Molecular and Neuroendocrine Determinants of Seasonal Body Weight Regulation

This thesis deals with the characterisation of neuroendocrine pathways involved in seasonal body regulation revealed by the Siberian hamster (Phodopus sungorus). In the neuronal centre of body weight regulation, the hypothalamus, central signal transduction of the "adiposity signals" leptin and insulin (both inhibit food intake) and of the food intake stimulating hormone ghrelin was investigated comprehensively. Another aim of this thesis was the identification of the molecular identity underlying the phenomenon of leptin resistance, a key event in the onset of obesity. Furthermore, possible convergence of central leptin- and insulin signalling pathways was investigated. The hypothalamic signal transduction of both hormones was strikingly seasonally regulated implying a central role of these humoral signals in mediating seasonal body weight changes. The molecular identity of seasonally induced leptin resistance could be unravelled: It is caused by modulation of the signal transduction cascade distal to the leptin receptor. Moreover, the results of this thesis contradict to the popular opinion of possible synergistic effects conveyed by the anorexigenic hormones leptin and insulin which are related to their hypothalamic signalling. Ghrelin and its central signalling by the ghrelin receptor is very likely responsible for the acute regulation of food intake whereas it does not act on chronic changes in energy homeostasis (seasonal body weight cycles)

Molecular and Neuroendocrine Determinants of Seasonal Body Weight Regulation

This thesis deals with the characterisation of neuroendocrine pathways involved in seasonal body regulation revealed by the Siberian hamster (Phodopus sungorus). In the neuronal centre of body weight regulation, the hypothalamus, central signal transduction of the "adiposity signals" leptin and insulin (both inhibit food intake) and of the food intake stimulating hormone ghrelin was investigated comprehensively. Another aim of this thesis was the identification of the molecular identity underlying the phenomenon of leptin resistance, a key event in the onset of obesity. Furthermore, possible convergence of central leptin- and insulin signalling pathways was investigated. The hypothalamic signal transduction of both hormones was strikingly seasonally regulated implying a central role of these humoral signals in mediating seasonal body weight changes. The molecular identity of seasonally induced leptin resistance could be unravelled: It is caused by modulation of the signal transduction cascade distal to the leptin receptor. Moreover, the results of this thesis contradict to the popular opinion of possible synergistic effects conveyed by the anorexigenic hormones leptin and insulin which are related to their hypothalamic signalling. Ghrelin and its central signalling by the ghrelin receptor is very likely responsible for the acute regulation of food intake whereas it does not act on chronic changes in energy homeostasis (seasonal body weight cycles)

Photoperiodic and diurnal regulation of WNT signalling in the arcuate nucleus of the 1 female Djungarian hamster, Phodopus sungorus

yesThe WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal

Photoperiodic and diurnal regulation of WNT signalling in the arcuate nucleus of the 1 female Djungarian hamster, Phodopus sungorus

yesThe WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal

Die Weltgeschichte ist das Weltgericht : Schillers Vers und die deutsche Geschichtsphilosophie des 19. Jahrhunderts

<p><b>A.</b><i>SerpinA3N</i> gene expression in the liver was significantly up-regulated in a HF diet at all times tested 3 days (<i>P</i><0.01), 1 week (<i>P</i><0.001) and 16 weeks (<i>P</i><0.01). <b>B.</b><i>Haptoglobin</i> gene expression was unchanged in HF fed mice liver after 3 days on diet but was increased after 1 week (<i>P</i><0.01) and 16 weeks (<i>P</i><0.01). <b>C.</b><i>SAA</i> gene expression was significantly higher on a HF diet after 3 days (<i>P</i><0.01) and 1 week (<i>P</i><0.001) but was not different after 16 weeks. <b>D.</b><i>ApoA-IV</i> gene expression was significantly down-regulated in the liver of HF fed mice after 3 days (<i>P</i><0.05) and 1 week (<i>P</i><0.01) on diet but was unchanged after 16 weeks on diet. <b>E.</b> In comparison <i>ApoA-IV</i> gene expression was significantly up-regulated in the ileum of HF fed mice after 3 days, 1 week and 16 weeks on diet (<i>P</i><0.05). <b>F.</b><i>SerpinA3N</i> gene expression in the liver was significantly down-regulated with time on a LF diet between 3 days and 16 weeks (<i>P</i><0.001). Units are fold expression changes between experimental groups relative to <i>B2M</i> and calculated from the ΔΔ<i>C</i>_t values (n = 6–8).</p

SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice

Background: Energy homeostasis is regulated by the hypothalamus but fails when animals are fed a high-fat diet (HFD), and leptin insensitivity and obesity develops. To elucidate the possible mechanisms underlying these effects, a microarray-based transcriptomics approach was used to identify novel genes regulated by HFD and leptin in the mouse hypothalamus. Results: Mouse global array data identified serpinA3N as a novel gene highly upregulated by both a HFD and leptin challenge. In situ hybridisation showed serpinA3N expression upregulation by HFD and leptin in all major hypothalamic nuclei in agreement with transcriptomic gene expression data. Immunohistochemistry and studies in the hypothalamic clonal neuronal cell line, mHypoE-N42 (N42), confirmed that alpha 1-antichymotrypsin (α1AC), the protein encoded by serpinA3, is localised to neurons and revealed that it is secreted into the media. SerpinA3N expression in N42 neurons is upregulated by palmitic acid and by leptin, together with IL-6 and TNFα, and all three genes are downregulated by the anti-inflammatory monounsaturated fat, oleic acid. Additionally, palmitate upregulation of serpinA3 in N42 neurons is blocked by the NFκB inhibitor, BAY11, and the upregulation of serpinA3N expression in the hypothalamus by HFD is blunted in IL-1 receptor 1 knockout (IL-1R1−/−) mice. Conclusions: These data demonstrate that serpinA3 expression is implicated in nutritionally mediated hypothalamic inflammation

Impact of physiological rhythms on energy homeostasis in rodents

Obesity and related metabolic disorders such as type 2 diabetes are a major health issue of our modern society. The brain has been identified to play an essential role in the pathogenesis of these diseases. Disruptions of the neuroendocrine system, such as the development of hypothalamic leptin resistance, are strongly correlated with the manifestation of diet-induced obesity (DIO). To date, the molecular mechanisms underlying these metabolic derangements are incompletely understood. Over the last decade, a close connection of energy metabolism and the circadian clock has been established, but the link between DIO and disruptions of physiological rhythms still needs further investigation. Therefore, the aim of this thesis was to gain new insights into neuroendocrine mechanisms that lead to the development of leptin resistance and the role of physiological rhythms in the disruption of energy metabolism. In this study, we investigated the implication of the adipocyte-derived hormone adiponectin in neuroendocrine control of energy metabolism. We detected expression of all investigated genes involved in the adiponectin signalling pathway in the hypothalamus of mice. Expression levels of adiponectin were reduced during states of food deprivation, potentially presenting a regulatory mechanism to counteract the anorexigenic traits that had been previously described for central adiponectin signalling and that were confirmed by us in this study, in order to prevent further reduction in body weight. In both fasted control mice as well as DIO mice, gene expression of the adiponectin receptor AdipoR1 was elevated, suggesting multiple regulatory mechanisms to maintain sufficient adiponectin signal transduction. The upregulation of AdipoR1 during DIO might be an attempt to support the beneficial effects of the hormone on metabolic health that have been reported for peripheral adiponectin. In line with this, we demonstrated that adiponectin holds insulin-sensitising, blood glucose-lowering and anti-inflammatory properties in control as well as DIO mice and that these effects are mediated via central signal transduction. We furthermore investigated the role of the WNT/β-catenin pathway in the neuroendocrine control of energy metabolism. Here, we found gene expression of members of the WNT pathway on all regulatory levels (ligands, intracellular pathway enzymes, target genes) in the hypothalamus of adult Djungarian hamsters, Phodopus sungorus, a seasonal rodent that exhibits profound annual changes in body weight and leptin sensitivity. Expression of all ligands as well as target genes was upregulated in hamsters acclimated to long day (LD) relative to short day (SD) conditions. Confirming our results from these transcriptional studies, we furthermore found increased phosphorylation of the WNT pathway co-receptor LRP-6, demonstrating elevated activation of canonical WNT signalling, in LD