Erik Schéle

The Gut Microbiota Reduces Leptin Sensitivity and the Expression of the Obesity-Suppressing Neuropeptides Proglucagon (Gcg) and Brain-Derived Neurotrophic Factor (Bdnf) in the Central Nervous System

The gut microbiota contributes to fat mass and the susceptibility to obesity. However, the underlying mechanisms are not completely understood. To investigate whether the gut microbiota affects hypothalamic and brainstem body fat-regulating circuits, we compared gene expression of food intake-regulating neuropeptides between germ-free and conventionally raised (CONV-R) mice. We found that CONV-R mice had decreased expression of the antiobesity neuropeptide glucagon-like peptide-1 (GLP-1) precursor proglucagon (Gcg) in the brainstem. Moreover, in both the hypothalamus and the brainstem, CONV-R mice had decreased expression of the antiobesity neuropeptide brain-derived neurotrophic factor (Bdnf). CONV-R mice had reduced expression of the pro-obesity peptides neuropeptide-Y (Npy) and agouti-related protein (Agrp), and increased expression of the antiobesity peptides proopiomelanocortin (Pomc) and cocaine- and amphetamine-regulated transcript (Cart) in the hypothalamus. The latter changes in neuropeptide expression could be secondary to elevated fat mass in CONV-R mice. Leptin treatment caused less weight reduction and less suppression of orexigenic Npy and Agrp expression in CONV-R mice compared with germ-free mice. The hypothalamic expression of leptin resistance-associated suppressor of cytokine signaling 3 (Socs-3) was increased in CONV-R mice. In conclusion, the gut microbiota reduces the expression of 2 genes coding for body fat-suppressing neuropeptides, Gcg and Bdnf, an alteration that may contribute to fat mass induction by the gut microbiota. Moreover, the presence of body fat-inducing gut microbiota is associated with hypothalamic signs of Socs-3-mediated leptin resistance, which may be linked to failed compensatory body fat reduction.

Interleukin‐6 Gene Knockout Influences Energy Balance Regulating Peptides in the Hypothalamic Paraventricular and Supraoptic Nuclei

Interleukin (IL)‐6 is a pro‐inflammatory cytokine that also affects metabolic function because IL‐6 depleted (IL‐6−/−) mice develop late‐onset obesity. IL‐6 appears to act in the central nervous system, presumably in the hypothalamus, to increase energy expenditure that appears to involve stimulation of the sympathetic nervous system. In the present study, we explored possible central mechanisms for the effects exerted by IL‐6 on body fat. Therefore, we measured the effects of IL‐6 depletion in IL‐6−/− mice on expression of key hypothalamic peptide genes involved in energy balance by the real time polymerase chain reaction. Additionally, co‐localisation between such peptides and IL‐6 receptor α was investigated by immunohistochemistry. IL‐6 deficiency decreased the expression of several peptides found in the paraventricular nucleus (PVN), which is a nucleus that has been attributed an adipostatic function. For example, corticotrophin‐releasing hormone (CRH), which is reported to stimulate the sympathetic nervous system, was decreased by 40% in older IL‐6−/− mice. Oxytocin, which is reported to prevent obesity, was also decreased in older IL‐6−/− animals, as was arginine vasopressin (AVP). The IL‐6 receptor α was abundantly expressed in the PVN, but also in the supraoptic nucleus, and was shown to be co‐expressed to a high extent with CRH, AVP, oxytocin and thyrotrophin‐releasing hormone. These data indicate that depletion of endogenous IL‐6, a body fat suppressing cytokine, is associated with the decreased expression of CRH and oxytocin (i.e. energy balance regulating peptides) as well as AVP in the PVN. Because IL‐6 receptor α is co‐expressed with CRH, oxytocin and AVP, IL‐6 could stimulate the expression of these peptides directly.

Inter-relation between Interleukin (IL)-1, IL-6 and Body Fat Regulating Circuits of the Hypothalamic Arcuate Nucleus

