AgRP Neurons Control Systemic Insulin Sensitivity via Myostatin Expression in Brown Adipose Tissue.

Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis.

Neonatal insulin action impairs hypothalamic neurocircuit formation in response to maternal high-fat feeding.

Maternal metabolic homeostasis exerts long-term effects on the offspring's health outcomes. Here, we demonstrate that maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is associated with an impairment of the hypothalamic melanocortin circuitry. Whereas the number and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiological properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These experiments reveal a critical timing, when altered maternal metabolism disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.

Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

Parallel pathways for serotonin biosynthesis and metabolism in C. elegans.

The neurotransmitter serotonin plays a central role in animal behavior and physiology, and many of its functions are regulated via evolutionarily conserved biosynthesis and degradation pathways. Here we show that in Caenorhabditis elegans, serotonin is abundantly produced in nonneuronal tissues via phenylalanine hydroxylase, in addition to canonical biosynthesis via tryptophan hydroxylase in neurons. Combining CRISPR-Cas9 genome editing, comparative metabolomics and synthesis, we demonstrate that most serotonin in C. elegans is incorporated into N-acetylserotonin-derived glucosides, which are retained in the worm body and further modified via the carboxylesterase CEST-4. Expression patterns of CEST-4 suggest that serotonin or serotonin derivatives are transported between different tissues. Last, we show that bacterial indole production interacts with serotonin metabolism via CEST-4. Our results reveal a parallel pathway for serotonin biosynthesis in nonneuronal cell types and further indicate that serotonin-derived metabolites may serve distinct signaling functions and contribute to previously described serotonin-dependent phenotypes.

Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b.

Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism. MicroRNA (miRNA)-dependent post-transcriptional gene silencing has been recognized recently to control gene expression in disease development and progression, including that of insulin-resistant type 2 diabetes. The deregulation of miRNAs miR-143 (ref. 4), miR-181 (ref. 5), and miR-103 and miR-107 (ref. 6) alters hepatic insulin sensitivity. Here we report that the expression of miR-802 is increased in the liver of two obese mouse models and obese human subjects. Inducible transgenic overexpression of miR-802 in mice causes impaired glucose tolerance and attenuates insulin sensitivity, whereas reduction of miR-802 expression improves glucose tolerance and insulin action. We identify Hnf1b (also known as Tcf2) as a target of miR-802-dependent silencing, and show that short hairpin RNA (shRNA)-mediated reduction of Hnf1b in liver causes glucose intolerance, impairs insulin signalling and promotes hepatic gluconeogenesis. In turn, hepatic overexpression of Hnf1b improves insulin sensitivity in Lepr(db/db) mice. Thus, this study defines a critical role for deregulated expression of miR-802 in the development of obesity-associated impairment of glucose metabolism through targeting of Hnf1b, and assigns Hnf1b an unexpected role in the control of hepatic insulin sensitivity.

A neurotransmitter atlas of C. elegans males and hermaphrodites.

Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the C. elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel neurons that uptake monoaminergic neurotransmitters. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.

Starvation-induced changes in somatic insulin/IGF-1R signaling drive metabolic programming across generations.

Exposure to adverse nutritional and metabolic environments during critical periods of development can exert long-lasting effects on health outcomes of an individual and its descendants. Although such metabolic programming has been observed in multiple species and in response to distinct nutritional stressors, conclusive insights into signaling pathways and mechanisms responsible for initiating, mediating, and manifesting changes to metabolism and behavior across generations remain scarce. By using a starvation paradigm in Caenorhabditis elegans, we show that starvation-induced changes in dauer formation-16/forkhead box transcription factor class O (DAF-16/FoxO) activity, the main downstream target of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling, are responsible for metabolic programming phenotypes. Tissue-specific depletion of DAF-16/FoxO during distinct developmental time points demonstrates that DAF-16/FoxO acts in somatic tissues, but not directly in the germline, to both initiate and manifest metabolic programming. In conclusion, our study deciphers multifaceted and critical roles of highly conserved insulin/IGF-1 receptor signaling in determining health outcomes and behavior across generations.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

SLC17A6/7/8 Vesicular Glutamate Transporter Homologs in Nematodes.

