdie variant blyk om die genetiese fout te wees wat verantwoordelik is vir die ysteroorlading in die betrokke Indiese familie. Die erge juvenielehemochromatose fenotipe wat meestal geassosieer word met HAMP-mutasies, is nie waargeneem in hierdie familie nie. Hul simptome kom ooreen met die simptome van die klassieke Tipe 1 OH. Dit blyk moontlik te wees dat die variante identifiseer in die HMOX1 en HFE gene die impak van die HAMP variant modifiseer en die matiger siekte-fenotipe tot gevolg het. Alhoewel hierdie variant slegs in een Indiese familie geïdentifiseer is, kan dit lig werp op die soektog na die veroorsakende ysterladingsgeen in Indië.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / Hereditary haemochromatosis (HH), a common autosomal recessive disease, is characterized by increased iron absorption leading to progressive iron accumulation in organs such as the liver, heart and pancreas. In the South African population the disease is prevalent in individuals of Caucasian origin, with a carrier frequency of one in six for the C282Y mutation in the HFE gene. We investigated the role of genes implicated in iron metabolism, including the high-iron gene (HFE), haem oxgenase-1 gene (HMOX1), solute carrier family 40 (iron-regulated transporter) member 1 gene (SLC40A1), cytochrome b reductase gene (CYBRD1), hepcidin antimicrobial peptide gene (HAMP) and the hemojuvelin gene (HJV) in a patient cohort with non-HFE iron overload. DNA analysis was performed on samples from 36 unrelated South African Caucasian patients presenting with primary iron overload, who tested either negative or heterozygous for C282Y. In this study, mutation screening was performed by PCR amplification and HEX-SSCP analysis. Sixteen previously described and two novel variants were identified by semi-automated DNA sequencing. Common variants identified in the HFE gene included C282Y, H63D, IVS2+4T→C, IVS4-44T→C, IVS4+48G→A and IVS5-47G→A. The Q127H mutation in exon 3 of the HFE gene was identified in one patient, who tested negative for both C282Y and H63D. Mutation S65C was identified only in the population-matched controls and was absent in the patient group. Other previously described polymorphisms identified included the IVS5+51delTGGCTGTCTGACT deletion in HMOX1, I109 and V221 in SLC40A1, IVS1-4C→G, IVS2+8T→C and S266N, in the CYBRD1 gene and, S264 and A310G in the HJV gene. The novel variants, -89C→T, in the promoter region of the CYBRD1 gene, was detected in only one patient, while S333 in exon 4 of the HJV gene was present in three patients. These variants were not identified in any of the population-matched controls screened and could explain the non-HFE iron overload presented by these patients. This study clearly demonstrates the importance of modifier genes in patients with iron overload that cannot be explained by the common C282Y mutation. Studies on iron-related genes and the identification of mutations in these genes in non-HFE patients could lead to improved diagnosis and counselling of South African patients presenting with primary iron overload.

Orientadores: Sarah Monte Alegre, Elza Olga Ana Muscelli Berardi / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T19:48:11Z (GMT). No. of bitstreams: 1 Carvalho_CamilaPuzzide_M.pdf: 2368362 bytes, checksum: feeaaef28d0f62b028795119992d2b1c (MD5) Previous issue date: 2008 / Resumo: A obesidade é conceituada como um quadro crônico que se caracteriza pelo acúmulo excessivo de gordura de modo a comprometer a saúde. Atualmente o tecido adiposo além de sua função como depósito de gordura, passou a ser considerado um órgão endócrino (Trayhurn, 2001). Os adipócitos secretam uma série de substâncias, como leptina, adiponectina, citocinas inflamatórias (TNF-alfa, IL-6) e PCR, que estão envolvidas em várias funções, incluindo, homeostase da glicose, inflamação, balanço energético, metabolismo dos lipídios e sistema fibrinolítico/homeostase vascular (Arner, 2003). Além dos hormônios secretados pelo tecido adiposo, sabe-se que o sistema digestório também secreta hormônios como as incretinas, uma delas é o GLP-1, secretado pelas células-L intestinais e possui efeito anti-diabético. Há também a secreção de grelina, que é um hormônio produzida no fundo gástrico, relacionada ao controle da fome, e à regulação do balanço energético a longo prazo (Cummings DE., 2001). No presente estudo avaliou-se o efeito do emagrecimento por dieta e o induzido por cirurgia bariátrica sobre os níveis plasmáticos de: GLP-1, grelina, adiponectina, citocinas inflamatórias e PCR, além da análise da correlação entre estas variáveis, em indivíduos obesos e diabéticos obesos. Foram avaliados 11 pacientes portadores de obesidade mórbida com intolerância à glicose normal (grupo NGT) e 8 pacientes, que além da obesidade apresentam diagnóstico de diabetes ou intolerância à glicose (grupo AMG: com 4 pacientes