Expression of orexin peptide in CAG/orexin mouse
Previous results with CAG/orexin
transgenic mice revealed multifold increases in both orexin-A and orexin-B peptides in whole brain extracts (Mieda et al., 2004
). Immunohistochemical localization of orexin-A in the brain of CAG/orexin
mice demonstrates ectopic peptide production in medial, basal, lateral, and suprachiasmatic hypothalamic nuclei, nucleus accumbens, globus pallidus, hippocampal formation, ventral tegmental area, and locus coerulus (Figures S1, 2, Table S1
). All of these locations have previously been implicated as participants in networks controlling various homeostatic, circadian, learned, and/or hedonistic aspects of food intake, taste preference, or energy homeostasis (Saper et al., 2002
; Willie and Woolsey, 2008
). Previous results demonstrated that CAG/orexin
transgene insertion was sufficient to rescue the narcolepsy/cataplexy phenotype of mice lacking endogenous orexinergic neurons (Mieda et al., 2004
). Thus, the CAG/orexin
transgene produces functional peptides that can activate orexin receptors.
The physiological relevance of peripheral actions of orexins, if any, remains controversial (Heinonen et al., 2008
). In spite of the use of a general promoter for orexin overexpression, we found that CAG/orexin
mice exhibited ectopic orexin-A immunoreactivity in a limited set of peripheral tissues including thyroid gland, adrenal cortex, and some pancreatic islets. No evidence of ectopic expression was encountered in other metabolic tissues such as brown and white adipose, liver, or skeletal muscle (Figure S3, Table S2, 3
CAG/orexin Mice are Resistant to Diet-Induced Obesity
To examine the effect of increased orexin on body weight, CAG/orexin transgenic mice and wild-type littermate mice were fed either a low or a high fat diet. In both male and female mice, the body weights of wild-type mice were significantly higher when fed a high fat diet compared to a low fat diet. However, mice overexpressing orexin did not show a significant difference in body weight growth between a low fat diet and a high fat diet (). Thus, wild-type mice are susceptible to diet-induced obesity, whereas CAG/orexin mice are quite resistant.
Growth curves of genetically modified mice fed low or high fat diet
To determine which receptor pathway mediates the anti-obesity effect of orexin overexpression, we crossed CAG/orexin transgenic mice to OX1R−/− and OX2R−/− lines. We compared the effects of isolated orexin-OX2R signaling (in OX1R−/− mice and OX1R−/−; CAG/orexin mice) versus isolated orexin-OX1R signaling (in OX2R−/− mice and OX2R−/−; CAG/orexin mice) upon growth curves. show that increased OX2R activation is sufficient to mediate the preponderance of resistance to diet-induced obesity. On the other hand, in both sexes, increased OX1R activation alone does not significantly protect from development of obesity (). Unlike differences in body weight, there were no significant differences in linear growth among the various genotypic groups (data not shown). Thus, OX2R signaling selectively mediates the anti-obesity effect of orexin overexpression in mice challenged with a high fat diet.
CAG/orexin transgenic male mice were also resistant to ageing-related adiposity while wild-type male mice fed a low fat diet showed continuous weight gain during ageing (). In spite of similar growth curves before 18 weeks of age, the growth curve between 19 weeks and 30 weeks of age of wild-type mice fed a low fat diet was significantly larger than that of CAG/orexin transgenic mice (p=0.0016). Likewise, OX1R−/− male mice fed a low fat diet showed larger body weight growth between 17 weeks and 30 weeks of age than OX1R−/−; CAG/orexin mice despite no significant difference in the growth curves before 16 weeks of age (p=0.036). OX2R−/− male mice fed a low fat diet showed larger body weight than OX2R−/−; CAG/orexin mice through the whole observation period (p=0.005), however, the fat mass and serum leptin of OX2R−/−; CAG/orexin male mice were similar to those of OX2R−/− male mice ().
