Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice
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- Tsuneki, H., Murata, S., Anzawa, Y. et al. Diabetologia (2008) 51: 657. doi:10.1007/s00125-008-0929-8
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Orexin/hypocretin is a hypothalamic neuropeptide that regulates motivated behaviours, such as feeding and arousal, and, importantly, is also involved in energy homeostasis. The aim of this study was to reveal the role of orexin in the regulation of insulin sensitivity for glucose metabolism.
Orexin knockout mice fasted overnight underwent oral glucose tolerance testing and insulin tolerance testing. The impact of orexin deficiency on insulin signalling was studied by Western blotting to measure levels of Akt phosphorylation and its upstream and downstream molecules in the hypothalamus, muscle and liver in orexin knockout mice.
We found that orexin deficiency caused the age-related development of impaired glucose tolerance and insulin resistance in both male mice without obesity and female mice with mild obesity, fed a normal chow diet. When maintained on a high-fat diet, these abnormalities became more pronounced exclusively in female orexin knockout mice that developed severe obesity. Insulin signalling through Akt was disrupted in peripheral tissues of middle-aged (9-month-old) but not young adult (2-to-3-month-old) orexin knockout mice fed a normal chow diet. Moreover, basal levels of hypothalamic Akt phosphorylation were abnormally elevated in orexin knockout mice at every age studied, and insulin stimulation failed to increase the level of phosphorylation. Similar abnormalities were observed with respect to GSK3β phosphorylation in the hypothalamus and peripheral tissues of middle-aged orexin knockout mice.
Our results demonstrate a novel role for orexin in hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with age.
KeywordsHypocretinHypothalamusImpaired glucose toleranceInsulin resistanceInsulin signallingOrexin
glycogen synthase kinase
The hypothalamus contains multiple neuronal systems responsible for the regulation of energy homeostasis. Among them, considerable attention has been focused on orexin, also known as hypocretin, because of its potent regulation of wakefulness, feeding behaviour and energy metabolism . Orexin-A and -B are a pair of neuropeptides cleaved from a common precursor peptide, prepro-orexin [2, 3]. Orexin neurons are localised in the posterolateral hypothalamus and project into the entire brain except the cerebellum . Electrophysiological studies on hypothalamic slices have shown that orexin neurons are responsive to peripheral metabolic cues [4, 5]. The excitability of orexin neurons was inhibited by glucose and leptin, indicators of high energy levels, whereas ghrelin, a signal for energy depletion, caused a stimulatory effect. The glucosensing machinery of orexin neurons is sufficiently sensitive to translate physiological glucose variations into different firing rates through tandem-pore K+ channels . In fact, in vivo, orexin neurons effectively respond to nutritional status , and levels of mRNA transcripts for prepro-orexin are increased upon fasting or insulin-induced hypoglycaemia in rats [3, 8]. Thus, hypothalamic orexin neurons are a central site for integrating peripheral metabolic signals.
An association between short habitual sleep duration and increased BMI has been found in a large cohort study, suggesting interaction between the homeostatic systems regulating sleep/wakefulness and energy balance . Orexin deficiency has been shown to be involved in the pathogenesis of narcolepsy in human and animal models, such as mice with knockout mutations in the gene for prepro-orexin (prepro-orexin knockout mice, referred to throughout the remainder of this article as orexin knockout mice) and orexin neuron-deficient mice [10, 11]. In humans, narcolepsy is accompanied by energy imbalance, such as decreased energy intake and increased BMI, resulting in an increase in the incidence of type 2 diabetes [12–15]. Mice deficient in orexin neurons also exhibit late-onset obesity, despite eating less , and on a high-fat diet, become heavier than wild-type mice . Thus, orexin neurons appear to provide a crucial link between arousal and energy balance [1, 11, 18].
Circulating levels of glucose, a major source of energy, are tightly regulated by a sophisticated mechanism in which insulin plays a critical role by reducing hepatic glucose production and stimulating glucose transport into muscle and fat [19, 20]. Insulin is also present in mammalian brain , and hypothalamic insulin actions cause inhibition of hepatic glucose production, which regulates fasting blood glucose levels [22, 23]. We have previously reported that central administration of orexin reduces fasting blood glucose levels in mice, without changing serum insulin levels . It is therefore possible that orexin enhances the actions of insulin in the hypothalamus to reduce fasting glucose, although the interaction between orexin and insulin in this region of the brain has not been clarified. In this study, we investigated the effect of orexin deficiency on glucose/insulin homeostasis using orexin knockout mice, and provide the first evidence that orexin is an essential factor for maintaining hypothalamic and peripheral insulin sensitivity for glucose metabolism, particularly with advancing age.