hamsters. These findings provide strong evidence for increased WNT signalling during LD compared with SD photoperiod. We found a 24-hour rhythm in the hypothalamic expression of WNT target genes, with decreasing levels during the light and increasing levels during the dark phase in both LD and SD hamsters. Moreover, leptin administration led to a further increase in LRP-6 activation in hamsters from both photoperiods. Taken together, we demonstrate a novel integration site for the leptin signal in the hypothalamus, potentially linking the WNT pathway to body weight regulation. Furthermore, our results suggest an important role of canonical WNT signalling in the seasonal as well as daily neuroendocrine control of energy metabolism in Djungarian hamsters. By examining whether hypothalamic leptin signalling and whole body metabolism are modulated by a daily rhythm, we detected a 24-hour rhythm of STAT3 phosphorylation, a marker for activated leptin signalling on a molecular level, in the hypothalamus of wild-type mice. Both basal as well as leptin-induced leptin sensitivity were highest at the end of the dark (active) phase and lowest at the end of the light (inactive) phase. Furthermore, we found that leptin sensitivity on a behavioural level followed the same rhythm, with mice showing a greater response to exogenous leptin at the end of the dark phase at Zeitgeber time (ZT) 0 compared with the end of the light phase at ZT12. Throughout the 24-hour cycle, mice displayed a robust rhythm in food intake, locomotor activity as well as oxygen consumption and energy expenditure, with reduced whole body metabolism during their inactive and increased metabolic rate during their active phase. In DIO mice that were subjected to high-fat diet (HFD) feeding, we found a disruption of the 24-hour rhythmic regulation of leptin pathway activation on a molecular level for both basal and leptin-induced leptin sensitivity. Intriguingly, we demonstrated that this hypothalamic leptin resistance is a temporary phenomenon that persists only at specific times during the day. Responsiveness to leptin was deteriorated during the second part of the dark and the first half of the light phase (ZT21 – ZT6), but identical to mice fed low-fat diet (LFD) at all other times on both the molecular and behavioural level. Furthermore, DIO mice displayed a disruption of the daily rhythms in food intake, locomotor activity, oxygen consumption and energy expenditure. We found that the daily caloric overconsumption observed in mice fed HFD was restricted to the phase when DIO mice were leptin resistant relative to mice fed LFD. In conclusion, these findings provide strong evidence for a crucial role of the 24-hour rhythm of leptin sensitivity in the control of energy metabolism. We furthermore demonstrated that mice with access to HFD exclusively during their leptin resistant phase (ZT21 – ZT3) displayed impairments in a variety of parameters that indicate metabolic health, such as compromised rhythms of locomotor activity, metabolic rate, and energy expenditure as well as increased circulating insulin levels. Restricting HFD exclusively to the leptin sensitive phase (ZT9 – ZT15), on the other hand, protected mice from the development of these severe metabolic impairments. To date it is still largely unknown whether HFD-induced development of metabolic diseases results from an increase in body fat content, diet composition or disrupted circadian rhythms. We observed these differences between TRF groups despite an identical reduction in body weight and plasma leptin levels in all TRF mice, suggesting that they are based on the time of food intake during the 24-hour rhythm of leptin sensitivity, but independent from factors such as body composition or HFD content. Nonetheless, all mice fed HFD displayed a reduction in the absolute values of average metabolic rate and energy expenditure relative to mice fed LFD, demonstrating that also the HFD itself affects energy metabolism. In conclusion, these results demonstrate that TRF is efficient in the reduction of body weight and the amelioration of metabolic health. However, our findings also highlight the importance of synchronising food intake with daily physiological rhythms to maintain metabolic health. Taken together, this thesis identifies novel pathways that are involved in the neuroendocrine regulation of energy metabolism and provides new insights into the connection between physiological rhythms and the development of metabolic diseases

