Substantial progress has been made identifying central neuronal circuits that are crucial for controlling feeding behavior and energy homeostasis. Understanding the cellular and molecular mechanisms mediating information processing in neuronal circuits that regulate energy homeostasis is essential to devise specific pharmacological strategies, which assist obese patients in starting and maintaining a program of weight loss with no or minimized side effects on other neuronal systems (Gouni-Berthold et al., 2013; Holes-Lewis et al., 2013; Misra, 2013). Important microcircuits in control of energy balance are localized in the arcuate nucleus of the hypothalamus (ARH), which contains two main neuron populations with antagonistic functions: Neuropeptide Y and agouti-related peptide (NPY/AgRP) co-expressing neurons signaling hunger, promoting food intake, and pro-opiomelanocortin (POMC) - expressing neurons which signal satiety (Sohn et al., 2013; Varela and Horvath, 2012).

Many important fuel-sensing endocrine and metabolic factors, such as insulin, leptin, glucose, serotonin, dopamine, free fatty acids and uridine diphosphate (Gao and Horvath, 2007; Jo et al., 2009; Jordan et al., 2010; Sohn et al., 2011; Steculorum et al., 2015; Varela and Horvath, 2012; Zhang and van den Pol, 2016) have been identified and various immediate actions of these modulators and nutrient components on the POMC and AgRP neurons are well understood (Belgardt et al., 2008; Claret et al., 2007; Jo et al., 2009; Parton et al., 2007; Spanswick et al., 1997, 2000). However, the effect of the catecholamine noradrenalin (NA) on the ARH is not well defined. Noradrenalin is associated with a number of important CNS functions (Szabadi, 2013) including energy homeostasis and food intake (Commins et al., 1999; Ste Marie et al., 2005; Thomas and Palmiter, 1997; Wellman, 2005, 2000). Potentially, NA could be a direct modulator of the energy homeostasis-regulatory POMC- and AgRP neurons in the ARH, since noradrenergic projections from the hindbrain (A1, A2 noradrenergic cell groups) and the locus coeruleus (LC) to the ARH have been described in rats (Fraley, 2006; Fraley and Ritter, 2003; Yoon et al., 2013) and manipulation of the noradrenergic system in the hypothalamus has been found to modify feeding behavior and food intake. Impairment of the dorsal or the ventral noradrenergic bundle, which contain efferent projections from the LC to the forebrain, caused hyper- or hypophagia, respectively (Ahlskog and Hoebel, 1973; Hoebel et al., 1989). In line with these lesion experiments, local NA injections into defined hypothalamus regions either elicits or inhibits feeding in rodents (Booth, 1967; Leibowitz, 1978). These findings not only demonstrate noradrenergic modulation of food intake, but also indicate that the NA effect on feeding behavior is versatile and site-specific. Additional evidence that NA is associated with the regulation of the homeostatic system in the ARH came from extracellular recordings of unidentified ARH neurons in rats, which responded to NA application (Kang et al., 2000). Furthermore, NA increases the expression of Agrp and Npy mRNA in the rat ARH in response to glucoprivation (Fraley and Ritter, 2003). This effect is abolished if NA terminals in the ARH are eliminated by the injection of saporin-conjugated anti-dopamine-β-hydroxylase (anti-dβh), a selective NA immunotoxin. Specifically, the immunotoxin also eliminated a large number of neurons in the ventrolateral medulla (A1/C1 cell groups), which have been identified as mediators of glucoprivic feeding. Together, these findings provide strong evidence that NA modulates eating behavior by changing functional properties of ARH neurons.

Recent work by the Bouret lab provides a plausible hypothesis in regard to the question in which behavioral context the noradrenergic LC projections into the ARH play a key role and what response they facilitate. Based on in vivo electrophysiological recordings in primates, it is hypothesized that the LC plays a key role in mobilizing the required energy to face anticipated physical challenges, such as to obtain rewards (Bouret and Richmond, 2015; Varazzani et al., 2015). In this model, the LC activity is directly related to the energetic need that is required to master the challenge. NA release in the ARH might then promote compensatory energy intake.

On the cellular level NA action can be mediated by a variety of ARs that can inhibit or excite neurons. NA and adrenalin activate three major classes of G protein-coupled receptors (GPCRs): α₁-, α₂- and β-ARs (Hein, 2006). These receptor types can be further subdivided in a number of different isoforms. The intracellular signaling cascades of ARs are well investigated. While excitatory α₁-ARs are coupled to Gq/11 and mediate an increase in intracellular Ca²⁺ levels and closure of G protein-coupled inwardly rectifying potassium channels (GIRKs), inhibitory α₂-ARs couple to Gi/o leading to inhibition of voltage-gated Ca²⁺ channels (VGCCs) and opening of GIRKs. β-ARs mediate excitation via activation of Gs proteins and subsequent elevation of internal Ca²⁺ concentrations and opening of VGCCs (Marzo et al., 2009).

In this study, we assessed if NA can directly modulate orexigenic NPY/AgRP and anorexigenic POMC neurons, which are key components of the control circuits in ARH that regulate food intake and energy homeostasis. In electrophysiological recordings, NA had clear opposite effects on these neurons, increasing the activity of NPY/AgRP neurons and inhibiting POMC neurons. Cell type-specific transcriptomics and pharmacological experiments revealed that the activation of NPY/AgRP neurons is predominantly mediated by α_1A-ARs, while POMC neurons are inhibited via α_2A-ARs. Collectively, our data indicate a strong orexigenic influence of NA on the ARH circuitry that controls energy balance.

Here we asked, if and how NA directly modulates the activity of NPY/AgRP and POMC expressing neurons of the ARH. This question was addressed in three steps. First, electrophysiological recordings were performed to analyze the modulatory effect of NA on these neurons. Second, we used cell type-specific transcriptomics to build an inventory of adrenergic receptors that are expressed in NPY/AgRP and POMC neurons and potentially could mediate the modulation of these neurons. Third, pharmacological tools were used to test which adrenergic receptors are functionally expressed and how they contribute to the net modulatory effect of NA.

