EPAS1 Attenuates Atherosclerosis Initiation at Disturbed Flow Sites Through Endothelial Fatty Acid Uptake.

BACKGROUND: Atherosclerotic plaques form unevenly due to disturbed blood flow, causing localized endothelial cell (EC) dysfunction. Obesity exacerbates this process, but the underlying molecular mechanisms are unclear. The transcription factor EPAS1 (HIF2A) has regulatory roles in endothelium, but its involvement in atherosclerosis remains unexplored. This study investigates the potential interplay between EPAS1, obesity, and atherosclerosis. METHODS: Responses to shear stress were analyzed using cultured porcine aortic EC exposed to flow in vitro coupled with metabolic and molecular analyses and by en face immunostaining of murine aortic EC exposed to disturbed flow in vivo. Obesity and dyslipidemia were induced in mice via exposure to a high-fat diet or through Leptin gene deletion. The role of Epas1 in atherosclerosis was evaluated by inducible endothelial Epas1 deletion, followed by hypercholesterolemia induction (adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9]; high-fat diet). RESULTS: En face staining revealed EPAS1 enrichment at sites of disturbed blood flow that are prone to atherosclerosis initiation. Obese mice exhibited substantial reduction in endothelial EPAS1 expression. Sulforaphane, a compound with known atheroprotective effects, restored EPAS1 expression and concurrently reduced plasma triglyceride levels in obese mice. Consistently, triglyceride derivatives (free fatty acids) suppressed EPAS1 in cultured EC by upregulating the negative regulator PHD2. Clinical observations revealed that reduced serum EPAS1 correlated with increased endothelial PHD2 and PHD3 in obese individuals. Functionally, endothelial EPAS1 deletion increased lesion formation in hypercholesterolemic mice, indicating an atheroprotective function. Mechanistic insights revealed that EPAS1 protects arteries by maintaining endothelial proliferation by positively regulating the expression of the fatty acid-handling molecules CD36 (cluster of differentiation 36) and LIPG (endothelial type lipase G) to increase fatty acid beta-oxidation. CONCLUSIONS: Endothelial EPAS1 attenuates atherosclerosis at sites of disturbed flow by maintaining EC proliferation via fatty acid uptake and metabolism. This endothelial repair pathway is inhibited in obesity, suggesting a novel triglyceride-PHD2 modulation pathway suppressing EPAS1 expression. These findings have implications for therapeutic strategies addressing vascular dysfunction in obesity.

Preclinical evaluation of tissue-selective gene therapies for congenital generalised lipodystrophy.

Lipodystrophy is a rare disorder which can be life-threatening. Here individuals fail to develop or maintain appropriate adipose tissue stores. This typically causes severe metabolic complications, including hepatic steatosis and lipoatrophic diabetes. There is no cure for lipodystrophy, and treatment options remain very limited. Here we evaluate whether tissue-selective adeno-associated virus (AAV) vectors can provide a targeted form of gene therapy for lipodystrophy, using a preclinical lipodystrophic mouse model of Bscl2 deficiency. We designed AAV vectors containing the mini/aP2 or thyroxine-binding globulin promoter to selectively target adipose or liver respectively. The AAV-aP2 vectors also contained the liver-specific microRNA-122 target sequence, restricting hepatic transgene expression. Systemic delivery of AAV-aP2 vectors overexpressing human BSCL2 restored adipose tissue development and metabolic health in lipodystrophic mice without detectable expression in the liver. High doses (1 × 1012 GCs) of liver-selective vectors led to off target expression and adipose tissue development, whilst low doses (1 × 1010 GCs) expressed selectively and robustly in the liver but did not improve metabolic health. This reveals that adipose tissue-selective, but not liver directed, AAV-mediated gene therapy is sufficient to substantially recover metabolic health in generalised lipodystrophy. This provides an exciting potential new avenue for an effective, targeted, and thereby safer therapeutic intervention.

Disentangling the detrimental effects of local from systemic adipose tissue dysfunction on articular cartilage in the knee.

