ABSTRACT
The TGFß cytokine family member, GDF-15, reduces food intake and body weight and represents a potential treatment for obesity. Because the brainstem-restricted expression pattern of its receptor, GDNF Family Receptor α-like (GFRAL), presents an exciting opportunity to understand mechanisms of action for area postrema neurons in food intake; we generated GfralCre and conditional GfralCreERT mice to visualize and manipulate GFRAL neurons. We found infection or pathophysiologic states (rather than meal ingestion) stimulate GFRAL neurons. TRAP-Seq analysis of GFRAL neurons revealed their expression of a wide range of neurotransmitters and neuropeptides. Artificially activating GfralCre -expressing neurons inhibited feeding, decreased gastric emptying, and promoted a conditioned taste aversion (CTA). GFRAL neurons most strongly innervate the parabrachial nucleus (PBN), where they target CGRP-expressing (CGRPPBN) neurons. Silencing CGRPPBN neurons abrogated the aversive and anorexic effects of GDF-15. These findings suggest that GFRAL neurons link non-meal-associated pathophysiologic signals to suppress nutrient uptake and absorption.
Subject(s)
Avoidance Learning/drug effects , Eating/drug effects , Feeding Behavior/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Growth Differentiation Factor 15/pharmacology , Neurons/physiology , Parabrachial Nucleus/physiology , Animals , Body Weight , Female , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Male , Mice , Neurons/drug effects , Parabrachial Nucleus/drug effects , Rats , Rats, Long-EvansABSTRACT
Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grains, promotes nausea, emesis and anorexia. With DON exposure, circulating levels of intestinally derived satiation hormones, including glucagon-like peptide 1 (GLP-1) are elevated. To directly test whether GLP-1 signaling mediates the effects of DON, we examined the response of GLP-1 or GLP-1R-deficient mice to DON injection. We found comparable anorectic and conditioned taste avoidance learning responses in GLP-1/GLP-1R deficient mice compared to control littermates, suggesting that GLP-1 is not necessary for the effects of DON on food intake and visceral illness. We then used our previously published data from translating ribosome affinity purification with RNA sequencing (TRAP-seq) analysis of area postrema neurons that express the receptor for the circulating cytokine growth differentiation factor (GDF15), growth differentiation factor a-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, calcium sensing receptor (CaSR), is heavily enriched in GFRAL neurons. Given that GDF15 potently reduces food intake and can cause visceral illness by signaling through GFRAL neurons, we hypothesized that DON may also signal by activating CaSR on GFRAL neurons. Indeed, circulating GDF15 levels are elevated after DON administration but both GFRAL knockout and GFRAL neuron-ablated mice exhibited similar anorectic and conditioned taste avoidance responses compared to WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.
ABSTRACT
Growth differentiation factor 15 (GDF15), a TGFß superfamily cytokine, acts through its receptor, cell line-derived neurotrophic factorfamily receptor α-like (GFRAL), to suppress food intake and promote nausea. GDF15 is broadly expressed at low levels but increases in states of disease such as cancer, cachexia, and sepsis. Whether GDF15 is necessary for inducing sepsis-associated anorexia and body weight loss is currently unclear. To test this we used a model of moderate systemic infection in GDF15KO and GFRALKO mice with lipopolysaccharide (LPS) treatment to define the role of GDF15 signaling in infection-mediated physiologic responses. Since physiological responses to LPS depend on housing temperature, we tested the effects of subthermoneutral and thermoneutral conditions on eliciting anorexia and inducing GDF15. Our data demonstrate a conserved LPS-mediated increase in circulating GDF15 levels in mouse, rat, and human. However, we did not detect differences in LPS-induced anorexia between WT and GDF15KO or GFRALKO mice. Furthermore, there were no differences in anorexia or circulating GDF15 levels at either thermoneutral or subthermoneutral housing conditions in LPS-treated mice. These data demonstrate that GDF15 is not necessary to drive food intake suppression in response to moderate doses of LPS.NEW & NOTEWORTHY Although many responses to LPS depend on housing temperature, the anorexic response to LPS does not. LPS results in a potent and rapid increase in circulating levels of GDF15 in mice, rats, and humans. Nevertheless, GDF15 and its receptor (GFRAL) are not required for the anorexic response to systemic LPS administration. The anorexic response to LPS likely involves a myriad of complex physiological alterations.