Noradrenalin modulates electrophysiological activity differentially in NPY/AgRP and POMC neurons

In the first set of experiments, we probed if NA modulates the electrophysiological activity of NPY/AgRP and POMC neurons. To address this question directly, we analyzed identified NPY/AgRP-GFP and POMC-GFP neurons of the ARH in acute brain slices of adult mice. Recordings were performed in the perforated patch clamp configuration to minimize effects on intracellular signaling pathways.

As described previously, most NPY/AgRP and POMC neurons were spontaneously active under control conditions (NPY/AgRP neurons: (Baver et al., 2014; Steculorum et al., 2015; Tsaousidou et al., 2014; Wei et al., 2015); POMC neurons: (Ernst et al., 2009; Koch et al., 2015; Newton et al., 2013; Plum et al., 2006; Zhang and van den Pol, 2013). Bath-applied 10 µM NA had clear opposite effects on these functionally antagonistic neuron types. NA depolarized orexigenic NPY/AgRP neurons and increased their action potential frequency from 2.0 ± 0.9 Hz to 5.0 ± 1.0 Hz (Figure 1A,B; NPY/AgRP neurons, Wilcoxon matched pairs signed rank test, n = 8, p=0.0012). In contrast, NA clearly hyperpolarized anorexigenic POMC neurons and drastically reduced or even abolished spontaneous generation of action potentials from 3.5 ± 1.1 to 0.7 ± 0.7 Hz (Figure 1A,B; POMC neurons, Wilcoxon matched pairs signed rank test, n = 8, p=0.0156). In both neuron types, the NA effect had a rapid onset and was readily reversible. To better define and understand the cause-effect relationship of the NA modulation, we measured concentration-response curves for both neuron types.

Figure 1

Download asset Open asset

NPY/AgRP neurons, NA concentration-response relation

NA depolarized the membrane potential and increased the action potential frequency starting at a concentration of 100 nM (Figure 2A). The maximal responses were observed at 10 µM where NA increased the average firing frequency from 2.2 ± 0.8 Hz to 4.7 ± 0.8 Hz (Figure 2B; paired t-test, n = 8, p<0.0001) and depolarized the membrane by 5.1 ± 1.0 mV (Figure 2C; paired t-test, n = 8, p=0.0011). The normalized concentration-response relations for the membrane depolarization and increase in firing frequency were well fit with a sigmoidal relation (Equation 1). The concentration-response relations had EC₅₀s of 1.9 µM (1.1–3.2 µM; n = 8) and 1.5 µM (0.8–2.8 µM; n = 8), respectively. The effective concentration range is in line with previous studies that analyzed NA modulation in other cell types (Hayar et al., 1997). To rule out that the observed NA effect is caused by secondary modulation, which is mediated by neurons that are presynaptic to NPY/AgRP neurons, we analyzed the NA effect on NPY/AgRP neurons in which GABAergic and glutamatergic input was pharmacologically blocked. Synaptically isolated NPY/AgRP neurons responded similarly to the application of NA as without synaptic blockers (Figure 2D,E), demonstrating that the excitatory NA effect is mediated by cell-intrinsic ARs and the applied GABA and glutamate antagonists do not directly interfere with the AR.

Figure 2 with 1 supplement see all

Download asset Open asset

POMC neurons, NA concentration-response relation

10 µM NA clearly inhibited POMC neurons (Figure 1A–C). In neurons with low control action potential frequencies, this often caused an all or nothing effect. Therefore, we used the membrane potential and membrane conductance densities to quantify the NA effect and to establish concentration-response relations.

The NA inhibition started at 100 nM and the maximal inhibitory effect was reached at 10 µM where NA hyperpolarized the membrane potential by 18.6 ± 3.2 mV (Figure 3B; paired t-test, n = 8; p=0.0007) and increased the conductance from 647 ± 106 pS to 1546 ± 266 pS (Figure 3C; paired t-test, n = 8; p=0.0029). The NA effect developed rapidly and was reversible. However, after application of high concentration, the wash out of NA induced a slow, sustained (~10 min) rebound excitation. NA concentrations higher than 10 µM did not further increase the effect.

Figure 3

Download asset Open asset

The normalized concentration-response relations for conductance increase and membrane hyperpolarization were well fit with a sigmoidal relation (Equation 1), with EC₅₀s of 0.9 µM (0.6–1.5 µM; n = 8) and 1.3 µM (1.0–1.9 µM; n = 8) for the hyperpolarization and the conductance density, respectively. The effective concentration range is in line with previous studies that analyzed NA modulation in other cell types (Jurgens et al., 2007).

In experiments in which the GABAergic and glutamatergic input of the POMC neurons was pharmacologically blocked, the NA response was slightly decreased (~16%) compared to recordings without synaptic blockers present, suggesting a small presynaptic contribution by NA responsive neurons, which project on POMC neurons (Figure 3D,E). Given the excitatory effect of NA on NPY/AgRP neurons, this effect is likely caused by their mono-directional inhibitory synaptic innervation of POMC neurons.

Adrenergic receptors are expressed in NPY/AgRP and POMC neurons of the ARH

To understand how NA modulates NPY/AgRP and POMC neurons at the molecular level, we performed cell type-specific transcriptomics to analyze AR gene expression in these neurons. Using enhanced green fluorescent protein (eGFP)-based fluorescence-activated cell sorting (FACS), we isolated eGFP-expressing NPY/AgRP or POMC neurons from coronal slices, which contained the hypothalamus, of 6-week-old mice (four mice per sample; NPY-GFP: n = 3; POMC-GFP: n = 2). Subsequently, we performed total RNA isolation and whole transcriptome analysis from these neuronal populations. Comparing POMC versus NPY transcriptional profiles yielded 4482 significantly differentially expressed genes (FC≥2, p≤0.01). Within this pool, Pomc was enriched 147-fold in the corresponding population, with Npy and Agrp being higher expressed in NPY-derived samples, but not in a statistically significant way (Figure 4A, supplemental file 1). NPY/AgRP neurons expressed all subtypes of ARs, except α_1D- and α_2B- ARs. POMC neurons only expressed α_1A- and α_2A- ARs, albeit to a lower magnitude (Figure 4A).