OBJECTIVE: Obesity increases osteoarthritis (OA) risk due to adipose tissue dysfunction with associated metabolic syndrome and excess weight. Lipodystrophy syndromes exhibit systemic metabolic and inflammatory abnormalities similar to obesity without biomechanical overloading. Here, we used lipodystrophy mouse models to investigate the effects of systemic versus intra-articular adipose tissue dysfunction on the knee. METHODS: Intra-articular adipose tissue development was studied using reporter mice. Mice with selective lipodystrophy of intra-articular adipose tissue were generated by conditional knockout (cKO) of Bscl2 in Gdf5-lineage cells, and compared with whole-body Bscl2 knockout (KO) mice with generalised lipodystrophy and associated systemic metabolic dysfunction. OA was induced by surgically destabilising the medial meniscus (DMM) and obesity by high-fat diet (HFD). Gene expression was analysed by quantitative RT-PCR and tissues were analysed histologically. RESULTS: The infrapatellar fat pad (IFP), in contrast to overlying subcutaneous adipose tissue, developed from a template established from the Gdf5-expressing joint interzone during late embryogenesis, and was populated shortly after birth by adipocytes stochastically arising from Pdgfrα-expressing Gdf5-lineage progenitors. While female Bscl2 KO mice with generalised lipodystrophy developed spontaneous knee cartilage damage, Bscl2 cKO mice with intra-articular lipodystrophy did not, despite the presence of synovial hyperplasia and inflammation of the residual IFP. Furthermore, male Bscl2 cKO mice showed no worse cartilage damage after DMM. However, female Bscl2 cKO mice showed increased susceptibility to the cartilage-damaging effects of HFD-induced obesity. CONCLUSION: Our findings emphasise the prevalent role of systemic metabolic and inflammatory effects in impairing cartilage homeostasis, with a modulatory role for intra-articular adipose tissue.

Up-regulation of SLC7A11/xCT creates a vulnerability to selenocystine-induced cytotoxicity.

The SLC7A11/xCT cystine and glutamate antiporter has emerged as an attractive target for cancer therapy due to its selective overexpression in multiple cancers and its role in preventing ferroptosis. Utilizing pharmacological and genetic approaches in hepatocellular carcinoma cell lines, we demonstrate that overexpression of SLC7A11 engenders hypersensitivity towards l-selenocystine, a naturally occurring diselenide that bears close structural similarity to l-cystine. We find that the abundance of SLC7A11 positively correlates with sensitivity to l-selenocystine, but surprisingly, not to Erastin, an inhibitor of SLC7A11 activity. Our data indicate that SLC7A11 acts as a transport channel for l-selenocystine, which preferentially incites acute oxidative stress and damage eventuating to cell death in cells that highly express SLC7A11. Hence, our findings raise the prospect of l-selenocystine administration as a novel strategy for targeting cancers that up-regulate SLC7A11 expression.

The altered lipidome of hepatocellular carcinoma.

Alterations in metabolic pathways are a hallmark of cancer. A deeper understanding of the contribution of different metabolites to carcinogenesis is thus vitally important to elucidate mechanisms of tumor initiation and progression to inform therapeutic strategies. Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide and its altered metabolic landscape is beginning to unfold with the advancement of technologies. In particular, characterization of the lipidome of human HCCs has accelerated, and together with biochemical analyses, are revealing recurrent patterns of alterations in glycerophospholipid, sphingolipid, cholesterol and bile acid metabolism. These widespread alterations encompass a myriad of lipid species with numerous roles affecting multiple hallmarks of cancer, including aberrant growth signaling, metastasis, evasion of cell death and immunosuppression. In this review, we summarize the current trends and findings of the altered lipidomic landscape of HCC and discuss their potential biological significance for hepatocarcinogenesis.

Functional activity of the caudate mediates the relation between early childhood microstructural variations and elevated metabolic syndrome scores.

BACKGROUND: Metabolic syndrome score in children assesses the risk of developing cardiovascular disease in future. We aim to probe the role of the caudate in relation to the metabolic syndrome score. Furthermore, using both functional and structural neuroimaging, we aim to examine the interplay between functional and structural measures. METHODS: A longitudinal birth cohort study with functional and structural neuroimaging data obtained at 4.5, 6.0 and 7.5 years and metabolic syndrome scores at 8.0 years was used. Pearson correlation and linear regression was used to test for correlation fractional anisotropy (FA) and fractional amplitude of low frequency fluctuations (fALFF) of the caudate with metabolic syndrome scores. Mediation analysis was used to test if later brain measures mediated the relation between earlier brain measures and metabolic syndrome scores. Inhibitory control was also tested as a mediator of the relation between caudate brain measures and metabolic syndrome scores. RESULTS: FA at 4.5 years and fALFF at 7.5 years of the left caudate was significantly correlated with metabolic syndrome scores. Post-hoc mediation analysis showed that fALFF at 7.5 years fully mediated the relation between FA at 4.5 years and metabolic syndrome scores. Inhibitory control was significantly correlated with fALFF at 7.5 years, but did not mediate the relation between fALFF at 7.5 years and metabolic syndrome scores. CONCLUSIONS: We found that variations in caudate microstructure at 4.5 years predict later variation in functional activity at 7.5 years. This later variation in functional activity fully mediates the relation between microstructural changes in early childhood and metabolic syndrome scores at 8.0 years.