Neuroendokrine Regulation der Energie und Glukosehomöostase- Vom zentralen WNT- Signalweg über hypothalamische Inflammation und Leptinsensitivität -

Adipositas ist eines der größten Gesundheitsprobleme der modernen Wohlstandsgesellschaft und einer der Hauptrisikofaktoren für die Entstehung von Diabetes mellitus Typ II. Beiden metabolischen Störungen liegen Veränderungen des neuroendokrinen Systems zu Grunde. Hierbei ist vor allem der Verlust der Leptinsensitivität zu nennen, die für die Verknüpfung von Adipositas und Diabetes Typ II verantwortlich sein könnte. Mittlerweile ist das zentrale Nervensystem, insbesondere der mediobasale Hypothalamus (MBH), als Hauptregulationszentrum der zentralen Energie- und Glukosehomöostase anzusehen. Daher ist der Zusammenhang von Adipositas und Diabetes mellitus Typ II auf eine Modulation der beteiligten Signalwege im zentralen Nervensystem zurückzuführen. In dieser Arbeit konnte gezeigt werden, dass der Verlust der zentralen Leptinsignalweiterleitung adipöser Individuen maßgeblich an dem Zusammenhang von Adipositas und Diabetes mellitus Typ II beteiligt ist. Darüber hinaus interagierte Leptin mit dem zentralen WNT/β Catenin- (WNT) Signalweg und inhibierte dabei das Schlüsselenzym, die Glykogen- Synthase- Kinase- 3β (GSK-3β), in Neuronen des MBH. Des Weiteren waren die positiven Effekte von Leptin auf den Glukosemetabolismus von einem intakten WNT- Signalweg abhängig. Im Einklang mit diesen Ergebnissen ging eine zentrale Inhibition der GSK-3β mit einer verstärkten Aktivierung des hypothalamischen Insulin- Signalweges einher, verbesserte die Glukosetoleranz und verringerte die Nahrungsaufnahme von leptindefizienten Mäusen. Eine erhöhte Aktivität im MBH hingegen war mit Adipositas assoziiert, steigerte die Nahrungsaufnahme, führte zu Regulationsstörungen der Energie- und Glukosehomöostase und war mit einer verminderten Leptinsensitivität assoziiert. Diese Daten legen nahe, dass Leptin seine katabolen Eigenschaften über die hypothalamische GSK-3β vermittelt und, dass dieses Enzym als zentraler Regulator des Energie- und Glukosemetabolismus angesehen werden kann. Neben dem zentralen Verlust der Leptinsignalweiterleitung spielen auch noch andere, mit dem Körperfett assoziierte, Faktoren eine entscheidende Rolle bei der zentralen Regulation des Glukosemetabolismus. Neuere Forschungsergebnisse weisen darauf hin, dass die Veränderungen der zentralen Signalwege auf eine hypothalamische Inflammation zurückzuführen sein könnten. Die Zunahme des Fettgewebes geht mit einer erhöhten Sekretion von Zytokinen einher, die pro-inflammatorische Signalwege im Hypothalamus induzieren können. Es konnte gezeigt werden, dass eine erhöhte Aktivität der pro-inflammatorischen c-Jun N-terminale Kinase (JNK) in verschiedenen Nuclei des MBH mit Adipositas assoziiert ist. Die akute Inhibition dieser zentralen Kinase führte zu einer erhöhten Aktivierung des zentralen Insulin- Signalweges und verbesserte die Glukosetoleranz. Die Daten deuten darauf hin, dass die beobachteten Effekte auf eine Inhibition der zentralen GSK-3β zurückzuführen waren, wodurch die Rolle dieser Kinase in der zentralen Regulation der Glukosehomöostase weiter bekräftigt wurde. Neben der JNK ist auch der zentrale pro-inflammatorische NF-κB- Signalweg mit Adipositas verknüpft und eine Aktivierung wird mit einer aktiven GSK-3β in Verbindung gebracht. Eine akute Inhibition dieses Signalweges, mit dem Flavonoid Butein, brachte eine Verbesserung der Glukosetoleranz von diabetischen und adipösen Mäusen mit sich. Des Weiteren verminderte die chronische Inhibition des NF-κB- Signalweges (mittels Gentherapie) im MBH die Zunahme des Körpergewichtes und der Körperfettmasse von Mäusen auf einer hochkalorischen Diät. Dabei verbesserte diese Behandlung die Glukosetoleranz der Tiere, erhöhte die Sauerstoffaufnahme, den Energieverbrauch sowie die basale metabolische Rate und war mit einer erhöhten Leptinsensitivität verbunden. Mit dieser Arbeit konnten neue Aspekte der zentralen Regulation der Energie- und Glukosehomöostase entschlüsselt werden. Unsere Daten deuten darauf hin, dass die zentrale GSK-3β ein Schlüsselenzym ist, welches die metabolischen Eigenschaften von Leptin und Insulin vermittelt. Die Aktivierung dieser hypothalamischen Kinase durch pro-inflammatorische Signalwege scheint die Entstehung einer zentralen Leptinresistenz zu vermitteln, was eine Dysregulation der Glukosehomöostase nach sich zieht. Die zentrale Inhibition dieses Enzyms oder der zugrundeliegenden Inflammation liefert somit neue therapeutische Interventionsmöglichkeiten bei der Entstehung von Adipositas und Diabetes mellitus Typ II

Hyperleptinemia as a contributing factor for the impairment of glucose intolerance in obesity

Obesity has emerged as a major risk factor for insulin resistance leading to the development of type 2 diabetes (T2D). The condition is characterized by high circulating levels of the adipose-derived hormone leptin and a state of chronic low-grade inflammation. Pro-inflammatory signaling in the hypothalamus is associated with a decrease of central leptin- and insulin action leading to impaired systemic glucose tolerance. Intriguingly, leptin not only regulates body weight and glucose homeostasis but also acts as a pro-inflammatory cytokine. Here we demonstrate that increasing leptin levels (62,5 µg/kg/d, PEGylated leptin) in mice fed a high-fat diet (HFD) exacerbated body weight gain and aggravated hypothalamic micro- as well as astrogliosis. In contrast, administration of a predetermined dose of a long-acting leptin antagonist (100 µg/kg/d, PESLAN) chosen to block excessive leptin signaling during diet-induced obesity (DIO) showed the opposite effect and significantly improved glucose tolerance as well as decreased the total number of microglia and astrocytes in the hypothalamus of mice fed HFD. These results suggest that high levels of leptin, such as in obesity, worsen HFD-induced micro-and astrogliosis, whereas the partial reduction of hyperleptinemia in DIO mice may have beneficial metabolic effects and improves hypothalamic gliosis