Figure 4 with 1 supplement see all

Download asset Open asset

To verify our gene expression data related to the ARs expressed in these neuronal populations, we performed RNA in situ hybridizations, using probes against Adra1a, Adra2a, Adrb1 and Adrb2. Both NPY/AgRP and POMC neurons were positive for Adra1a and Adra2a expression, with Adra1a being expressed in more NPY/AgRP neurons, compared to POMC neurons (Figure 4B,C). Adra2a was however expressed to a similar extent between the two populations (Figure 4C). Adrb1 and Adrb2 were expressed only in NPY/AgRP neurons (Figure 4—figure supplement 1). Taken together, our data suggest that ARs are differentially expressed between NPY and POMC neurons within the hypothalamus, possibly mediating any distinct modulatory effects of NA on these populations.

NA excites NPY/AgRP neurons predominantly via the activation of α_1A-ARs

While NPY/AgRP neurons expressed excitatory α_1A,B- and β-ARs, and inhibitory α_2A -AR, NA clearly excited all recorded NPY/AgRP neurons. To assess the AR classes that contribute to the excitatory electrophysiological response we used pharmacological tools, which were selective for AR subclasses.

In the first series of experiments α-ARs were blocked by the α₁-AR antagonist prazosine (5 µM) and the α₂-AR antagonist yohimbine (5 µM; Figure 5A,B). During the combined application of prazosine and yohimbine the excitatory NA effect was completely blocked in 44% (4 of 9; Figure 5A) and significantly (~60%) attenuated in 55% (5 of 9; Figure 5B) of the NPY/AgRP neurons. Together, these experiments suggest that the excitatory NA effect in NPY/AgRP neurons can be mediated solely by α₁-ARs or by the co-activation of α₁-ARs and β-ARs.

Figure 5

Download asset Open asset

Next, we asked which α₁-AR subtype(s) mediates the excitatory NA effect. The selective α_1A-AR agonist A-61603 (1 µM) depolarized the membrane potential and increased the AP frequency in NPY/AgRP neurons similarly as a subsequent NA application (Figure 6A,B). Both the A-61603 and the NA effect were fully reversible. In line with these data, we found that the selective α_1A-AR antagonist WB4101 (100 nM) abolished the excitatory NA response (Figure 6C,D). Application of WB4101 resulted in an inhibitory effect, suggesting baseline activation of α_1A-AR. The subtype-specific α1_B,D-AR antagonist chloro-ethyl-clonidine (CEC; 100 nM) did not block or alter the NA action (Figure 6E,F), suggesting that α_1B,D-ARs do not contribute to the excitation. Taken together, these data show that NA excites NPY/AgRP neurons via activation of α_1A-ARs.

Figure 6 with 1 supplement see all

Download asset Open asset

Of note, the α_1A-AR antagonist WB4101 not only blocked the NA excitation (as described above). In the presence of WB4101 NA even had an inhibitory effect on the NPY/AgRP membrane potential (Figure 6C,D). A subsequent block of inhibitory α_2A-ARs by the specific antagonist BRL44408 reversed the inhibition to a small excitation. These data show that the inhibitory α_2A-ARs are functional in NPY/AgRP neurons and also support the notion that excitatory β-ARs are activated by NA, as indicated in the first set of experiments.

Collectively, our data indicate that all receptors, which were identified by cell-type specific transcriptomics, are functional in NPY/AgRP neurons. The excitatory NA effect that we observed in virtually all neurons is predominantly mediated by α_1A-ARs and might be in some neurons co-mediated by β-ARs. The activation of excitatory AR interferes with the activation of inhibitory α_2A-ARs.

NA inhibits POMC neurons via the activation of α2_A-ARs

POMC neurons express excitatory α_1A-ARs and inhibitory α_2A-ARs, and were markedly inhibited by NA. First, we tested, if the inhibitory NA effect is mediated by α_2A-AR, as suggested by our transcriptomics data. The α_2A-AR antagonist BRL 44408 (10 µM) blocked the NA mediated inhibitory effect. Application of 10 µM BRL 44408 alone, did not significantly alter the recorded electrophysiological properties, suggesting no baseline activity of α_2A-ARs (Figure 7A). The specific α_2B-AR antagonist ARC 239 (1 µM) did not change the inhibitory effect of NA, which suggests that the inhibitory NA action is solely mediated by α_2A-ARs (Figure 7B).

Figure 7

Download asset Open asset

Next, we asked if the excitatory α_1A-ARs are functional in POMC neurons (Figure 7C–E). When the inhibitory α₂-ARs were blocked by yohimbine (5 µM), NA excited POMC neurons (Figure 7D; paired t-test, n = 10, p=0.0168). This excitation was completely abolished by the α₁-AR antagonist prazosine (5 µM), showing the functionality of α₁-ARs in POMC neurons (Figure 7E; paired t-test, n = 6, p=0.2467).

Taken together, our results show that the NA-induced inhibition of POMC neurons is mediated by α_2A-AR and can mask the activation of excitatory α₁-ARs.

NA neurons of the brainstem give rise to a complex system of fiber tracts innervating most of the forebrain including the hypothalamus and the ARH (Szabadi, 2013). Because of the extensive and complex projection pattern, the NA system is associated with a number of important CNS functions including control of wake-sleep-cycles, nociception and memory consolidation (Berridge and Waterhouse, 2003; Samuels and Szabadi, 2008a, 2008b). One important role that has been clearly linked to the NA system by a combination of anatomical, pharmacological, lesion and behavioral studies is the modulation of energy homeostasis and food intake (Wellman, 2005, 2000)

Here, we show for the first time that NA directly modulates NPY/AgRP and POMC neurons of the ARH, which are key components of the neuronal circuits that control food intake and energy homeostasis. Interestingly, NA changes the activity of these functionally antagonistic neurons in opposite ways, increasing the activity of the orexigenic NPY/AgRP neurons and decreasing the activity of the anorexigenic POMC neurons. The strong differential modulation, which was reproducible in virtually all recorded neurons, is remarkable, since other important fuel sensing modulators like insulin, leptin, glucose, oleic acid and UDP typically affect only a subpopulation of these neurons (Jo et al., 2009; Parton et al., 2007; Steculorum et al., 2015; Williams et al., 2010). On the network and systems level, this clear differential NA modulation of functionally antagonistic neuron types should generate a strong orexigenic drive on the homeostatic circuits of the ARH. This conclusion is consistent with early behavioral experiments from Clark et al. (1988) showing that injection of NA into the third ventricle increased food intake. Furthermore, the authors showed that blockade of α₂-AR attenuated the NA-induced feeding behavior, which is in line with our finding that satiety signaling POMC- neurons are inhibited by NA via α₂-AR.