Interleukin (IL)‐1 and IL‐6 are immune modulating cytokines that also affect metabolic function because both IL‐1 receptor I deficient (IL‐1RI−/−) and IL‐6 deficient (IL‐6−/−) mice develop late‐onset obesity and leptin resistance. Both IL‐1 and IL‐6 appear to target the central nervous system (CNS) to increase energy expenditure. The hypothalamic arcuate nucleus (ARC) is a major relay between the periphery and CNS in body fat regulation (e.g. by being a target of leptin). The present study aimed to investigate the possible mechanisms responsible for the effects exerted by endogenous IL‐1 and IL‐6 on body fat at the level of the ARC, as well as possible interactions between IL‐1 and IL‐6. Therefore, we measured the gene expression of neuropeptides of the ARC involved in energy balance in IL‐1RI−/− and IL‐6−/− mice. We also investigated the interactions between expression of IL‐1 and IL‐6 in these mice, and mapped IL‐6 receptor α (IL‐6Rα) in the ARC. The expression of the obesity promoting peptide neuropeptide Y (NPY), found in the ARC, was increased in IL‐1RI−/− mice. The expression of NPY and agouti‐related peptide (AgRP), known to be co‐expressed with NPY in ARC neurones, was increased in cold exposed IL‐6−/− mice. IL‐6Rα immunoreactivity was densely localised in the ARC, especially in the medial part, and was partly found in NPY positive cell bodies and also α‐melanocyte‐stimulating hormone positive cell bodies. The expression of hypothalamic IL‐6 was decreased in IL‐1RI−/− mice, whereas IL‐1ß expression was increased in IL‐6−/− mice. The results of the present study indicate that depletion of the activity of the fat suppressing cytokines IL‐1 and IL‐6 in knockout mice can increase the expression of the obesity promoting neuropeptide NPY in the ARC. Depletion of IL‐1 activity suppresses IL‐6 expression, and IL‐6Rα‐like immunoreactivity is present in neurones in the medial ARC, including neurones containing NPY. Therefore, IL‐6, IL‐1 and NPY/AgRP could interact at the level of the hypothalamic ARC in the regulation of body fat.

Interleukin-6 Receptor α is Co-localised with Melanin-Concentrating Hormone in Human and Mouse Hypothalamus

Interleukin (IL)‐6 deficient mice develop mature‐onset obesity. Furthermore, i.c.v. administration of IL‐6 increases energy expenditure, suggesting that IL‐6 centrally regulates energy homeostasis. To investigate whether it would be possible for IL‐6 to directly influence the energy homeostasis via hypothalamic regulation in humans and rodents, we mapped the distribution of the ligand binding IL‐6 receptor α (IL‐6Rα) in this brain region. In the human hypothalamus, IL‐6Rα‐immunoreactivity was detected in perikarya and first‐order dendrites of neurones. The IL‐6Rα‐immunoreactive (‐IR) neurones were observed posterior to the level of the interventricular foramen. There, IL‐6Rα‐IR neurones were located in the lateral hypothalamic, perifornical, dorsal and posterior hypothalamic areas, the hypothalamic dorsomedial nucleus and in the zona incerta. In the caudal part of the hypothalamus, the density of the IL‐6Rα‐IR neurones gradually increased. Double‐labelling immunofluorescent studies demonstrated that IL‐6Rα immunoreactivity was localised in the same neurones as the orexigenic neuropeptide, melanin‐concentrating hormone (MCH). By contrast, IL‐6Rα‐immunoreactivity was not observed in the orexin B‐IR neurones. To determine whether the observed expression of IL‐6Rα is evolutionary conserved, we studied the co‐localisation of IL‐6Rα with MCH and orexin in the mouse hypothalamus, where IL‐6Rα‐immunoreactivity was present in numerous MCH‐IR and orexin‐IR neurones. Our data demonstrate that the MCH neurones of the human hypothalamus, as well as the MCH and orexin neurones of the mouse hypothalamus, contain IL‐6Rα. This opens up the possibility that IL‐6 influences the energy balance through the MCH neurones in humans, and both MCH and orexin neurones in mice.

Centrally Administered Ghrelin Acutely Influences Food Choice in Rodents

We sought to determine whether the orexigenic hormone, ghrelin, is involved in the intrinsic regulation of food choice in rats. Ghrelin would seem suited to serve such a role given that it signals hunger information from the stomach to brain areas important for feeding control, including the hypothalamus and reward system (e.g. ventral tegmental area, VTA). Thus, in rats offered a choice of palatable foods (sucrose pellets and lard) superimposed on regular chow for 2 weeks, we explored whether acute central delivery of ghrelin (intracerebroventricular (ICV) or intra-VTA) is able to redirect their dietary choice. The major unexpected finding is that, in rats with high baseline lard intake, acute ICV ghrelin injection increased their chow intake over 3-fold, relative to vehicle-injected controls, measured at both 3 hr and 6 hr after injection. Similar effects were observed when ghrelin was delivered to the VTA, thereby identifying the VTA as a likely contributing neurobiological substrate for these effects. We also explored food choice after an overnight fast, when endogenous ghrelin levels are elevated, and found similar effects of dietary choice to those described for ghrelin. These effects of fasting on food choice were suppressed in models of suppressed ghrelin signaling (i.e. peripheral injection of a ghrelin receptor antagonist to rats and ghrelin receptor (GHSR) knock-out mice), implicating a role for endogenous ghrelin in the changes in food choice that occur after an overnight fast. Thus, in line with its role as a gut-brain hunger hormone, ghrelin appears to be able to acutely alter food choice, with notable effects to promote “healthy” chow intake, and identify the VTA as a likely contributing neurobiological substrate for these effects.