Orexin knockout mice with a mixed C57BL/6J–129/SvEv background, which completely lack both orexin-A and -B, were generated using a standard method, as described previously . N5 or N10 generations of heterozygous (+/−) mice (backcrossed to C57BL/6J mice) were mated to obtain null (−/−) mutants and wild-type (+/+) littermates. The N5 mice were used in this study unless otherwise indicated. The offspring were genotyped by PCR using the primer set shown in Electronic supplementary material (ESM) Table 1. The animals were housed under standard light (12 h light/dark cycle) and temperature conditions, and were given free access to a normal diet (CE-2; Clea Japan, Tokyo, Japan) and water. Certain groups of mice were given a high-fat diet containing 30% fat (Rodent Quick Fat; Clea Japan) for 6 months or containing 60% fat (D12492; Research Diets, New Brunswick, NJ, USA) for 6 weeks. All experimental procedures were approved by the University of Toyama Animal Research Committee.
OGTT and insulin tolerance test
For the OGTT, mice fasted for 10 h were orally loaded with glucose (2 g/kg). For the insulin tolerance test, mice fasted for 10 h were intraperitoneally injected with human regular insulin (Humalin R; Eli Lilly, Indianapolis, IN, USA; 1 U/kg). Blood was collected from the tail vein, and blood glucose concentrations were determined using a glucose analyser (Antsense II, Horiba Industry, Kyoto, Japan).
Measurement of locomotor activity
Mice were placed individually in an acrylic cage, and after 1 h habituation, the locomotor activity (number of movements) was recorded at 30 min intervals for 24 h using a Scanet MV-20plus system (Melquest, Toyama, Japan).
Measurement of serum parameters
Blood was collected from the orbital sinus of fasted mice under halothane anaesthesia, and then kept on ice for 2 h before being centrifuged at 16,000 ×g for 1 min at 4°C. Serum was stored at −80°C until use. The serum insulin and leptin concentrations were analysed using an ELISA kit (Morinaga Seikagaku, Tokyo, Japan).
Intracerebroventricular administration of insulin
Mice were anaesthetised with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and positioned in a stereotaxic frame. A 10 mm long guide cannula was implanted into the lateral ventricle (0.3 mm posterior to bregma, 0.9 mm lateral from the central suture) and fixed to the skull with dental cement. The injection depth was 2.4 mm from the skull surface. After a 7 day recovery period, the mice were intracerebroventricularly injected with 3.0 μl of human regular insulin (10 mU per mouse).
Mice fasted overnight were given an i.v. tail vein injection (4 U/kg) or intracerebroventricular injection (10 mU) of human regular insulin, and killed 15 or 5 min after the injection, respectively. PBS was injected as vehicle control. Subsequently, the hypothalamus, hindlimb muscle and liver were dissected, frozen in liquid nitrogen, and stored at −80°C until use. The tissues were lysed and subjected to immunoprecipitation and/or Western blotting, as described previously . Primary antibodies against Akt1, insulin receptor β (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phosphotyrosine (PY99; Santa Cruz Biotechnology); glycogen synthase kinase 3β (GSK3β) (BD Transduction Laboratories, San Jose, CA, USA); IRS-1 and IRS-2 (Upstate Biotechnology, Lake Placid, NY, USA); AMP kinase α, phospho-Akt (Ser473), phospho-Akt (Thr308), phospho-AMP kinase α (Thr172), phospho-GSK3β (Ser9), p44/p42 mitogen-activated protein (MAP) kinase and phospho-p44/p42 MAP kinase (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA, USA); GLUT4 (Abcam, Cambridge, MA, USA); and β-actin (Sigma, St Louis, MO, USA) were used.
Data are represented as means±SEM. The significance of differences between two groups was assessed by Student’s t test, and differences between multiple groups were assessed by one-way ANOVA and Bonferroni test, using Statview software (SAS Institute, Cary, NC, USA). A p value of less than 0.05 was considered significant.
Impaired glucose tolerance and insulin resistance in orexin deficiency
To investigate whether orexin deficiency affects glucose metabolism, orexin knockout mice at 2, 6 and 9 months of age underwent an OGTT. Fasting blood glucose levels were not significantly different between orexin knockout mice and wild-type mice at any age (Fig. 1c–h, at 0 min). After oral administration of glucose, no abnormalities in glucose tolerance were observed in either male (Fig. 1c) or female (Fig. 1d) 2-month-old orexin knockout mice, or in either male (Fig. 1e) or female (Fig. 1f) 6-month-old orexin knockout mice, compared with wild-type controls. However, in 9-month-old orexin knockout male mice, blood glucose levels were 1.2-fold and 1.3-fold higher at 15 and 120 min after glucose administration, respectively, compared with those in wild-type male mice (Fig. 1g), although the differences in the glucose AUCs did not reach significance (Fig. 1i). In addition, the glucose levels in the 9-month-old orexin knockout female mice were 1.3-fold higher at 60 and 120 min after glucose administration than those in wild-type female mice (Fig. 1h), and the glucose AUC was significantly increased (Fig. 1j). These results indicate that glucose tolerance was moderately impaired with age in orexin knockout mice.