Since the modulatory effects of NA were observed in synaptically isolated neurons, the data suggest that NA directly activated intrinsically expressed ARs. Our cell type-specific transcriptomic data showed expression of excitatory and inhibitory receptor subtypes in both NPY/AgRP and POMC neurons, which is in line with recent data from Henry et al. (2015). NPY/AgRP neurons expressed excitatory α_1A-, β-ARs and inhibitory α_2A-ARs, while POMC neurons only express excitatory α_1A-ARs and inhibitory α_2A-ARs. Expression of other AR-subtypes was not detected in these neurons.

To unravel which of the expressed AR-subtypes contribute to the cell type-specific physiological effects of NA on NPY/AgRP and POMC neurons, we used pharmacological tools that have been previously shown to be selective for AR subtypes. While in NPY/AgRP the robust excitation is mediated by excitatory α_1A- and β-ARs, α_2A-ARs mediated the strong inhibition in POMC neurons.

The functional consequences of these diverse expression and activation profiles on the systems and behavioral level is not entirely clear and has to be investigated in future studies. Previous studies in other neuron types revealed similar phenomena and suggested that simultaneous expression of excitatory and inhibitory (NA) receptors might derive from developmental effects or localized, differential expression of ARs in different functional compartments.

Simultaneous expression of excitatory and inhibitory adrenergic receptors has been found in neurons of the LC. In juvenile mice, mRNA for excitatory α_1A,B,D- and inhibitory α_2A,B,C-ARs has been detected and application of NA caused excitation, while in adult mice NA hyperpolarizes LC neurons. Here, α₁-ARs attenuate the NA-induced hyperpolarization (Osborne et al., 2002). As in LC neurons, POMC neurons might change their response to NA during development. In the ARH developmental changes of electrophysiological responses to leptin have been described for NPY/AgRP neurons (Baquero et al., 2014; Nilsson et al., 2005). Simultaneous expression of excitatory and inhibitory ARs has also been found in dentate neurons of the human cerebellum (Schambra et al., 2005). Here, α_2B-ARs are thought to modulate excitatory glutamatergic input, while α_1A,B-ARs may play a role in timing and computation of inhibitory input. Previous studies in dentate neurons of the cerebellum suggested that α₁-ARs are expressed on the cell body while α₂-ARs are expressed on the terminals, in order to locally regulate transmitter release (Milner et al., 2000).

This raises the question regarding the NA source and under which physiological conditions NA is active to modulate food intake. While the blood brain barrier in adult animals is considered mostly impermeable for catecholamines, there is evidence from early work that catecholamines can cross the blood brain barrier in the hypothalamus (Weil-Malherbe et al., 1959). However, the site specific effects of local lesions and local NA injections into the hypothalamus might argue for local, site specific NA release from neurons. In rats, projections from the hindbrain/brainstem and the LC to the ARH have been identified (Fraley, 2006; Fraley and Ritter, 2003; Yoon et al., 2013). Specific deletion of projections from the ventro-lateral medulla (A1/C1 cell group) is sufficient to eliminate an increase in AgRP and NPY mRNA expression in response to glucoprivation induced by 2-DG. Furthermore, homeostatic feeding in response to local hypoglycemia in the hindbrain was decreased (Fraley and Ritter, 2003). In this context, recent work in primates from the Bouret group suggested that the LC plays a key role in mobilizing the required energy in order to face anticipated physical challenges, such as obtaining rewards (Bouret and Richmond, 2015; Varazzani et al., 2015). In this model, the LC activity is directly related to the energetic need that is required to master the challenge. Consistent with this hypothesis, our data suggest that NA release into the ARH upon LC activation generates an orexigenic drive to compensate for the required energy. In line with this hypothesis, previous studies revealed that NA injections into the hypothalamus elicit food intake (Booth, 1967; Leibowitz, 1978).

Together, these behavioral experiments are strong evidence for the orexigenic nature of NA signaling in the control of food intake. In this context, the orexigenic hormones insulin and leptin may modulate NA signaling in the ARH. While insulin selectively decreased α₂-AR expression in the ARH and dorsomedial hypothalamus (Levin et al., 1998), leptin inhibits NA release in rats (Brunetti et al., 1999; Francis et al., 2004), Both effects would augment the net anorexigenic signal. In support of the latter relation, obese mice that lack the gene to produce leptin (Lep^ob/ob) exhibit increased NA levels within the hypothalamus, which further decreases the anorexigenic signal (Oltmans, 1983).

Several drugs that were developed to modify food intake, act on the monoaminergic transmitter system in the brain. Since the molecular and cellular targets that mediate the effects of these drugs are often not clearly identified, they often cause strong side effects (Hainer et al., 2006). To successfully support obese patients to manage their food intake with minimized side effects, it is important to precisely define the targets of these drugs. Since the current findings reveal cell type-specific pathways that mediate NA modulation in the ARH they might help to better define specific targets for anti-obesity drugs.

1. 1. Ahlskog JE
   2. Hoebel BG

   (1973) Overeating and obesity from damage to a noradrenergic system in the brain

   Science 182:166–169.

   https://doi.org/10.1126/science.182.4108.166

   • PubMed
   • Google Scholar
2. 1. Akaike N
   2. Harata N

   (1994) Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms

   The Japanese Journal of Physiology 44:433–473.

   https://doi.org/10.2170/jjphysiol.44.433

   • PubMed
   • Google Scholar
3. 1. Baquero AF
   2. de Solis AJ
   3. Lindsley SR
   4. Kirigiti MA
   5. Smith MS
   6. Cowley MA
   7. Zeltser LM
   8. Grove KL

   (2014) Developmental switch of leptin signaling in arcuate nucleus neurons

   Journal of Neuroscience 34:9982–9994.

   https://doi.org/10.1523/JNEUROSCI.0933-14.2014

   • PubMed
   • Google Scholar
4. 1. Baver SB
   2. Hope K
   3. Guyot S
   4. Bjørbaek C
   5. Kaczorowski C
   6. O'Connell KM