Fat mass, serum leptin levels, and lean mass of orexin signaling-modified mice
CAG/orexin Transgene Reduces Fat Mass and Leptin
Consistent with body weight data, at 28 weeks of age, CAG/orexin
male mice showed a significant reduction of fat mass on a low fat diet as compared with wild-type male mice (). The fat mass of CAG/orexin
male mice and OX1R−/−
mice fed a high fat diet was significantly less than those of wild-type mice and OX1R−/−
mice, respectively, for both sexes (). There was no significant difference in fat mass between OX2R−/−
mice and OX2R−/−
mice on both a low fat and a high fat diet for either sex. OX2R−/−
male mice exhibited a significant tendency toward increased fat mass under high fat, and OX2R−/−
female mice exhibited a mild but significant tendency toward increased fat mass under even low fat conditions compared to wild-type mice, which is consistent with previously described adiposity of narcoleptic mice (Hara et al., 2001
) and a physiological role of OX2R signaling in suppressing adiposity.
Next, we measured serum leptin which is typically correlates with fat mass. Concordance between fat mass and leptin levels was confirmed in each genotype. Specifically, the leptin levels of CAG/orexin mice and of OX1R−/−; CAG/orexin mice were significantly lower than those of wild-type mice and of OX1R−/− mice fed a high fat diet, respectively, whereas there was no significant difference in serum leptin levels between OX2R−/−; CAG/orexin mice and OX2R−/−mice on both low fat and high fat diets for both sexes (). Compared to differences observed in fat mass, the CAG/orexin transgene was associated with small but significant reductions in lean mass of male mice having functional orexin receptors and those deficient in OX1R under high fat conditions (). The CAG/orexin transgene was similarly associated with a significant mild reduction in lean mass of female mice having functional receptors under high fat conditions, but a significant mild reduction of lean mass by the transgene under OX2R deficient low fat conditions was also observed ().
Increased Energy Expenditure of CAG/orexin mice
To explore the underlying cause of differential resistance to diet-induced obesity in mice overexpressing orexin, we housed mice from each genotypic group in metabolic cages in order to measure oxygen consumption, carbon dioxide production, and locomotor activity. The effective mass-corrected energy expenditures of CAG/orexin
male mice and OX1R−/−
mice on a high fat diet were consistently elevated over those of wild-type mice and OX1R−/−
mice, respectively (), while the energy expenditures of OX2R−/−
mice resembled those of OX2R−/−
mice (). In contrast, we observed no consistent differences in respiratory quotient (an indirect indicator of lipid versus carbohydrate utilization) among different genotypic groups on a high fat diet (). The CAG/orexin
transgene induced no differences in energy expenditure or respiratory quotient among any genotypic groups on a low fat diet, regardless of the presence or absence of orexin receptors (Figure S4
). Low fat-fed OX1R−/−
mice showed reduced energy expenditure compared to wild-type controls (Figure S4G
). Importantly, CAG/orexin
transgenic mice did not exhibit hyperactivity, regardless of diet or receptor status (Figure S5
), although OX2R−/−
mice fed a low fat diet showed some reduced locomotion compared to wild-type mice (Figure S5G
), which is consistent with previous data from narcoleptic mice (Hara et al., 2001
). Basal core body temperature in CAG/orexin
mice on a high fat diet tended to be higher than in wild-type controls, but this difference did not reach significance (wild-type low fat diet: 36.6±0.1°C; CAG/orexin
low fat diet: 36.7±0.1°C; wild-type high fat diet: 36.8±0.1°C; CAG/orexin
high fat diet: 37.0±0.1°C; n = 5–6).
The metabolic parameters of orexin signaling-modified mice on a high fat diet
Both total high fat diet intake for 14 days () and body weight-adjusted daily food intake (data not shown) were significantly reduced in CAG/orexin
mice compared to wild-type controls. Critically, this did not result from abnormal taste preferences: compared to wild-type mice, CAG/orexin
and wild-type mice similarly exhibited greater preferences for high fat over low fat chow and for 10% sucrose over 1% sucrose solutions (Figure S6
Glucose Metabolism of CAG/orexin Mice
To examine the effect of orexin overexpression on glucose metabolism, we first measured blood glucose and serum insulin of fed mice at 30 weeks of age. When maintained on a low fat diet, we observed no significant difference in fed glucose level among genotypes (). On a high fat diet, however, wild-type mice exhibit hyperglycemia that is attenuated in CAG/orexin, OX1R−/−, and OX1R−/−; CAG/orexin mice, but not OX2R−/− or OX2R−/−; CAG/orexin mice. Thus, the protective effect depends upon functional OX2R, but can be mediated by endogenous orexin levels even without orexin overexpression. Notably, these data also show that OX1R deficiency alone can prevent high fat diet-induced hyperglycemia (see below).