com DM tipo 2 e 4 pacientes com intolerância à glicose), que foram submetidos à cirurgia bariátrica pela técnica de tipo Fobi- Capella (Fobi,2001). Os grupos NGT e AMG foram caracterizados antes da cirurgia por apresentarem IMC 46,1 ± 2,27 kg/m2 e 51,20 ± 4,6 kg/m2 respectivamente, bem como o alto porcentual de gordura e circunferência da cintura. Os indivíduos no início do estudo, apresentavam níveis elevados de insulina de jejum 21,85 ± 2,93 uU/ml e 32,95 ± 9,66 uU/ml para NGT e AMG respectivamente, houve uma redução, que foi mais acentuada após 9 meses da realização da cirurgia chegando a valores dentro da faixa de normalidade, T5 12,08 ± 0,91 uU/ml NGT e 10,2 ± 1,55 uU/ml AMG. Quanto à glicose plasmática houve redução dos valores principalmente no grupo AMG, que apresentou resultados semelhantes ao grupo NGT, os valores no período T5 foram 77,7 ± 1,88 mg/dl para NGT e 70,93 ± 6,01 mg/dl para AMG. Em relação ao HOMA-IR, houve redução dos valores no período pós-operatório, sendo a diferença mais acentuada no grupo AMG, T1: 9,85 ± 3,67; T2: 4,67 ± 1,34 e T5: 1,73±0,24. Demonstramos que o aumento nos níveis de GLP-1 foi significativo no período pós-operatório (9° mês) tanto para o grupo NGT como AMG, momento no qual observamos melhora na sensibilidade à insulina e o grupo AMG apresentou uma maior secreção de GLP-1 no período T5 aos 30 minutos 34,06 ± 6,18 pmol/l quando comparados aos NGT 22,69 ± 4,04 pmol/l. Os níveis de adiponectina aumentaram ao longo do emagrecimento, sendo o aumento mais pronunciado após a realização da cirurgia, T1 6,43 ± 1,23 ug/ml; T2 8,35 ± 1,48 ug/ml; T5 17,17 ± 4,89 ug/ml para grupo NGT e T1 5,04 ± 1,2 ug/ml; T2 7,12 ± 1,73 ug/ml; T5 15,43 ± 5,52 ug/ml para AMG. A partir destes resultados o presente estudo permitiu concluir que a perda de peso por dieta necessitaria ser mais expressiva para que ocorresse melhora metabolismo glicose como também na secreção de GLP-1 e adiponectina, como a que ocorreu no emagrecimento cirúrgico. Demonstramos que o emagrecimento, em especial, em indivíduos do grupo AMG apresentaram normalização da curva de glicose, insulina e peptídeo-C, diminuição da resistência à insulina, como também aumento expressivo de GLP-1, e adiponectina mesmo não tendo ocorrido à estabilização de peso, e esta perda, conduziu a melhora no metabolismo de carboidratos, em relação à sensibilidade à insulina, contribuindo para a diminuição do risco de desenvolver DM tipo 2. Esta melhora, também, se expressou na redução do quadro inflamatório, assim como no aumento nos níveis de adiponectina e GLP-1. / Abstract: Obesity is a chronic disease characterized by extreme accumulation of fat that compromise health. Currently adipose tissue is known as an endocrine organ (Trayhurn, 2001). Adipocyte cells secrets several adipokines like: leptin, adiponectin, inflammatory cytokine (TNF-alpha, IL-6) and PCR that can change: glucose homeostasis, energy homeostasis, lipid metabolism, vascular homeostasis and stimulate inflammatory state (Arner, 2003). Besides adipose tissue secretion it is proposed that gut tract secret incretins. One of them is GLP-1, which is secreted by hindgut and has anti-diabetic effect. There is also ghrelin secretion that is a hormone produced by the stomach, and it is related to hunger control and regulation of energy homeostasis (Cummings, 2001). In the present study we evaluated the effect of weight loss by diet restriction and induced by bariatric surgery analyzing levels of: insulin, glucose, C-peptide GLP-1, inflammatory cytokines, ghrelin, adiponectin, PCR besides analysis of correlation between these variables, in obese and diabetic obese. We studied 11 morbidly obese patients with normal glucose tolerance (group NGT) and 8 patients who besides obesity had type 2 Diabetes Mellitus or were glucose intolerant (group AMG: with 4 patients with type 2 DM and 4 intolerant patients). Both groups were submitted to bariatric surgery Fobi-Capella technique and OGTT at 3 periods: first period, pre-operative, and 9° post-operative. At 3° and 6° months of post-operative period had only fasting levels. Before surgery the patients were characterized by having a BMI 46.1 ± 2.27 kg/m2 for NGT group and 51.20 ± 4.6 kg/m2 for AMG, as well as the high percentage of fat mass and waist circumference. The individuals at the beginning of the study, presented high insulin levels 21.85 ± 2.93 uU/ml and 32.95 ± 9.66 uU/ml for NGT and AMG respectively, and both had a major reduction 9 months after of surgery. Glucose levels reduced mainly in AMG group and presented resulted similar to group NGT at T5: 77,7 ± 1,88 mg/dl for NGT and 70,93 ± 6,01 mg/dl for AMG. HOMA-IR reduced at post-operative period, and the major reduction was in AMG group, T1: 9,85 ± 3,67; T2: 4,67 ± 1,34 and T5: 1,73±0,24 . GLP-1 levels had a significant increased at post-operative period (9° month) for NGT and AMG