   (2014) Leptin modulates the intrinsic excitability of AgRP/NPY neurons in the arcuate nucleus of the hypothalamus

   Journal of Neuroscience 34:5486–5496.

   https://doi.org/10.1523/JNEUROSCI.4861-12.2014

   • PubMed
   • Google Scholar
5. 1. Belgardt BF
   2. Husch A
   3. Rother E
   4. Ernst MB
   5. Wunderlich FT
   6. Hampel B
   7. Klöckener T
   8. Alessi D
   9. Kloppenburg P
   10. Brüning JC

   (2008) PDK1 deficiency in POMC-expressing cells reveals FOXO1-dependent and -independent pathways in control of energy homeostasis and stress response

   Cell Metabolism 7:291–301.

   https://doi.org/10.1016/j.cmet.2008.01.006

   • PubMed
   • Google Scholar
6. 1. Berridge CW
   2. Waterhouse BD

   (2003) The locus coeruleus-noradrenergic system: modulation of behavioral state and state-dependent cognitive processes

   Brain Research Reviews 42:33–84.

   https://doi.org/10.1016/S0165-0173(03)00143-7

   • PubMed
   • Google Scholar
7. 1. Booth DA

   (1967) Localization of the adrenergic feeding system in the rat diencephalon

   Science 158:515–517.

   https://doi.org/10.1126/science.158.3800.515

   • PubMed
   • Google Scholar
8. 1. Bouret S
   2. Richmond BJ

   (2015) Sensitivity of locus ceruleus neurons to reward value for goal-directed actions

   Journal of Neuroscience 35:4005–4014.

   https://doi.org/10.1523/JNEUROSCI.4553-14.2015

   • PubMed
   • Google Scholar
9. 1. Brunetti L
   2. Michelotto B
   3. Orlando G
   4. Vacca M

   (1999) Leptin inhibits norepinephrine and dopamine release from rat hypothalamic neuronal endings

   European Journal of Pharmacology 372:237–240.

   https://doi.org/10.1016/S0014-2999(99)00255-1

   • PubMed
   • Google Scholar
10. 1. Claret M
    2. Smith MA
    3. Batterham RL
    4. Selman C
    5. Choudhury AI
    6. Fryer LG
    7. Clements M
    8. Al-Qassab H
    9. Heffron H
    10. Xu AW
    11. Speakman JR
    12. Barsh GS
    13. Viollet B
    14. Vaulont S
    15. Ashford ML
    16. Carling D
    17. Withers DJ

    (2007) AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons

    Journal of Clinical Investigation 117:2325–2336.

    https://doi.org/10.1172/JCI31516

    • PubMed
    • Google Scholar
11. 1. Clark JT
    2. Gist RS
    3. Kalra SP
    4. Kalra PS

    (1988) Alpha ₂-adrenoceptor blockade attenuates feeding behavior induced by neuropeptide Y and epinephrine

    Physiology & Behavior 43:417–422.

    https://doi.org/10.1016/0031-9384(88)90113-8

    • PubMed
    • Google Scholar
12. 1. Commins SP
    2. Marsh DJ
    3. Thomas SA
    4. Watson PM
    5. Padgett MA
    6. Palmiter R
    7. Gettys TW

    (1999) Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue

    Endocrinology 140:4772–4778.

    https://doi.org/10.1210/endo.140.10.7043

    • PubMed
    • Google Scholar
13. 1. Dodt HU
    2. Zieglgänsberger W

    (1990) Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy

    Brain Research 537:333–336.

    https://doi.org/10.1016/0006-8993(90)90380-T

    • PubMed
    • Google Scholar
14. 1. Ernst MB
    2. Wunderlich CM
    3. Hess S
    4. Paehler M
    5. Mesaros A
    6. Koralov SB
    7. Kleinridders A
    8. Husch A
    9. Münzberg H
    10. Hampel B
    11. Alber J
    12. Kloppenburg P
    13. Brüning JC
    14. Wunderlich FT

    (2009) Enhanced Stat3 activation in POMC neurons provokes negative feedback inhibition of leptin and insulin signaling in obesity

    Journal of Neuroscience 29:11582–11593.

    https://doi.org/10.1523/JNEUROSCI.5712-08.2009

    • PubMed
    • Google Scholar
15. 1. Fraley GS
    2. Ritter S

    (2003) Immunolesion of norepinephrine and epinephrine afferents to medial hypothalamus alters basal and 2-deoxy-D-glucose-induced neuropeptide Y and agouti gene-related protein messenger ribonucleic acid expression in the arcuate nucleus

    Endocrinology 144:75–83.

    https://doi.org/10.1210/en.2002-220659

    • PubMed
    • Google Scholar
16. 1. Fraley GS

    (2006) Immunolesions of glucoresponsive projections to the arcuate nucleus alter glucoprivic-induced alterations in food intake, luteinizing hormone secretion, and GALP mRNA, but not sex behavior in adult male rats

    Neuroendocrinology 83:97–105.

    https://doi.org/10.1159/000094375

    • PubMed
    • Google Scholar
17. 1. Francis J
    2. MohanKumar SM
    3. MohanKumar PS

    (2004) Leptin inhibits norepinephrine efflux from the hypothalamus in vitro: role of gamma aminobutyric acid

    Brain Research 1021:286–291.

    https://doi.org/10.1016/j.brainres.2004.07.010

    • PubMed
    • Google Scholar
18. 1. Gao Q
    2. Horvath TL

    (2007) Neurobiology of feeding and energy expenditure

    Annual Review of Neuroscience 30:367–398.

    https://doi.org/10.1146/annurev.neuro.30.051606.094324

    • PubMed
    • Google Scholar
19. 1. Gouni-Berthold I
    2. Brüning JC
    3. Berthold HK

    (2013) Novel approaches to the pharmacotherapy of obesity

    Current Pharmaceutical Design 19:4938–4952.

    https://doi.org/10.2174/13816128113199990302

    • PubMed
    • Google Scholar
20. 1. Hainer V
    2. Kabrnova K
    3. Aldhoon B
    4. Kunesova M
    5. Wagenknecht M

    (2006) Serotonin and norepinephrine reuptake inhibition and eating behavior

    Annals of the New York Academy of Sciences 1083:252–269.