Genetic Ablation of LAT1 Inhibits Growth of Liver Cancer Cells and Downregulates mTORC1 Signaling.

Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer.

222-Nanometer Far-UVC Exposure Results in DNA Damage and Transcriptional Changes to Mammalian Cells.

Ultraviolet (UV) germicidal tools have recently gained attention as a disinfection strategy against the COVID-19 pandemic, but the safety profile arising from their exposure has been controversial and impeded larger-scale implementation. We compare the emerging 222-nanometer far UVC and 277-nanometer UVC LED disinfection modules with the traditional UVC mercury lamp emitting at 254 nm to understand their effects on human retinal cell line ARPE-19 and HEK-A keratinocytes. Cells illuminated with 222-nanometer far UVC survived, while those treated with 254-nanometer and 277-nanometer wavelengths underwent apoptosis via the JNK/ATF2 pathway. However, cells exposed to 222-nanometer far UVC presented the highest degree of DNA damage as evidenced by yH2AX staining. Globally, these cells displayed transcriptional changes in cell-cycle and senescence pathways. Thus, the introduction of 222-nanometer far UVC lamps for disinfection purposes should be carefully considered and designed with the inherent dangers involved.

Reprogramming of mitochondrial proline metabolism promotes liver tumorigenesis.

Dysregulated cellular energetics has recently been recognized as a hallmark of cancer and garnered attention as a potential targeting strategy for cancer therapeutics. Cancer cells reprogram metabolic activities to meet bio-energetic, biosynthetic and redox requirements needed to sustain indefinite proliferation. In many cases, metabolic reprogramming is the result of complex interactions between genetic alterations in well-known oncogenes and tumor suppressors and epigenetic changes. While the metabolism of the two most abundant nutrients, glucose and glutamine, is reprogrammed in a wide range of cancers, accumulating evidence demonstrates that additional metabolic pathways are also critical for cell survival and growth. Proline metabolism is one such metabolic pathway that promotes tumorigenesis in multiple cancer types, including liver cancer, which is the fourth main cause of cancer mortality in the world. Despite the recent spate of approved treatments, including targeted therapy and combined immunotherapies, there has been no significant gain in clinical benefits in the majority of liver cancer patients. Thus, exploring novel therapeutic strategies and identifying new molecular targets remains a top priority for liver cancer. Two of the enzymes in the proline biosynthetic pathway, pyrroline-5-carboxylate reductase (PYCR1) and Aldehyde Dehydrogenase 18 Family Member A1 (ALDH18A1), are upregulated in liver cancer of both human and animal models, while proline catabolic enzymes, such as proline dehydrogenase (PRODH) are downregulated. Here we review the latest evidence linking proline metabolism to liver and other cancers and potential mechanisms of action for the proline pathway in cancer development.

Metabolic pathway analyses identify proline biosynthesis pathway as a promoter of liver tumorigenesis.

BACKGROUND & AIM: Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis. METHODS: We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo. RESULTS: The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome. CONCLUSION: Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. LAY SUMMARY: Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer.

Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle.

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

Multimodal imaging approach to monitor browning of adipose tissue in vivo.

The discovery that white adipocytes can undergo a browning process to become metabolically active beige cells has attracted significant interest in the fight against obesity. However, the study of adipose browning has been impeded by a lack of imaging tools that allow longitudinal and noninvasive monitoring of this process in vivo. Here, we report a preclinical imaging approach to detect development of beige adipocytes during adrenergic stimulation. In this approach, we expressed near-infrared fluorescent protein, iRFP720, driven under an uncoupling protein-1 (Ucp1) promoter in mice by viral transduction, and used multispectral optoacoustic imaging technology with ultrasound tomography (MSOT-US) to assess adipose beiging during adrenergic stimulation. We observed increased photoacoustic signal at 720 nm, coupled with attenuated lipid signals in stimulated animals. As a proof of concept, we validated our approach against hybrid positron emission tomography combined with magnetic resonance (PET/MR) imaging modality, and quantified the extent of adipose browning by MRI-guided segmentation of 2-deoxy-2-18F-fluoro-d-glucose uptake signals. The browning extent detected by MSOT-US and PET/MR are well correlated with Ucp1 induction. Taken together, these systems offer great opportunities for preclinical screening aimed at identifying compounds that promote adipose browning and translation of these discoveries into clinical studies of humans.

Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis.

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca(2+)-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural-functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.

Synaptotagmin-7 phosphorylation mediates GLP-1-dependent potentiation of insulin secretion from ß-cells.

Glucose stimulates insulin secretion from ß-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca(2+)-triggered exocytosis by direct phosphorylation of a synaptotagmin.

Epigenomic Control of Thermogenic Adipocyte Differentiation and Function.

Obesity and its associated metabolic disorders are spreading at a fast pace throughout the world; thus, effective therapeutic approaches are necessary to combat this epidemic. Obesity develops when there is a greater caloric intake than energy expenditure. Promoting energy expenditure has recently attracted much attention as a promising approach for the management of body weight. Thermogenic adipocytes are capable of burning fat to dissipate chemical energy into heat, thereby enhancing energy expenditure. After the recent re-discovery of thermogenic adipocytes in adult humans, much effort has focused on understanding the molecular mechanisms, especially the epigenetic mechanisms, which regulate thermogenic adipocyte development and function. A number of chromatin signatures, such as histone modifications, DNA methylation, chromatin accessibilities, and interactions, have been profiled at the genome level and analyzed in various murine and human thermogenic fat cell systems. Moreover, writers and erasers, as well as readers of the epigenome are also investigated using genomic tools in thermogenic adipocytes. In this review, we summarize and discuss the recent advance in these studies and highlight the insights gained into the epigenomic regulation of thermogenic program as well as the pathogenesis of human metabolic diseases.

An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.

A Critical Role for ß-Catenin in Modulating Levels of Insulin Secretion from ß-Cells by Regulating Actin Cytoskeleton and Insulin Vesicle Localization.

The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic ß-cells are only partly understood. Here we investigate the involvement of ß-catenin in these processes. Reducing ß-catenin levels using siRNA knockdown attenuated GSIS in a range of ß-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both ß-cell models and isolated islets by short-term exposure to the ß-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases ß-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that ß-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of ß-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating ß-catenin from the plasma membrane pool to the nucleus. We show that ß-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that ß-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that ß-catenin levels modulate Ca2+-dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion.

MEMRI detects neuronal activity and connectivity in hypothalamic neural circuit responding to leptin.

Hypothalamus plays the central role in regulating energy homeostasis. To understand the hypothalamic neurocircuit in responding to leptin, Manganese-Enhanced MRI (MEMRI) was applied. Highly elevated signal could be mapped in major nuclei of the leptin signaling pathway, including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH) in fasted mice and the enhancement was reduced by leptin administration. However, whether changes in MEMRI signal reflect Ca2+ channel activity, neuronal activation or connectivity in the leptin signaling pathway are not clear. By blocking L-type Ca2+ channels, the signal enhancement in the ARC, PVN and DMH, but not VMH, was reduced. By disrupting microtubule with colchicine, signal enhancement of the secondary neural areas like DMH and PVN was delayed which is consistent with the known projection density from ARC into these regions. Finally, strong correlation between c-fos expression and MEMRI signal increase rate was observed in the ARC, VMH and DMH. Together, we provide experimental evidence that MEMRI signal could represent activity and connectivity in certain hypothalamic nuclei and hence may be used for mapping activated neuronal pathway in vivo. This understanding would facilitate the application of MEMRI for evaluation of hypothalamic dysfunction in metabolic diseases.

Molecular regulation of insulin granule biogenesis and exocytosis.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia, insulin resistance and hyperinsulinemia in early disease stages but a relative insulin insufficiency in later stages. Insulin, a peptide hormone, is produced in and secreted from pancreatic ß-cells following elevated blood glucose levels. Upon its release, insulin induces the removal of excessive exogenous glucose from the bloodstream primarily by stimulating glucose uptake into insulin-dependent tissues as well as promoting hepatic glycogenesis. Given the increasing prevalence of T2DM worldwide, elucidating the underlying mechanisms and identifying the various players involved in the synthesis and exocytosis of insulin from ß-cells is of utmost importance. This review summarizes our current understanding of the route insulin takes through the cell after its synthesis in the endoplasmic reticulum as well as our knowledge of the highly elaborate network that controls insulin release from the ß-cell. This network harbors potential targets for anti-diabetic drugs and is regulated by signaling cascades from several endocrine systems.