Members of the superfamily of solute carrier (SLC) transmembrane proteins transport diverse substrates across distinct cellular membranes. Three SLC protein families transport distinct neurotransmitters into synaptic vesicles to enable synaptic transmission in the nervous system. Among them is the SLC17A6/7/8 family of vesicular glutamate transporters, which endows specific neuronal cell types with the ability to use glutamate as a neurotransmitter. The genome of the nematode Caenorhabditis elegans encodes three SLC17A6/7/8 family members, one of which, eat-4/VGLUT, has been shown to be involved in glutamatergic neurotransmission. Here, we describe our analysis of the two remaining, previously uncharacterized SLC17A6/7/8 family members, vglu-2 and vglu-3 These two genes directly neighbor one another and are the result of a recent gene duplication event in C. elegans, but not in other Caenorhabditis species. Compared to EAT-4, the VGLU-2 and VGLU-3 protein sequences display a more distant similarity to canonical, vertebrate VGLUT proteins. We tagged both genomic loci with gfp and detected no expression of vglu-3 at any stage of development in any cell type of both C. elegans sexes. In contrast, vglu-2::gfp is dynamically expressed in a restricted set of distinct cell types. Within the nervous system, vglu-2::gfp is exclusively expressed in a single interneuron class, AIA, where it localizes to vesicular structures in the soma, but not along the axon, suggesting that VGLU-2 may not be involved in synaptic transport of glutamate. Nevertheless, vglu-2 mutants are partly defective in the function of the AIA neuron in olfactory behavior. Outside the nervous system, VGLU-2 is expressed in collagen secreting skin cells where VGLU-2 most prominently localizes to early endosomes, and to a lesser degree to apical clathrin-coated pits, the trans-Golgi network, and late endosomes. On early endosomes, VGLU-2 colocalizes most strongly with the recycling promoting factor SNX-1, a retromer component. Loss of vglu-2 affects the permeability of the collagen-containing cuticle of the worm, and based on the function of a vertebrate VGLUT1 protein in osteoclasts, we speculate that vglu-2 may have a role in collagen trafficking in the skin. We conclude that C. elegans SLC17A6/7/8 family members have diverse functions within and outside the nervous system.

Obesity exacerbates colitis-associated cancer via IL-6-regulated macrophage polarisation and CCL-20/CCR-6-mediated lymphocyte recruitment.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide in which the vast majority of cases exhibit little genetic risk but are associated with a sedentary lifestyle and obesity. Although the mechanisms underlying CRC and colitis-associated colorectal cancer (CAC) remain unclear, we hypothesised that obesity-induced inflammation predisposes to CAC development. Here, we show that diet-induced obesity accelerates chemically-induced CAC in mice via increased inflammation and immune cell recruitment. Obesity-induced interleukin-6 (IL-6) shifts macrophage polarisation towards tumour-promoting macrophages that produce the chemokine CC-chemokine-ligand-20 (CCL-20) in the CAC microenvironment. CCL-20 promotes CAC progression by recruiting CC-chemokine-receptor-6 (CCR-6)-expressing B cells and γδ T cells via chemotaxis. Compromised cell recruitment as well as inhibition of B and γδ T cells protects against CAC progression. Collectively, our data reveal a function for IL-6 in the CAC microenvironment via lymphocyte recruitment through the CCL-20/CCR-6 axis, thereby implicating a potential therapeutic intervention for human patients.

Mild Impairment of Mitochondrial OXPHOS Promotes Fatty Acid Utilization in POMC Neurons and Improves Glucose Homeostasis in Obesity.

Mitochondrial oxidative phosphorylation (OXPHOS) and substrate utilization critically regulate the function of hypothalamic proopiomelanocortin (POMC)-expressing neurons. Here, we demonstrate that inactivation of apoptosis-inducing factor (AIF) in POMC neurons mildly impairs mitochondrial respiration and decreases firing of POMC neurons in lean mice. In contrast, under diet-induced obese conditions, POMC-Cre-specific inactivation of AIF prevents obesity-induced silencing of POMC neurons, translating into improved glucose metabolism, improved leptin, and insulin sensitivity, as well as increased energy expenditure in AIFΔPOMC mice. On a cellular level, AIF deficiency improves mitochondrial morphology, facilitates the utilization of fatty acids for mitochondrial respiration, and increases reactive oxygen species (ROS) formation in POMC neurons from obese mice, ultimately leading to restored POMC firing upon HFD feeding. Collectively, partial impairment of mitochondrial function shifts substrate utilization of POMC neurons from glucose to fatty acid metabolism and restores their firing properties, resulting in improved systemic glucose and energy metabolism in obesity.

Olfactory Imprinting: A Worm's Memory of Things Past.

Exposure to distinct stimuli during critical periods of development can affect behavior long-term. A new study in Caenorhabditis elegans demonstrates that changes in neuronal activity of one synaptic connection following early-life pheromone exposure are sufficient to permanently enhance specific avoidance responses.

Single and Transient Ca2+ Peaks in Podocytes do not induce Changes in Glomerular Filtration and Perfusion.

Chronic alterations in calcium (Ca2+) signalling in podocytes have been shown to cause proteinuria and progressive glomerular diseases. However, it is unclear whether short Ca2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca2+ peaks contribute to podocyte disease.

Central insulin signaling modulates hypothalamus-pituitary-adrenal axis responsiveness.