    https://doi.org/10.1196/annals.1367.017

    • PubMed
    • Google Scholar
21. 1. Hayar A
    2. Feltz P
    3. Piguet P

    (1997) Adrenergic responses in silent and putative inhibitory pacemaker-like neurons of the rat rostral ventrolateral medulla in vitro

    Neuroscience 77:199–217.

    https://doi.org/10.1016/S0306-4522(96)00445-9

    • PubMed
    • Google Scholar
22. 1. Hein L

    (2006) Adrenoceptors and signal transduction in neurons

    Cell and Tissue Research 326:541–551.

    https://doi.org/10.1007/s00441-006-0285-2

    • PubMed
    • Google Scholar
23. 1. Henry FE
    2. Sugino K
    3. Tozer A
    4. Branco T
    5. Sternson SM

    (2015) Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss

    eLife 4:e09800.

    https://doi.org/10.7554/eLife.09800

    • PubMed
    • Google Scholar
24. 1. Hoebel BG
    2. Hernandez L
    3. Schwartz DH
    4. Mark GP
    5. Hunter GA

    (1989) Microdialysis studies of brain norepinephrine, serotonin, and dopamine release during ingestive behavior. Theoretical and clinical implications

    Annals of the New York Academy of Sciences 575:171–193.

    https://doi.org/10.1111/j.1749-6632.1989.tb53242.x

    • PubMed
    • Google Scholar
25. 1. Holes-Lewis KA
    2. Malcolm R
    3. O'Neil PM

    (2013) Pharmacotherapy of obesity: clinical treatments and considerations

    The American Journal of the Medical Sciences 345:284–288.

    https://doi.org/10.1097/MAJ.0b013e31828abcfd

    • PubMed
    • Google Scholar
26. 1. Horn R
    2. Marty A

    (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method

    The Journal of General Physiology 92:145–159.

    https://doi.org/10.1085/jgp.92.2.145

    • PubMed
    • Google Scholar
27. 1. Jo YH
    2. Su Y
    3. Gutierrez-Juarez R
    4. Chua S

    (2009) Oleic acid directly regulates POMC neuron excitability in the hypothalamus

    Journal of Neurophysiology 101:2305–2316.

    https://doi.org/10.1152/jn.91294.2008

    • PubMed
    • Google Scholar
28. 1. Jordan SD
    2. Könner AC
    3. Brüning JC

    (2010) Sensing the fuels: glucose and lipid signaling in the CNS controlling energy homeostasis

    Cellular and Molecular Life Sciences 67:3255–3273.

    https://doi.org/10.1007/s00018-010-0414-7

    • PubMed
    • Google Scholar
29. 1. Jurgens CW
    2. Hammad HM
    3. Lichter JA
    4. Boese SJ
    5. Nelson BW
    6. Goldenstein BL
    7. Davis KL
    8. Xu K
    9. Hillman KL
    10. Porter JE
    11. Doze VA

    (2007) Alpha2A adrenergic receptor activation inhibits epileptiform activity in the rat hippocampal CA3 region

    Molecular Pharmacology 71:1572–1581.

    https://doi.org/10.1124/mol.106.031773

    • PubMed
    • Google Scholar
30. 1. Kang YM
    2. Ouyang W
    3. Chen JY
    4. Qiao JT
    5. Dafny N

    (2000) Norepinephrine modulates single hypothalamic arcuate neurons via alpha(1)and beta adrenergic receptors

    Brain Research 869:146–157.

    https://doi.org/10.1016/S0006-8993(00)02380-5

    • PubMed
    • Google Scholar
31. 1. Koch M
    2. Varela L
    3. Kim JG
    4. Kim JD
    5. Hernández-Nuño F
    6. Simonds SE
    7. Castorena CM
    8. Vianna CR
    9. Elmquist JK
    10. Morozov YM
    11. Rakic P
    12. Bechmann I
    13. Cowley MA
    14. Szigeti-Buck K
    15. Dietrich MO
    16. Gao XB
    17. Diano S
    18. Horvath TL

    (2015) Hypothalamic POMC neurons promote cannabinoid-induced feeding

    Nature 519:45–50.

    https://doi.org/10.1038/nature14260

    • PubMed
    • Google Scholar
32. 1. Kyrozis A
    2. Reichling DB

    (1995) Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration

    Journal of Neuroscience Methods 57:27–35.

    https://doi.org/10.1016/0165-0270(94)00116-X

    • PubMed
    • Google Scholar
33. 1. Leibowitz SF

    (1978) Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking

    Pharmacology Biochemistry and Behavior 8:163–175.

    https://doi.org/10.1016/0091-3057(78)90333-7

    • Google Scholar
34. 1. Levin BE
    2. Israel P
    3. Lattemann DP

    (1998) Insulin selectively downregulates alpha2-adrenoceptors in the arcuate and dorsomedial nucleus

    Brain Research Bulletin 45:179–181.

    https://doi.org/10.1016/S0361-9230(97)00336-5

    • PubMed
    • Google Scholar
35. 1. Lindau M
    2. Fernandez JM

    (1986) IgE-mediated degranulation of mast cells does not require opening of ion channels

    Nature 319:150–153.

    https://doi.org/10.1038/319150a0

    • PubMed
    • Google Scholar
36. 1. Marzo A
    2. Bai J
    3. Otani S

    (2009) Neuroplasticity regulation by noradrenaline in mammalian brain

    Current Neuropharmacology 7:286–295.

    https://doi.org/10.2174/157015909790031193

    • PubMed
    • Google Scholar
37. 1. Milner TA
    2. Shah P
    3. Pierce JP

    (2000) beta-adrenergic receptors primarily are located on the dendrites of granule cells and interneurons but also are found on astrocytes and a few presynaptic profiles in the rat dentate gyrus

    Synapse 36:178–193.

    https://doi.org/10.1002/(SICI)1098-2396(20000601)36:3<178::AID-SYN3>3.0.CO;2-6

    • PubMed
    • Google Scholar
38. 1. Misra M

    (2013) Obesity pharmacotherapy: current perspectives and future directions

    Current Cardiology Reviews 9:33–54.

