Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

University of Pittsburgh

Importance of Substrate-Coupled Proton Antiport by the Vesicular Monoamine Transporter in the Actions of Amphetamines in  Brain.

Antipsychotic drugs are widely prescribed medications, used for numerous psychiatric illnesses. However, antipsychotic drugs cause serious metabolic side effects that can lead to substantial weight gain and increased risk for type 2 diabetes. While individual drugs differ, all antipsychotic drugs may cause these important side effects to varying degrees. Given that the single unifying property shared by these medications is blockade of dopamine D and D receptors, these receptors likely play a role in antipsychotic drug-induced metabolic side effects. Dopamine D and dopamine D receptors are expressed in brain regions critical for metabolic regulation and appetite. Surprisingly, these receptors are also expressed peripherally in insulin-secreting pancreatic beta cells. By inhibiting glucose-stimulated insulin secretion, dopamine D and dopamine D receptors are important mediators of pancreatic insulin release. Crucially, antipsychotic drugs disrupt this peripheral metabolic regulatory mechanism. At the same time, disruptions to circadian timing have been increasingly recognized as a risk factor for metabolic disturbance. Reciprocal dopamine and circadian signaling is important for the timing of appetitive/feeding behaviors and insulin release, thereby coordinating cell metabolism with caloric intake. In particular, circadian regulation of dopamine D receptor/dopamine D receptor signaling may play a critical role in metabolism. Therefore, we propose that antipsychotic drugs' blockade of dopamine D receptor and dopamine D receptors in pancreatic beta cells, hypothalamus, and striatum disrupts the cellular timing mechanisms that regulate metabolism. Ultimately, understanding the relationships between the dopamine system and circadian clocks may yield critical new biological insights into mechanisms of antipsychotic drug action, which can then be applied into clinical practice.

Dopamine D receptors and the circadian clock reciprocally mediate antipsychotic drug-induced metabolic disturbances.

Among the critically ill, infectious diseases can play a significant role in the etiology of neuropsychiatric disturbances. All critical care physicians are familiar with delirium as a secondary complication of systemic infection. This article focuses on key infectious diseases that commonly and directly produce neuropsychiatric symptoms, including direct infection of the central nervous system, human immunodeficiency virus infection, and AIDS.

Neuropsychiatric Aspects of Infectious Diseases: An Update.

For decades, there have been observations demonstrating significant metabolic disturbances in people with schizophrenia including clinically relevant weight gain, hypertension, and disturbances in glucose and lipid homeostasis. Many of these findings pre-date the use of antipsychotic drugs (APDs) which on their own are also strongly associated with metabolic side effects. The combination of APD-induced metabolic changes and common adverse environmental factors associated with schizophrenia have made it difficult to determine the specific contributions of each to the overall metabolic picture. Data from drug-naïve patients, both from the pre-APD era and more recently, suggest that there may be an intrinsic metabolic risk associated with schizophrenia. Nevertheless, these findings remain controversial due to significant clinical variability in both psychiatric and metabolic symptoms throughout patients' disease courses. Here, we provide an extensive review of classic and more recent literature describing the metabolic phenotype associated with schizophrenia. We also suggest potential mechanistic links between signaling pathways associated with schizophrenia and metabolic dysfunction. We propose that, beyond its symptomatology in the central nervous system, schizophrenia is also characterized by pathophysiology in other organ systems directly related to metabolic control.

Intrinsic and Antipsychotic Drug-Induced Metabolic Dysfunction in Schizophrenia.

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the β-amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease.

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells.

Pathogen manipulation of host metabolism: A common strategy for immune evasion.

Parkinson's disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. Here, we addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.

Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons.

Second generation antipsychotics are prescribed for an increasing number of psychiatric conditions, despite variable associations with weight gain, dyslipidemia, and impaired glucose tolerance. The mechanism(s) of the apparent causal relationships between these medications and metabolic effects have been inadequately defined and are potentially confounded by genetic risk of mental illness, attendant lifestyle, and concomitant medications. Therefore, we conducted a study in which 24 healthy volunteers were randomized to olanzapine (highly weight-gain liability), iloperidone (less weight-gain liability), or placebo treatment for 28 days under double-blind conditions. We hypothesized that antipsychotics induce weight gain primarily through increased caloric intake, which causes secondary dyslipidemia and insulin resistance. Subjects were phenotyped pre- and post-treatment for body weight, adiposity by dual energy X-ray absorptiometry, energy expenditure by indirect calorimetry, food intake, oral glucose tolerance, plasma lipids, glucose, insulin, and other hormones. We found significantly increased food intake and body weight but no change in energy expenditure in olanzapine-treated subjects, with associated trends towards lipid abnormalities and insulin resistance the extent of which were presumably limited by the duration of treatment. Iloperidone treatment led to modest non-significant and placebo no weightgain, lipid increases and alterations in insulin metabolism. We conclude that second generation antipsychotic drugs, as represented by olanzapine, produce their weight and metabolic effects, predominantly, by increasing food intake which leads to weight gain that in turn induces metabolic consequences, but also through other direct effects on lipid and glucose metabolism independant of food intake and weight gain.

Pathophysiology of drug induced weight and metabolic effects: findings from an RCT in healthy volunteers treated with olanzapine, iloperidone, or placebo.

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods.

Zachary Freyberg

Department of Psychiatry,
Department of Cell Biology

University of Pittsburgh

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Zachary Freyberg

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Psychiatry,Department of Cell Biology

University of Pittsburgh

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Department of Psychiatry,
Department of Cell Biology