Mechanisms of the pro-lipolytic and anti-obesity effects of the VGF-derived peptide TLQP-21

University of Minnesota Ph.D. dissertation. October 2015. Major: Integrative Biology and Physiology. Advisors: Alessandro Bartolomucci, John Osborn. 1 computer file (PDF); ix, 134 pages.Obesity is a major public health problem; in the U.S. nearly one third of the population is obese. The adipose tissue is innervated by the sympathetic nervous system (SNS), which regulates fat mass by playing a key role in initiating lipolysis and regulating lipid mobilization. TLQP-21, a 21 amino acid peptide encoded by the pre-pro-peptide VGF (non acronymic) expressed in the brain and sympathetic neurons innervating the adipose organ. Complement 3a receptor 1, C3aR1, is the target receptor for TLQP-21. Based on previous finding where peripheral TLQP-21 decreases adipocyte diameter and enhance β-adrenergic receptors (β-ARs) lipolysis in adipocytes the hypothesis tested in my thesis is that C3aR1 and β-ARs are required for TLQP-21 anti-obesity effects and the pro-lipolytic effects are mediated by increased mobilization of [Ca2+]i. Departing from the first experimental structural analysis of TLQP-21 in solution and receptor-bound state, we tested our hypothesis using a combined in vitro and in vivo approach. Using 3T3L1 cells and pre-adipocytes as our in vitro model, we determined that TLQP-21 enhances lipolysis via increased intracellular Ca2+ concentration [Ca2+]i, and activation of the MAPK/ERK pathway. The physiological effects of TLQP-21 were investigated in vivo using wild type, β-less (β1, β2, β3-AR KO) and C3aR1 KO mice. Chronic TLQP-21 treatment in obese wild type mice significantly decreased body weight and fat mass promoting an overall healthier metabolic phenotype. Conversely β-less and C3aR1 KO resulted fully resistant to the anti-obesity effects. Herein we identified the mechanism of TLQP-21 pro-lipolytic and anti-obesity effect and could thus be regarded as a novel target for pharmacotherapies of obesity

The TLQP-21 Peptide Activates the G-Protein-Coupled Receptor C3aR1 via a Folding-upon-Binding Mechanism

TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled-receptor (GPCR) Complement-3a-Receptor1 (C3aR1). So far, the mechanisms of TLQP-21-induced receptor activation remained unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation, upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C-terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Interestingly, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions

Loss of Gnas Imprinting Differentially Affects REM/NREM Sleep and Cognition in Mice

It has been suggested that imprinted genes are important in the regulation of sleep. However, the fundamental question of whether genomic imprinting has a role in sleep has remained elusive up to now. In this work we show that REM and NREM sleep states are differentially modulated by the maternally expressed imprinted gene Gnas. In particular, in mice with loss of imprinting of Gnas, NREM and complex cognitive processes are enhanced while REM and REM–linked behaviors are inhibited. This is the first demonstration that a specific overexpression of an imprinted gene affects sleep states and related complex behavioral traits. Furthermore, in parallel to the Gnas overexpression, we have observed an overexpression of Ucp1 in interscapular brown adipose tissue (BAT) and a significant increase in thermoregulation that may account for the REM/NREM sleep phenotypes. We conclude that there must be significant evolutionary advantages in the monoallelic expression of Gnas for REM sleep and for the consolidation of REM–dependent memories. Conversely, biallelic expression of Gnas reinforces slow wave activity in NREM sleep, and this results in a reduction of uncertainty in temporal decision-making processes

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.ISSN:0092-8674ISSN:1097-417

Metabolic Consequences and Vulnerability to Diet-Induced Obesity in Male Mice under Chronic Social Stress

Social and psychological factors interact with genetic predisposition and dietary habit in determining obesity. However, relatively few pre-clinical studies address the role of psychosocial factors in metabolic disorders. Previous studies from our laboratory demonstrated in male mice: 1) opposite status-dependent effect on body weight gain under chronic psychosocial stress; 2) a reduction in body weight in individually housed (Ind) male mice. In the present study these observations were extended to provide a comprehensive characterization of the metabolic consequences of chronic psychosocial stress and individual housing in adult CD-1 male mice. Results confirmed that in mice fed standard diet, dominant (Dom) and Ind had a negative energy balance while subordinate (Sub) had a positive energy balance. Locomotor activity was depressed in Sub and enhanced in Dom. Hyperphagia emerged for Dom and Sub and hypophagia for Ind. Dom also showed a consistent decrease of visceral fat pads weight as well as increased norepinephrine concentration and smaller adipocytes diameter in the perigonadal fat pad. On the contrary, under high fat diet Sub and, surprisingly, Ind showed higher while Dom showed lower vulnerability to obesity associated with hyperphagia. In conclusion, we demonstrated that social status under chronic stress and individual housing deeply affect mice metabolic functions in different, sometime opposite, directions. Food intake, the hedonic response to palatable food as well as the locomotor activity and the sympathetic activation within the adipose fat pads all represent causal factors explaining the different metabolic alterations observed. Overall this study demonstrates that pre-clinical animal models offer a suitable tool for the investigation of the metabolic consequences of chronic stress exposure and associated psychopathologies

The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis

Objectives: Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. Methods: We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, β1, β2, β3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. Results: We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances β-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving β-adrenergic and C3a receptor activation without associated adverse metabolic effects. Conclusions: In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules. Author Video: Author Video Watch what authors say about their articles Keywords: Adipocyte, Complement 3a receptor, Ca2+, MAPK/ERK, β-AR, Isoproterenol, VG

Fear conditioning performance.

<p>(a) Representation of the FC protocol over three days and three different experimental phases (Conditioning, Context and Cue). (b) Mean percentage values of freezing behavior ± s.e.m. are plotted for each experimental phase. Statistics are reported as following: <i>t</i>-test; * = <i>P</i><0.05.</p

Behavioral consequences of chronic psychosocial social stress in mice.

<p>A) Aggressive behavior assessed on days 1 to 4, 10 and 20 of the stress phase. Graph clearly shows how dominants (Dom) and subordinates (Sub) are non-overlapping behavioral categories. B) Locomotor activity measured during baseline (4 days) and the stress phase (20 days). Dom showed increased and Sub showed decreased locomotor activity (F(1,18) = 21.9, p<0.01). C) Locomotor activity measured before and after the daily agonistic interaction. Dom showed increased activity both before and after the agonistic interaction while Sub showed increased activity before but not after the agonistic interaction (F(1,18) = 4.1, p = 0.054). * p<0.05 and ** p<0.001 vs. basal, # p<0.05 vs. Dom.</p