groups, and it was at the same moment we observe the improvement on insulin sensitivity, AMG group had major secretion of GLP-1 at period T5 at 30 minutes 34,06 ± 6,18 pmol/l when compared to 22,69 ± 4,04 pmol/l for NGT. Adiponectin levels increased after weight loss by surgery, T1 6,43 ± 1,23 ug/ml; T2 8,35 ± 1,48 ug/ml; T5 17,17 ± 4,89 ug/ml for NGT and T1 5,04 ± 1,2 ug/ml; T2 7,12 ± 1,73 ug/ml; T5 15,43 ± 5,52 ug/ml for AMG . From these results the present study concluded that weight loss by diet restriction need to be more expressive to notice some metabolic improvement as it was observed by surgical weight loss. We demonstrate that surgical weight loss, especially in AMG group,lead a normalization of glucose curve, insulin and C-peptide, and also a reduction of the insulin resistance, as well as GLP-1 and adiponectin improvement even though they hadn't weight stabilization. This also leads to the improvement of the carbohydrates metabolism, insulin sensitivity, contributing for the reduction type 2 DM risk. This improvement, also, was expressed by the reduction on inflammatory state, as well as in the improvement of adiponectin and GLP-1 levels. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

A Doença de Alzheimer (DA) é a causa mais comum de demência e é caracterizada clinicamente por comprometimentos cognitivos. Histologicamente é caracterizada pela formação de placas senis e de emaranhados neurofibrilares intracelulares resultantes de alterações do metabolismo do peptídeo A e da hiperfosforilação da proteína tau, respectivamente. Essas alterações parecem, em parte, ser uma decorrência de uma deficiência na sinalização da insulina e conseqüente resistência do encéfalo a esse hormônio, sugerindo que a DA esporádica tenha uma relação com o Diabetes mellitus. A estreptozotocina tem sido utilizada como modelo de indução do Diabetes, e mais recentemente como modelo experimental da DA quando administrado intracerebroventricular. Nosso objetivo nesse estudo foi o de caracterizar o esse modelo experimental da DA induzido pela estreptozotocina, avaliando as conseqüências agudas e a longo prazo. Foram utilizados ratos Wistar machos de quatro meses de idade que receberam injeções intracerebroventriculares bilaterais de estreptozotocina ou de veículo. Os animais foram avaliados aguda e cronicamente por testes comportamentais de memória de referência e operacional utilizando o modelo do labirinto aquático de Morris que visavam avaliar o curso temporal dos prejuízos cognitivos após a injeção da droga. Em diferentes tempos após as injeções, os ratos foram sacrificados e regiões do encéfalo submetidas à técnica de immunoblotting para avaliação de proteínas indicadoras de neurodegeneração ou à técnica histoquimica pelo método de Fluoro-Jade C. A avaliação da memória operacional em períodos agudos mostrou que os prejuízos cognitivos parecem se instalar a partir de 3 horas da injeção de estreptozotocina. A avaliação crônica das memórias operacional e de referência mostrou que os ratos exibiram um prejuízo marcante no desempenho dessas tarefas ao longo dos testes, embora seja correto afirmar que esses animais ainda são capazes de adquirir informação relevante com relação à execução da tarefa, particularmente na versão de memória de referência. A análise de immunoblotting mostrou haver aumento da expressão do peptídeo beta amilóide significante em regiões como amigdala, córtex entorrinal, núcleos da base e do hipotálamo. Também foi observado um aumento significativo da fosforilação da proteína tau na amigdala, cerebelo, córtex, prosencéfalo basal e núcleos da base. Foi observada uma diminuição da enzima de síntese de acetilcolina, a colina acetil-transferase apenas na amigdala. Fibras em degeneração foram observadas no hipotálamo, na área septal e em neurônios piramidais na região CA1 após 1 dia da injeção de estreptozotocina. Já após 15 dias da injeção podemos observar marcação em neurônios do estriado e da região CA1 do hipocampo e em fibras próximas ao giro denteado. Em resumo a injeção intracerebroventricular de estreptozotocina parece produzir um bom modelo experimental da DA, pois reproduz as características cognitivas e histológicas encontradas nos pacientes com a doença / Alzheimer´s disease (AD) is the most common cause of dementia in aged humans. Recent reports have suggested a relationship between the onset of AD and an insulin-resistant brain condition. In this context, this study aimed at evaluating the effects of intracerebroventricular (ICV) injection of streptozotocin (STZ) in rats on behavior and neurodegeneration. Four month-old adult male Wistar rats were subjected to bilateral ICV injections of either STZ or vehicle and were tested for both reference and working memories in Morris water maze. After different survival times, rats were subjected to immunoblotting (to evaluate neurodegeneration