Subject(s)
Anorexia/metabolism , Growth Differentiation Factor 15/drug effects , Growth Differentiation Factor 15/metabolism , Lipopolysaccharides/pharmacology , Animals , Eating/drug effects , Humans , Mice , Nausea/chemically induced , Rats , Weight Loss/drug effectsABSTRACT
Cytosolic calcium influx activates signaling pathways known to support pancreatic beta cell function and survival by modulating gene expression. Impaired calcium signaling leads to decreased beta cell mass and diabetes. To appreciate the causes of these cytotoxic perturbations, a more detailed understanding of the relevant signaling pathways and their respective gene targets is required. In this study, we examined the calcium-induced expression of the cytoprotective beta cell transcription factor Npas4. Pharmacological inhibition implicated the calcineurin, Akt/protein kinase B, and Ca(2+)/calmodulin-dependent protein kinase signaling pathways in the regulation of Npas4 transcription and translation. Both Npas4 mRNA and protein had high turnover rates, and, at the protein level, degradation was mediated via the ubiquitin-proteasome pathway. Finally, beta cell cytotoxicity of the calcineurin inhibitor and immunosuppressant tacrolimus (FK-506) was prevented by Npas4 overexpression. These results delineate the pathways regulating Npas4 expression and stability and demonstrate its importance in clinical settings such as islet transplantation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Calcium Signaling/drug effects , Cytotoxins/adverse effects , Gene Expression Regulation/drug effects , Insulin-Secreting Cells/metabolism , Tacrolimus/adverse effects , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Calcineurin/genetics , Calcineurin/metabolism , Cytotoxins/pharmacology , Insulin-Secreting Cells/pathology , Male , Mice , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Stability/drug effects , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Tacrolimus/pharmacology , Ubiquitin/genetics , Ubiquitin/metabolismABSTRACT
Infradian mood and sleep-wake rhythms with periods of 48 hr and beyond have been observed in bipolar disorder (BD) subjects that even persist in time isolation, indicating an endogenous origin. Here we show that mice exposed to methamphetamine (Meth) in drinking water develop infradian locomotor rhythms with periods of 48 hr and beyond which extend to sleep length and mania-like behaviors in support of a model for cycling in BD. This cycling capacity is abrogated upon genetic disruption of DA production in DA neurons of the ventral tegmental area (VTA) or ablation of nucleus accumbens (NAc) projecting, dopamine (DA) neurons. Chemogenetic activation of NAc-projecting DA neurons leads to locomotor period lengthening in clock deficient mice, while cytosolic calcium in DA processes of the NAc was found fluctuating synchronously with locomotor behavior. Together, our findings argue that BD cycling relies on infradian rhythm generation that depends on NAc-projecting DA neurons.
ABSTRACT
While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) 'Driver' AAV and a tetracycline response element-driven, Cre-dependent 'Payload' AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.
The brain contains hundreds of types of neurons, which differ in size, shape and behavior. But neuroscientists often wish to study individual neuronal types in isolation. They are able to do this with the aid of a toolkit made up of two parts: viral vectors and genetically modified mice. Viral vectors are viruses that have been modified so that they are no longer harmful and can instead be used to introduce genetic material into cells on demand. To create a viral vector, the virus' own genetic material is replaced with a 'cargo' gene, such as the gene for a fluorescent protein. The virus is then introduced into a new host such as a mouse. Importantly, the virus only produces the protein encoded by its 'cargo' gene if it is inside a cell that also contains one of two specific enzymes. These enzymes are called Cre and Flp. This is where the second part of the toolkit comes in. Mice can be genetically engineered to produce either Cre or Flp exclusively in specific cell types. By introducing a viral vector into mice that produce either Cre or Flp only in one particular type of neuron, researchers can limit the activity of the cargo gene to that neuronal type. But sometimes even this approach is not selective enough. Researchers may wish to limit the activity of the cargo gene to a subpopulation of cells that produce Cre or Flp. Or they may wish to target only Cre- or Flp-producing cells in a small area of the brain, while leaving cells in neighboring areas unaffected. Sabatini et al. have now overcome this limitation by developing and testing a new set of viral vectors that are active only in neurons that produce both Cre and Flp. The vectors are called tTARGIT AAVs and allow researchers to target cells more precisely than was possible with the previous version of the toolkit. Sabatini et al. show tTARGIT AAVs in action by using them to identify a group of neurons that control how much energy mice use and how much food they eat. As well as applying the vectors to their own research on obesity, Sabatini et al. have also made them freely available for other researchers to use in their own projects.
Subject(s)
Gene Expression , Neurons/physiology , Transgenes , Animals , Dependovirus/genetics , Female , Male , Mice , Mice, TransgenicABSTRACT
The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing translating ribosome affinity purification with RNA-sequencing (TRAP-seq) data with single-nucleus RNA-sequencing (snRNA-seq) data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of six major genetically and anatomically differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.