Glucose metabolism of orexin signaling-modified mice on different fat diets
Increased serum insulin levels with obesity or ageing indicate mounting insulin resistance and sensitively predict deteriorating glucose control in human metabolic syndrome. When compared to wild-type mice, the CAG/orexin transgene reduced serum insulin levels on low fat diet and conferred protection from hyperinsulinemia on high fat diet (). Notably, a similar protective effect occurred in OX1R−/− mice (), despite relative obesity under these conditions (; ), suggesting that endogenous orexin-OX1R signaling can play a specific permissive role in development of hyperinsulinemia. However, the CAG/orexin transgene conferred protection from hyperinsulinemia upon all three genetic backgrounds on a high fat diet, suggesting that both OX1R and OX2R mediate protective effects of orexin overexpression on insulin sensitivity.
We next examined the effects of orexin overexpression upon fasting glucose and glucose tolerance after glucose administration in mice. On a low fat diet, orexin overexpression did not significantly affect glucose homeostasis (p=0.47, Figure S7
). On a high fat diet, however, CAG/orexin
mice exhibited significantly reduced basal fasting glucose levels as well as improved glucose tolerance at all time points tested relative to wild-type controls (). Despite absence of basal differences in fasting serum glucose between OX1R−/−
mice, the CAG/orexin
transgene conferred mild but significant improvements in glucose tolerance onto the OX1R−/−
background at later time points (). Improved glucose tolerance in the setting of reduced insulin levels indicates that the transgene confers improved insulin sensitivity. By contrast, we observed no significant differences in fasting glucose or glucose tolerance between OX2R−/−
mice and OX2R−/−
mice (). Thus, while OX1R may also influence circulating insulin levels, orexin overexpression improves insulin sensitivity by a predominantly OX2R-dependent mechanism.
Effects of CAG/orexin Transgene on Peripheral Tissues
Ectopic orexin production in thyroid tissue raises the possibility that abnormal activity of the thyroid axis contributes to leanness in CAG/orexin
mice. We measured serum thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) on low and high fat diets. High fat diet increased serum T3 and T4 levels of CAG/orexin
mice to a similar extent as wild-type mice despite significant differences in adiposity between the groups (Figure S8
). Serum TSH levels of CAG/orexin
mice on high fat diet were significantly elevated over those on a low fat diet, while high fat diet did not significantly affect serum TSH levels of wild-type mice. Importantly, the levels of serum TSH, T3, and T4 of CAG/orexin
mice were similar to those of wild-type mice when maintained on a low fat diet.
To determine whether increased energy expenditure of CAG/orexin
mice was associated with increased mitochondrial uncoupling proteins, we examined mRNA levels of major uncoupling proteins in brown fat and skeletal muscle (Figure S9
). High fat diet resulted in comparable increases in UCP1
mRNA in brown fat but not skeletal muscle in both genotypes. In contrast, UCP2
mRNA levels did not differ significantly by genotype or dietary condition, consistent with previous report (Surwit et al., 1998
Despite detection of ectopic orexin peptide in adrenal gland, CAG/orexin
mice and wild-type mice had similar total daily urinary levels of epinephrine and norepinephrine, and similar serum corticosterone levels (Figure S10
). In addition, CAG/orexin
transgene did not affect systolic blood pressure either in low fat or high fat diet (Figure S10
OX2R Agonist Prevents Diet-Induced Obesity
Our genetic studies implicate the OX2R pathway as mediator of the effects of orexin overexpression upon energy homeostasis. To further test the hypothesis that central enhancement of orexin-OX2R signaling confers resistance to diet-induced obesity, an OX2R selective agonist [Ala11, D-Leu15] Orexin-B (Asahi et al., 2003
) was continuously infused in the lateral ventricles of wild-type mice for 14 days. The administration of the OX2R selective agonist suppressed weight gain on a high fat diet without altering weight homeostasis on a low fat diet (). Importantly, the OX2R selective agonist had no obvious effect upon OX2R-deficient mice on a high fat diet (n=4, weight gain 3.33±0.61 g, P=0.67), verifying the specificity of the agonist in vivo. Following 14 days, the agonist-infused wild-type mice gained significantly less fat mass than did the vehicle-injected mice on a high fat diet, and no effect was observed on a low fat diet (). When centrally-infused mice fed high fat diet were monitored in metabolic chambers, OX2R agonist infusions resulted in consistently greater energy expenditures () but not respiratory quotients () or locomotor activity (data not shown), over vehicle-infused controls.