markers) or to Fluoro-Jade C histochemistry. A marked disruption of performance in working memory was already observed after 3 hours of STZ injections. Immunoblotting analysis showed a significant increase of beta amyloid peptide expression in the amygdala, entorrinal cortex, basal ganglia, and hypothalamus. A significant increase of tau phosphorylation was also observed in the amygdala, cerebellum, cortex, basal forebrain and basal ganglia. Degenerating fibers were seen in the hypothalamus and septal area 1 day postinjection and in CA1 pyramidal neurons and close to the hippocampal dentate gyrus after 15 days. ICV injection of STZ seems therefore to produce an animal model of AD, as it reproduces the characteristic cognitive and histological changes of the disease

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Background: Cardiovascular diseases are one of the most common causes of death worldwide. This is not only a problem in developed countries, it is of major concern for public health in developing countries as well. Increased homocysteine is an independent risk factor for cardiovascular diseases. Nutritional deficiencies of folate, vitamin B6 and vitamin B12 are associated with hyperhomocysteinemia. MTHFR A1298C, a single nucleotide polymorphism, is similarly linked with higher concentrations of homocysteine. The aim of this study was to determine the prevalence of MTHFR A1298C in a black elderly population, along with folate, vitamin B6 and vitamin B12 and to evaluate the effect on homocysteine levels. Methodology: The research design was an observational cross-sectional study and was ethically approved. A total of 84 elderly who attend a day-care centre (also met inclusion criteria) were purposively selected. DNA was extracted and frozen on the day of blood collection. The MTHFR A1298C genotype was determined with real time PCR. Homocysteine, folate, vitamin B6 and vitamin B12 serum levels were detected with commercial assay kits. Results: Homocysteine was found to be elevated with a median of 17.78 µmol/L (interquartile range 13.98-21.03 µmol/L). Serum folate, vitamin B6 and vitamin B12 medians were in the normal range. Although, 5.95% and 22.62% of the population were deficient and possibly deficient for vitamin B12, respectively. MTHFR A1298C frequency was as follow: 89.29% (AA), 9.52% (AC) and 1.19% (CC), with no significant correlation (p>0.05) with homocysteine. Vitamin B12 correlated significantly with homocysteine levels. Conclusion: Vitamin B12 deficiency had an effect on homocysteine levels. Overall, nutritional deficiencies are not responsible for the hyperhomocysteinemia in this population. In conclusion from this study showed MTHFR A1298C frequency in black South Africans does not contribute to homocysteine as a risk factor for cardiovascular disease. Keywords: Cardiovascular disease, elderly, folate, homocysteine, MTHFR A1298C, vitamin B6, vitamin B12.

No description available.

Brown adipose tissue (BAT) serves as a crucial thermogenic organ, extracting glucose and lipids from circulation to generate heat. Enhancing BAT activity holds potential as a therapy for treating metabolic diseases, such as obesity and diabetes. The sympathetic nervous system (SNS) is the main regulator of BAT activity by increasing extraction and oxidation of substrates. However, the SNS role in metabolic disorders is complex. In obesity, there is increased sympathetic tone, yet reduced BAT responsiveness. Furthermore, increasing systemic sympathetic tone in individuals already at heightened cardiovascular risk leads to adverse complications, as demonstrated by recent clinical trials. As a result, BAT’s impact on overall health in humans has been challenged in recent years, largely due to the lack of methods to selectively activate BAT without affecting other organs. Here, I used chemogenetics and retrograde viral injections in the interscapular BAT (iBAT) of mice to selectively activate only the neurons projecting to this tissue. Targeted activation of BAT did increase thermogenesis and improved glucose homeostasis. Leveraging on the single-cell RNA sequencing from our laboratory, we identified two sympathetic populations innervating iBAT: one primarily targets the small arterioles, while the other innervates the parenchyma. These populations mediate non-overlapping sympathetic-functions in iBAT: activating only the vascular projecting neurons lowers blood glucose without affecting thermogenesis, while activating the other population results in increased energy expenditure, local thermogenesis, and blood flow, with no effect on glycemia. The findings from this work could pave the way to the development of targeted strategies against metabolic disorders characterized by hyperglycemia, highlighting the potential of selectively activating specific SNS components to normalize blood glucose levels.