Subject(s)
Multigene Family , Neurons/physiology , Transcriptome , Ventromedial Hypothalamic Nucleus/physiology , Animals , Cluster Analysis , Databases, Genetic , Gene Expression Profiling , Genotype , Glutamic Acid/metabolism , Macaca mulatta , Mice, Transgenic , Neurons/metabolism , Phenotype , RNA-Seq , Receptors, Leptin/genetics , Receptors, Leptin/metabolism , Species Specificity , Steroidogenic Factor 1/genetics , Steroidogenic Factor 1/metabolism , Ventromedial Hypothalamic Nucleus/cytology , Ventromedial Hypothalamic Nucleus/metabolismABSTRACT
BACKGROUND: Leptin acts via its receptor, LepRb, on specialized neurons in the brain to modulate energy balance and glucose homeostasis. LepRbâSTAT3 signaling plays a crucial role in leptin action, but LepRb also mediates an additional as-yet-unidentified signal (Signal 2) that is important for leptin action. Signal 2 requires LepRb regions in addition to those required for JAK2 activation but operates independently of STAT3 and LepRb phosphorylation sites. METHODS: To identify LepRb sequences that mediate Signal 2, we used CRISPR/Cas9 to generate five novel mouse lines containing COOH-terminal truncation mutants of LepRb. We analyzed the metabolic phenotype and measures of hypothalamic function for these mouse lines. RESULTS: We found that deletion of LepRb sequences between residues 921 and 960 dramatically worsens metabolic control and alters hypothalamic function relative to smaller truncations. We also found that deletion of the regions including residues 1013-1053 and 960-1013 each decreased obesity compared to deletions that included additional COOH-terminal residues. CONCLUSIONS: LepRb sequences between residues 921 and 960 mediate the STAT3 and LepRb phosphorylation-independent second signal that contributes to the control of energy balance and metabolism by leptin/LepRb. In addition to confirming the inhibitory role of the region (residues 961-1013) containing Tyr985, we also identified the region containing residues 1013-1053 (which contains no Tyr residues) as a second potential mediator of LepRb inhibition. Thus, the intracellular domain of LepRb mediates multiple Tyr-independent signals.
Subject(s)
Receptors, Leptin/genetics , STAT3 Transcription Factor/metabolism , Amino Acid Sequence , Animals , CRISPR-Cas Systems/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Obesity/metabolism , Receptors, Leptin/metabolism , STAT3 Transcription Factor/genetics , Signal TransductionABSTRACT
To identify neurons that specifically increase blood glucose from among the diversely functioning cell types in the ventromedial hypothalamic nucleus (VMN), we studied the cholecystokinin receptor B-expressing (CCKBR-expressing) VMN targets of glucose-elevating parabrachial nucleus neurons. Activation of these VMNCCKBR neurons increased blood glucose. Furthermore, although silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose levels, silencing VMNCCKBR neurons decreased hIepatic glucose production, insulin-independently decreasing blood glucose without altering energy balance. Silencing VMNCCKBR neurons also impaired the counterregulatory response to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure. Hence, VMNCCKBR cells represent a specialized subset of VMN cells that function to elevate glucose. These cells not only mediate the allostatic response to hypoglycemia but also modulate the homeostatic setpoint for blood glucose in an insulin-independent manner, consistent with a role for the brain in the insulin-independent control of glucose homeostasis.
Subject(s)
Blood Glucose/metabolism , Neurons/metabolism , Ventromedial Hypothalamic Nucleus/metabolism , Animals , Female , Insulin/genetics , Insulin/metabolism , Male , Mice , Mice, TransgenicABSTRACT
BACKGROUND: The divalent cation Calcium (Ca2+) regulates a wide range of processes in disparate cell types. Within insulin-producing ß-cells, increases in cytosolic Ca2+ directly stimulate insulin vesicle exocytosis, but also initiate multiple signaling pathways. Mediated through activation of downstream kinases and transcription factors, Ca2+-regulated signaling pathways leverage substantial influence on a number of critical cellular processes within the ß-cell. Additionally, there is evidence that prolonged activation of these same pathways is detrimental to ß-cell health and may contribute to Type 2 Diabetes pathogenesis. SCOPE OF REVIEW: This review aims to briefly highlight canonical Ca2+ signaling pathways in ß-cells and how ß-cells regulate the movement of Ca2+ across numerous organelles and microdomains. As a main focus, this review synthesizes experimental data from in vitro and in vivo models on both the beneficial and detrimental effects of Ca2+ signaling pathways for ß-cell function and health. MAJOR CONCLUSIONS: Acute increases in intracellular Ca2+ stimulate a number of signaling cascades, resulting in (de-)phosphorylation events and activation of downstream transcription factors. The short-term stimulation of these Ca2+ signaling pathways promotes numerous cellular processes critical to ß-cell function, including increased viability, replication, and insulin production and secretion. Conversely, chronic stimulation of Ca2+ signaling pathways increases ß-cell ER stress and results in the loss of ß-cell differentiation status. Together, decades of study demonstrate that Ca2+ movement is tightly regulated within the ß-cell, which is at least partially due to its dual roles as a potent signaling molecule.