    https://doi.org/10.2174/1573403X11309010006

    • PubMed
    • Google Scholar
39. 1. Newton AJ
    2. Hess S
    3. Paeger L
    4. Vogt MC
    5. Fleming Lascano J
    6. Nillni EA
    7. Brüning JC
    8. Kloppenburg P
    9. Xu AW

    (2013) AgRP innervation onto POMC neurons increases with age and is accelerated with chronic high-fat feeding in male mice

    Endocrinology 154:172–183.

    https://doi.org/10.1210/en.2012-1643

    • PubMed
    • Google Scholar
40. 1. Nilsson I
    2. Johansen JE
    3. Schalling M
    4. Hökfelt T
    5. Fetissov SO

    (2005) Maturation of the hypothalamic arcuate agouti-related protein system during postnatal development in the mouse

    Developmental Brain Research 155:147–154.

    https://doi.org/10.1016/j.devbrainres.2005.01.009

    • PubMed
    • Google Scholar
41. 1. Oltmans GA

    (1983) Norepinephrine and dopamine levels in hypothalamic nuclei of the genetically obese mouse (ob/ob)

    Brain Research 273:369–373.

    https://doi.org/10.1016/0006-8993(83)90865-X

    • PubMed
    • Google Scholar
42. 1. Osborne PB
    2. Vidovic M
    3. Chieng B
    4. Hill CE
    5. Christie MJ

    (2002) Expression of mRNA and functional alpha(1)-adrenoceptors that suppress the GIRK conductance in adult rat locus coeruleus neurons

    British Journal of Pharmacology 135:226–232.

    https://doi.org/10.1038/sj.bjp.0704453

    • PubMed
    • Google Scholar
43. 1. Parton LE
    2. Ye CP
    3. Coppari R
    4. Enriori PJ
    5. Choi B
    6. Zhang CY
    7. Xu C
    8. Vianna CR
    9. Balthasar N
    10. Lee CE
    11. Elmquist JK
    12. Cowley MA
    13. Lowell BB

    (2007) Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity

    Nature 449:228–232.

    https://doi.org/10.1038/nature06098

    • PubMed
    • Google Scholar
44. 1. Plum L
    2. Ma X
    3. Hampel B
    4. Balthasar N
    5. Coppari R
    6. Münzberg H
    7. Shanabrough M
    8. Burdakov D
    9. Rother E
    10. Janoschek R
    11. Alber J
    12. Belgardt BF
    13. Koch L
    14. Seibler J
    15. Schwenk F
    16. Fekete C
    17. Suzuki A
    18. Mak TW
    19. Krone W
    20. Horvath TL
    21. Ashcroft FM
    22. Brüning JC

    (2006) Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity

    Journal of Clinical Investigation 116:1886–1901.

    https://doi.org/10.1172/JCI27123

    • PubMed
    • Google Scholar
45. 1. Rae J
    2. Cooper K
    3. Gates P
    4. Watsky M

    (1991) Low access resistance perforated patch recordings using amphotericin B

    Journal of Neuroscience Methods 37:15–26.

    https://doi.org/10.1016/0165-0270(91)90017-T

    • PubMed
    • Google Scholar
46. 1. Samuels ER
    2. Szabadi E

    (2008a) Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organisation

    Current Neuropharmacology 6:235–253.

    https://doi.org/10.2174/157015908785777229

    • PubMed
    • Google Scholar
47. 1. Samuels ER
    2. Szabadi E

    (2008b) Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part II: physiological and pharmacological manipulations and pathological alterations of locus coeruleus activity in humans

    Current Neuropharmacology 6:254–285.

    https://doi.org/10.2174/157015908785777193

    • PubMed
    • Google Scholar
48. 1. Schambra UB
    2. Mackensen GB
    3. Stafford-Smith M
    4. Haines DE
    5. Schwinn DA

    (2005) Neuron specific alpha-adrenergic receptor expression in human cerebellum: implications for emerging cerebellar roles in neurologic disease

    Neuroscience 135:507–523.

    https://doi.org/10.1016/j.neuroscience.2005.06.021

    • PubMed
    • Google Scholar
49. 1. Sohn JW
    2. Elmquist JK
    3. Williams KW

    (2013) Neuronal circuits that regulate feeding behavior and metabolism

    Trends in Neurosciences 36:504–512.

    https://doi.org/10.1016/j.tins.2013.05.003

    • PubMed
    • Google Scholar
50. 1. Sohn JW
    2. Xu Y
    3. Jones JE
    4. Wickman K
    5. Williams KW
    6. Elmquist JK

    (2011) Serotonin 2C receptor activates a distinct population of arcuate pro-opiomelanocortin neurons via TRPC channels

    Neuron 71:488–497.

    https://doi.org/10.1016/j.neuron.2011.06.012

    • PubMed
    • Google Scholar
51. 1. Spanswick D
    2. Smith MA
    3. Groppi VE
    4. Logan SD
    5. Ashford ML

    (1997) Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels

    Nature 390:521–525.

    https://doi.org/10.1038/37379

    • PubMed
    • Google Scholar
52. 1. Spanswick D
    2. Smith MA
    3. Mirshamsi S
    4. Routh VH
    5. Ashford ML

    (2000) Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats

    Nature Neuroscience 3:757–758.

    https://doi.org/10.1038/77660

    • PubMed
    • Google Scholar
53. 1. Ste Marie L
    2. Luquet S
    3. Curtis W
    4. Palmiter RD

    (2005) Norepinephrine- and epinephrine-deficient mice gain weight normally on a high-fat diet

    Obesity Research 13:1518–1522.

    https://doi.org/10.1038/oby.2005.185

    • PubMed
    • Google Scholar
54. 1. Steculorum SM
    2. Paeger L
    3. Bremser S
    4. Evers N
    5. Hinze Y
    6. Idzko M
    7. Kloppenburg P
    8. Brüning JC

    (2015) Hypothalamic UDP increases in obesity and promotes Feeding via P2Y6-Dependent activation of AgRP neurons

    Cell 162:1404–1417.

    https://doi.org/10.1016/j.cell.2015.08.032

    • PubMed
    • Google Scholar
55. 1. Szabadi E

    (2013) Functional neuroanatomy of the central noradrenergic system

    Journal of Psychopharmacology 27:659–693.