OBJECTIVE: Obesity is often accompanied by hyperactivity of the neuroendocrine stress axis and has been linked to an increased risk of psychiatric disorders. Insulin is reciprocally regulated with the stress hormone corticosterone (CORT), raising the possibility that insulin normally provides inhibitory tone to the hypothalamus-adrenal-pituitary (HPA) axis. Here we examined whether disrupting signaling via the insulin receptor (InsR) in hypothalamic subpopulations impacts the neuroendocrine response to acute psychological stress. METHODS: We used Nkx2.1-Cre, Sim1-Cre and Agrp-Cre transgenic driver lines to generate conditional knockouts of InsR signaling throughout the hypothalamus, paraventricular nucleus of the hypothalamus (PVH) and in neurons expressing Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARH), respectively. We used a combination of molecular, behavioral and neuroendocrine criteria to evaluate the consequences on HPA axis responsiveness. RESULTS: Endpoints related to body weight and glucose homeostasis were not altered in any of the conditional mutant lines. Consistent with observations in the neuronal Insr knockout mice (NIRKO), baseline levels of serum CORT were similar to controls in all three lines. In male mice with broad disruptions of InsR signals in Nkx2.1-expressing regions of the hypothalamus (IR(Nkx2.1) KO), we observed elevated arginine vasopressin (AVP) levels at baseline and heightened neuroendocrine responses to restraint stress. IR(Nkx2.1) KO males also exhibited increased anxiety-like behaviors in open field, marble burying, and stress-induced hyperthermia testing paradigms. HPA axis responsivity was not altered in IR(Sim1) KO males, in which InsR was disrupted in the PVH. In contrast to observations in the IR(Nkx2.1) KO males, disrupting InsR signals in ARH neurons expressing Agrp (IR(Agrp) KO) led to reduced AVP release in the median eminence (ME). CONCLUSIONS: We find that central InsR signals modulate HPA responsivity to restraint stress. InsR signaling in AgRP/NPY neurons appears to promote AVP release, while signaling in other hypothalamic neuron(s) likely acts in an opposing fashion. Alterations in InsR signals in neurons that integrate metabolic and psychiatric information could contribute to the high co-morbidity of obesity and mental disorders.

CNS insulin signaling in the control of energy homeostasis and glucose metabolism - from embryo to old age.

Central nervous system (CNS) insulin signaling regulates energy and glucose homeostasis by acting on hypothalamic neurocircuits and higher brain circuits such as the dopaminergic system. However, overnutrition, obesity, and type 2 diabetes mellitus (T2DM) induce insulin resistance selectively in different regions of the brain, thereby impairing energy homeostasis and augmenting disease progression. Moreover, fetal hyperinsulinemia in response to maternal overnutrition, obesity, and diabetes disrupts hypothalamic neurocircuit development and predisposes to metabolic disorders later in life. In light of the current obesity and diabetes epidemic, we review the molecular basis of insulin action and resistance in the CNS, mechanisms which are causal to the development of these metabolic disorders, both in the neonate and in the adult.

Perinatal programming of metabolic diseases: role of insulin in the development of hypothalamic neurocircuits.

It is increasingly accepted that the metabolic future of an individual can be programmed as early as at developmental stages. For instance, offspring of diabetic mothers have a greater risk of becoming obese and diabetic later in life. Animal studies have demonstrated that hyperinsulinemia and/or hyperglycemia during perinatal life permanently impair the organization and long-term function of hypothalamic networks that control appetite and glucose homeostasis. This review summarizes the main findings regarding the key regulatory roles of perinatal insulin and glucose levels on hypothalamic development and on long-term programming of metabolic diseases reported in different rodent models.

AgRP innervation onto POMC neurons increases with age and is accelerated with chronic high-fat feeding in male mice.

In many mammals, body weight increases continuously throughout adulthood until late middle age. The hormone leptin is necessary for maintaining body weight, in that high levels of leptin promote negative energy balance. As animals age, however, their increase in body weight is accompanied by a steady rise in circulating leptin levels, indicating the progressive development of counterregulatory mechanisms to antagonize leptin's anorexigenic effects. Hypothalamic neurons coexpressing agouti-related peptide (AgRP) and neuropeptide Y are direct leptin targets. These neurons promote positive energy balance, and they inhibit anorexigenic proopiomelanocortin (POMC) neurons via direct neuropeptide action and release of γ-aminobutyric acid. We show here that AgRP and neuropeptide Y innvervation onto POMC neurons increases dramatically with age in male mice. This is associated with progressive increase of inhibitory postsynaptic currents and decrease of POMC firing rate with age. Neuronal activity is significantly attenuated in POMC neurons that receive a high density of AgRP puncta. These high-density AgRP inputs correlate with leptin levels in normal mice and are nearly absent in mice lacking leptin. The progression of increased AgRP innervation onto POMC somas is accelerated in hyperleptinemic, diet-induced obese mice. Together our study suggests that modulation of hypothalamic AgRP innervation constitutes one mechanism to counter the effects of the age-associated rise in leptin levels, thus sustaining body weight and fat mass at an elevated level in adulthood.