Subject(s)
Calcium/metabolism , Diabetes Mellitus, Type 2/metabolism , Insulin-Secreting Cells/metabolism , Animals , Calmodulin/metabolism , Cations, Divalent , Cell Proliferation , Cell Survival , Humans , Insulin/metabolism , Mice , NFATC Transcription Factors/metabolism , PhosphorylationABSTRACT
Depolarization of neuroendocrine cells results in calcium influx, which induces vesicle exocytosis and alters gene expression. These processes, along with the restoration of resting membrane potential, are energy intensive. We hypothesized that cellular mechanisms exist to maximize energy production during excitation. Here, we demonstrate that NPAS4, an immediate early basic helix-loop-helix (bHLH)-PAS transcription factor, acts to maximize energy production by suppressing hypoxia-inducible factor 1α (HIF1α). As such, knockout of Npas4 from insulin-producing ß cells results in reduced OXPHOS, loss of insulin secretion, ß cell dedifferentiation, and type 2 diabetes. NPAS4 plays a similar role in the nutrient-sensing cells of the hypothalamus. Its knockout here results in increased food intake, reduced locomotor activity, and elevated peripheral glucose production. In conclusion, NPAS4 is critical for the coordination of metabolism during the stimulation of electrically excitable cells; its loss leads to the defects in cellular metabolism that underlie the cellular dysfunction that occurs in metabolic disease.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Hypothalamus/metabolism , Neuroendocrine Cells/metabolism , Oxidative Phosphorylation , Oxygen/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Hypothalamus/cytology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Transgenic , Neuroendocrine Cells/cytologyABSTRACT
As a sensory micro-organ, pancreatic ß-cells continually respond to nutritional signals and neuroendocrine input from other glucoregulatory organs. This sensory ability is essential for normal ß-cell function and systemic glucose homeostasis. Period circadian protein (Per)-aryl hydrocarbon receptor nuclear translocator protein (Arnt)-single-minded protein (Sim) (PAS) domain proteins have a conserved role as sensory proteins, critical in adaptation to changes in voltage, oxygen potential, and xenobiotics. Within ß-cells, PAS domain proteins such as hypoxia inducible factor 1α (Hif1α), Arnt, PAS kinase, Bmal1, and Clock respond to disparate stimuli, but act in concert to maintain proper ß-cell function. Elucidating the function of these factors in islets offers a unique insight into the sensing capacity of ß-cells, the consequences of impaired sensory function, and the potential to develop novel therapeutic targets for preserving ß-cell function in diabetes.
Subject(s)
Diabetes Mellitus/genetics , Insulin-Secreting Cells/pathology , Insulin-Secreting Cells/physiology , Transcription Factors/chemistry , Transcription Factors/physiology , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/chemistry , Aryl Hydrocarbon Receptor Nuclear Translocator/physiology , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/physiology , Diabetes Mellitus/metabolism , Diabetes Mellitus/physiopathology , Drosophila Proteins/chemistry , Drosophila Proteins/physiology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Nuclear Proteins/chemistry , Nuclear Proteins/physiology , Period Circadian Proteins/chemistry , Period Circadian Proteins/physiology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/physiology , Protein Structure, Tertiary , Von Hippel-Lindau Tumor Suppressor Protein/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/physiologyABSTRACT
Cellular homeostasis requires intrinsic sensing mechanisms to temper function in the face of prolonged activity. In the pancreatic ß-cell, glucose is likely a physiological trigger that activates an adaptive response to stimulation, thereby maintaining cellular homeostasis. Immediate early genes (IEGs) are activated as a first line of defense in cellular homeostasis and are largely responsible for transmitting an environmental cue to a cellular response. Here we examine the regulation and function of the novel ß-cell IEG, neuronal PAS domain protein 4 (Npas4). Using MIN6 cells, mouse and human islets, as well as in vivo infusions, we demonstrate that Npas4 is expressed within pancreatic islets and is upregulated by ß-cell depolarizing agents. Npas4 tempers ß-cell function through a direct inhibitory interaction with the insulin promoter and by blocking the potentiating effects of GLP-1 without significantly reducing glucose-stimulated secretion. Finally, Npas4 expression is induced by classical endoplasmic reticulum (ER) stressors and can prevent thapsigargin- and palmitate-induced dysfunction and cell death. These results suggest that Npas4 is a key activity-dependent regulator that improves ß-cell efficiency in the face of stress. We posit that Npas4 could be a novel therapeutic target in type 2 diabetes that could both reduce ER stress and cell death and maintain basal cell function.