    https://doi.org/10.1177/0269881113490326

    • PubMed
    • Google Scholar
56. 1. Thomas SA
    2. Palmiter RD

    (1997) Thermoregulatory and metabolic phenotypes of mice lacking noradrenaline and adrenaline

    Nature 387:94–97.

    https://doi.org/10.1038/387094a0

    • PubMed
    • Google Scholar
57. 1. Tsaousidou E
    2. Paeger L
    3. Belgardt BF
    4. Pal M
    5. Wunderlich CM
    6. Brönneke H
    7. Collienne U
    8. Hampel B
    9. Wunderlich FT
    10. Schmidt-Supprian M
    11. Kloppenburg P
    12. Brüning JC

    (2014) Distinct roles for JNK and IKK activation in Agouti-Related peptide neurons in the development of obesity and insulin resistance

    Cell Reports 9:1495–1506.

    https://doi.org/10.1016/j.celrep.2014.10.045

    • PubMed
    • Google Scholar
58. 1. Varazzani C
    2. San-Galli A
    3. Gilardeau S
    4. Bouret S

    (2015) Noradrenaline and dopamine neurons in the reward/effort trade-off: a direct electrophysiological comparison in behaving monkeys

    Journal of Neuroscience 35:7866–7877.

    https://doi.org/10.1523/JNEUROSCI.0454-15.2015

    • PubMed
    • Google Scholar
59. 1. Varela L
    2. Horvath TL

    (2012) Leptin and insulin pathways in POMC and AgRP neurons that modulate energy balance and glucose homeostasis

    EMBO Reports 13:1079–1086.

    https://doi.org/10.1038/embor.2012.174

    • PubMed
    • Google Scholar
60. 1. Wagle P
    2. Nikolić M
    3. Frommolt P

    (2015) QuickNGS elevates Next-Generation sequencing data analysis to a new level of automation

    BMC Genomics 16:487.

    https://doi.org/10.1186/s12864-015-1695-x

    • PubMed
    • Google Scholar
61. 1. Wei W
    2. Pham K
    3. Gammons JW
    4. Sutherland D
    5. Liu Y
    6. Smith A
    7. Kaczorowski CC
    8. O’Connell KMS

    (2015) Diet composition, not calorie intake, rapidly alters intrinsic excitability of hypothalamic AgRP/NPY neurons in mice

    Scientific Reports 5:1–10.

    https://doi.org/10.1038/srep16810

    • Google Scholar
62. 1. Weil-Malherbe H
    2. Axelrod J
    3. Tomchick R

    (1959) Blood-brain barrier for adrenaline

    Science 129:1226–1227.

    https://doi.org/10.1126/science.129.3357.1226

    • PubMed
    • Google Scholar
63. 1. Wellman PJ

    (2000) Norepinephrine and the control of food intake

    Nutrition 16:837–842.

    https://doi.org/10.1016/S0899-9007(00)00415-9

    • PubMed
    • Google Scholar
64. 1. Wellman PJ

    (2005) Modulation of eating by central catecholamine systems

    Current Drug Targets 6:191–199.

    https://doi.org/10.2174/1389450053174532

    • PubMed
    • Google Scholar
65. 1. Williams KW
    2. Margatho LO
    3. Lee CE
    4. Choi M
    5. Lee S
    6. Scott MM
    7. Elias CF
    8. Elmquist JK

    (2010) Segregation of acute leptin and insulin effects in distinct populations of arcuate proopiomelanocortin neurons

    Journal of Neuroscience 30:2472–2479.

    https://doi.org/10.1523/JNEUROSCI.3118-09.2010

    • PubMed
    • Google Scholar
66. 1. Ye JH
    2. Zhang J
    3. Xiao C
    4. Kong JQ

    (2006) Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons

    Journal of Neuroscience Methods 158:251–260.

    https://doi.org/10.1016/j.jneumeth.2006.06.006

    • PubMed
    • Google Scholar
67. 1. Yoon YS
    2. Lee JS
    3. Lee HS

    (2013) Retrograde study of CART- or NPY-neuronal projection from the hypothalamic arcuate nucleus to the dorsal raphe and/or the locus coeruleus in the rat

    Brain Research 1519:40–52.

    https://doi.org/10.1016/j.brainres.2013.04.039

    • PubMed
    • Google Scholar
68. 1. Zhang X
    2. van den Pol AN

    (2013) Direct inhibition of arcuate proopiomelanocortin neurons: a potential mechanism for the orexigenic actions of dynorphin

    The Journal of Physiology 591:1731–1747.

    https://doi.org/10.1113/jphysiol.2012.248385

    • PubMed
    • Google Scholar
69. 1. Zhang X
    2. van den Pol AN

    (2016) Hypothalamic arcuate nucleus tyrosine hydroxylase neurons play orexigenic role in energy homeostasis

    Nature Neuroscience 19:1341–1347.

    https://doi.org/10.1038/nn.4372

    • PubMed
    • Google Scholar

Author details

1. Lars Paeger

   1. Biocenter, Institute for Zoology, University of Cologne, Cologne, Germany
   2. Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases, Cologne, Germany

   Contribution

   LP, Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-8716-3483

2. Ismene Karakasilioti

   1. Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases, Cologne, Germany
   2. Max Planck Institute for Metabolism Research, Cologne, Germany
   3. Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany
   4. Center for Endocrinology, Diabetes and Preventive Medicine, University Hospital Cologne, Cologne, Germany

   Contribution

   IK, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Janine Altmüller

   1. Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany
   2. Cologne Center for Genomics, University of Cologne, Cologne, Germany

   Contribution

   JA, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

4. Peter Frommolt

   Bioinformatics Facility, Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases, Cologne, Germany

   Contribution

   PF, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

5. Jens Brüning

   1. Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases, Cologne, Germany
   2. Max Planck Institute for Metabolism Research, Cologne, Germany
   3. Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany
   4. Center for Endocrinology, Diabetes and Preventive Medicine, University Hospital Cologne, Cologne, Germany

   Contribution

   JB, Resources, Formal analysis, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

6. Peter Kloppenburg

   1. Biocenter, Institute for Zoology, University of Cologne, Cologne, Germany
   2. Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases, Cologne, Germany

   Contribution

   PK, Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   peter.kloppenburg@uni-koeln.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-4554-404X

• 3,207

  views

• 510

  downloads

• 37

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.