Effect of OX2R selective agonist on diet-induced obesity
As sleep/wake disturbances could affect food intake and energy expenditure, we recorded EEG/EMG signals during central OX2R agonist or vehicle infusions. Mice receiving OX2R agonist exhibited total wake or sleep times during both light and dark phases that closely resembled vehicle controls, irrespective of dietary condition (Figure S11
). As predicted from previous studies (Willie et al., 2003
), OX2R agonism continued to promote consolidation of behavioral states as demonstrated by increased wake and NREM episode durations in mice maintained on low fat diet. As this consolidation was not evident under high fat fed conditions, sleep/wake change cannot be the primary cause in metabolic effects of enhanced orexin signaling observed predominantly under high fat conditions.
We observed an expected homeostatic reduction of food intake in mice maintained on high fat diet compared to low fat diet (West et al., 1992
), and administration of the agonist significantly enhanced this effect by further suppressing food intake selectively in mice fed high fat diet (). After 14 days of OX2R agonist administration, we observed reduced hypothalamic mRNA expression of orexigenic factors, NPY
on a high fat diet, compared to those on a low fat diet (). Indeed, the number of c-Fos-positive cells in ARH region was significantly reduced in OX2R agonist-administered mice on a high fat diet (). The reduction of c-Fos-positive cell number was particularly notable in the ventromedial aspect of ARH () in which orexigenic NPY/AGRP neurons are located (Horvath, 2005
), consistent with the observed reduction in food intake and in NPY/AGRP
mRNAs we observed under this condition.
Leptin Mediates Anti-Obesity Effects of Orexin
Leptin negatively regulates body weight, suppresses food intake, and increases energy expenditure by inhibiting NPY/AGRP neurons and activating POMC neurons of ARH. Diet-induced obesity is associated with leptin resistance resulting from signal transduction abnormalities in ARH (Myers et al., 2008
). OX2R is highly expressed in ARH, and the effects of circulating leptin upon ARH resemble some effects of increased orexin-OX2R signaling that we observed. We hypothesized, therefore, that leptin signaling mediates some of the metabolic effects of orexin. To examine the consequences of orexin signaling enhancement on mice in the absence of leptin activity, we crossed CAG/orexin
transgenic and leptin-deficient ob/ob
lines. Remarkably, the CAG/orexin
transgene had no impact upon weight gain or fat mass of leptin-deficient ob/ob
mice (), suggesting that indeed the anti-obesity effect of CAG/orexin
depends upon leptin activity. We then centrally administered OX2R agonist to ob/ob
mice and similarly found no significant effect upon weight gain under low or high fat dietary conditions (). We also observed no effect of OX2R agonist compared to vehicle administration upon core body temperature of ob/ob
mice (data not shown).
No effect of orexin overexpression on the weight gain of ob/ob mouse
We directly examined whether orexin overexpression alters sensitivity to leptin. Leptin was continuously administered in the lateral ventricles of CAG/orexin and wild-type littermate pairs. Mice (3–4 month-old) were maintained on a low fat diet in order to initially match as to body weight (WT 29.7±3.6ng/ul and CAG/orexin 27.0±3.1ng/ul). Both wild-type mice and CAG/orexin mice lost weight during the administration of leptin, but CAG/orexin mice showed significantly enhanced weight loss and anorexia compared to wild-type mice on a low fat diet (), indicating that increased orexin signaling is associated with a more leptin-sensitive state.
Increased sensitivity of orexin overexpression mouse to leptin
Compared to control wild-type mice, 14 days of central leptin administration resulted in basal hypothalamic expression levels of NPY and AGRP and an expected induction of POMC mRNA (). In contrast, under basal conditions, CAG/orexin transgenic mice showed increased expression of NPY and AGRP, but not POMC mRNA. While we detected no significant changes in the expression of LEPR, SOCS3, or STAT3 gene products, the overall profile of altered hypothalamic gene expression we detected are consistent with the physiological state of anorexia and weight loss observed in mice undergoing leptin administration.