Regulation of body fat mass by the gut microbiota: Possible mediation by the brain

New insight suggests gut microbiota as a component in energy balance. However, the underlying mechanisms by which gut microbiota can impact metabolic regulation is unclear. A recent study from our lab shows, for the first time, a link between gut microbiota and energy balance circuitries in the hypothalamus and brainstem. In this article we will review this study further.

Perinatal lack of maternal IL-6 promotes increased adiposity during adulthood in mice.

The perinatal environment appears important in establishing metabolic phenotypes in adulthood. Mice deficient in IL-6 (IL-6(-/-)) tend to develop mature-onset obesity, but it is unknown whether perinatal exposure to IL-6 produced by the dam influences the metabolism of adult offspring. To address this issue, we monitored IL-6(-/-) offspring of IL-6(-/-) or IL-6(+/-) dams, as well as wild-type (WT) mice. At adult age, IL-6(-/-) mice weighed significantly more and had more body fat than WT mice, regardless of maternal genotype, and had lower insulin sensitivity. This phenotype was more pronounced in IL-6(-/-) offspring of IL-6(-/-) dams, because they gained weight significantly faster than IL-6(-/-) offspring of IL-6(+/-) dams and had more body fat and higher serum leptin levels at an earlier age. The leptin content was 2-fold higher in milk from IL-6(-/-) than WT dams. However, cross-fostering IL-6(-/-) mice with WT dams did not alter body weight, body composition, or adipocyte size at adult age compared with IL-6(-/-) mice fostered by IL-6(-/-) dams. Conversely, WT mice fostered by IL-6(-/-) dams weighed significantly more than those fostered by WT dams and had more body fat, larger adipocytes, and altered hypothalamic gene expression. We conclude that body fat of adult mice can be increased by perinatal exposure to factors affected by lack of maternal IL-6.

Body weight homeostat that regulates fat mass independently of leptin in rats and mice

Significance The only known homeostatic regulator of fat mass is the leptin system. We hypothesized that there is a second homeostat regulating body weight with an impact on fat mass. In this study we have added and removed weight loads from experimental animals and measured the effects on the biological body weight. The results demonstrate that there is a body weight homeostat that regulates fat mass independently of leptin. As the body weight-reducing effect of increased loading was dependent on osteocytes, we propose that there is a sensor for body weight in the long bones of the lower extremities acting as “body scales.” This is part of a body weight homeostat, “gravitostat,” that keeps body weight and body fat mass constant. Subjects spending much time sitting have increased risk of obesity but the mechanism for the antiobesity effect of standing is unknown. We hypothesized that there is a homeostatic regulation of body weight. We demonstrate that increased loading of rodents, achieved using capsules with different weights implanted in the abdomen or s.c. on the back, reversibly decreases the biological body weight via reduced food intake. Importantly, loading relieves diet-induced obesity and improves glucose tolerance. The identified homeostat for body weight regulates body fat mass independently of fat-derived leptin, revealing two independent negative feedback systems for fat mass regulation. It is known that osteocytes can sense changes in bone strain. In this study, the body weight-reducing effect of increased loading was lost in mice depleted of osteocytes. We propose that increased body weight activates a sensor dependent on osteocytes of the weight-bearing bones. This induces an afferent signal, which reduces body weight. These findings demonstrate a leptin-independent body weight homeostat (“gravitostat”) that regulates fat mass.

Dietary Polyunsaturated Fatty Acids Increase Survival and Decrease Bacterial Load during Septic Staphylococcus aureus Infection and Improve Neutrophil Function in Mice

ABSTRACT Severe infection, including sepsis, is an increasing clinical problem that causes prolonged morbidity and substantial mortality. At present, antibiotics are essentially the only pharmacological treatment for sepsis. The incidence of resistance to antibiotics is increasing; therefore, it is critical to find new therapies for sepsis. Staphylococcus aureus is a major cause of septic mortality. Neutrophils play an important role in the defense against bacterial infections. We have shown that a diet with high levels of dietary saturated fatty acids decreases survival in septic mice, but the mechanisms behind this remain elusive. The aim of the present study was to investigate how the differences in dietary fat composition affect survival and bacterial load after experimental septic infection and neutrophil function in uninfected mice. We found that, after S. aureus infection, mice fed a polyunsaturated high-fat diet (HFD-P) for 8 weeks had increased survival and decreased bacterial load during sepsis compared with mice fed a saturated high-fat diet (HFD-S), similar to mice fed a low-fat diet (LFD). Uninfected mice fed HFD-P had a higher frequency of neutrophils in bone marrow than mice fed HFD-S. In addition, mice fed HFD-P had a higher frequency of neutrophils recruited to the site of inflammation in response to peritoneal injection of thioglycolate than mice fed HFD-S. Differences between the proportion of dietary protein and carbohydrate did not affect septic survival at all. In conclusion, polyunsaturated dietary fat increased both survival and efficiency of bacterial clearance during septic S. aureus infection. Moreover, this diet increased the frequency and chemotaxis of neutrophils, key components of the immune response to S. aureus infections.

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.