Orexin is a protection factor against diet-induced obesity and insulin resistance
Orexin deficiency disrupts hypothalamic and peripheral insulin signalling
Levels of p44/p42 MAP kinase phosphorylation in the hypothalamus were not altered by i.v. insulin injection in either wild-type or orexin knockout mice at 9 months of age (Fig. 7g). Although basal levels of p44/p42 MAP kinase phosphorylation were elevated in skeletal muscle of orexin knockout mice, an increase in MAP kinase phosphorylation was induced in muscle of both wild-type and orexin knockout mice by insulin injection (Fig. 7h).
Basal levels of AMP kinase α phosphorylation in orexin knockout mice were significantly lower than those of wild-type mice (Fig. 7i), indicating that hypothalamic AMP kinase activity was reduced by orexin deficiency. These results were in agreement with the reduced food intake in orexin knockout mice, since the decreased AMP kinase activity in the hypothalamus is known to inhibit food intake .
Finally, we examined the influence of high-fat feeding on insulin signalling in male and female wild-type and orexin knockout mice. When the 2- to 3-month-old mice were fed a 60% high-fat diet for 6 weeks, the body weight gain was more evident in female orexin knockout mice than wild-type female mice, whereas the weight gain was comparable for the two groups of male mice (data not shown). After insulin stimulation, the wild-type and orexin knockout male mice exhibited a similar increase in levels of Akt phosphorylation in muscle (ESM Fig. 1a) and liver (ESM Fig. 1b), whereas no significant increases in phosphorylation levels were observed in the hypothalamus (data not shown). Similar results were obtained in the female wild-type and orexin knockout mice (ESM Fig. 1c,d). These results indicate that there is no difference between the sexes in terms of insulin-induced phosphorylation of Akt in orexin knockout mice fed a high-fat diet.
Orexin regulates motivated behaviours, such as arousal, feeding and reward-seeking, and has important effects on metabolic, autonomic and endocrine functions [28–30]. The findings that orexin-A increases oxygen consumption and body temperature even in anaesthetised rats , and that orexin neuron-deficient mice display reduced energy expenditure that is independent of sleep/wake, locomotion and food intake  indicate that orexin-induced increase in energy metabolism is not simply due to increased wakefulness and physical activity. In the present study, we investigated the involvement of orexin on glucose metabolism using orexin knockout mice, and found that orexin is essential for the maintenance of normal insulin sensitivity with increasing age, although the secondary influences of reduced chronic physical activity and energy expenditure on insulin resistance with age cannot be excluded using this animal model.
Insulin resistance is a major factor in the pathogenesis of type 2 diabetes, which occurs during the ageing process and results in impaired glucose metabolism [33, 34]. We observed that male orexin knockout mice exhibited impaired insulin sensitivity and hyperinsulinaemia at 9 months but not at 2–6 months of age. Glucose tolerance was not markedly impaired in middle-aged male orexin knockout mice, probably because insulin resistance could be compensated by hyperinsulinaemia to maintain glucose tolerance. Female orexin knockout mice exhibited impaired glucose tolerance, slight insulin resistance and moderate hyperinsulinaemia at 9 months but not at 2–6 months of age. Moreover, late-onset mild obesity was observed exclusively in female orexin knockout mice, consistent with previous reports [17, 35]. These results indicate that male orexin knockout mice developed age-related insulin resistance without obesity, whereas insulin resistance and some other factors related to obesity may synergistically cause abnormal glucose tolerance in female orexin knockout mice.
Both orexin receptors and insulin receptors are abundant in the hypothalamus, and promote peripheral energy metabolism, despite having opposite effects on food intake [23, 36, 37]. Brain insulin receptor deficiency causes obesity and mild insulin resistance in mice , similar to that seen in animals with orexin deficiency. Insulin receptor signalling is mediated by two main pathways: the PI3-kinase/Akt and MAP kinase pathway [20, 39]. The hypothalamic PI3-kinase/Akt pathway mediates the effect of insulin on food intake and energy expenditure via forkhead transcription factor Foxo1 , whereas insulin-dependent activation of hypothalamic MAP kinase does not have any effect on glucose homeostasis . We found that basal levels of Akt phosphorylation were elevated in the hypothalamus of orexin knockout mice, and central and peripheral injection of insulin failed to increase the phosphorylation levels. These results indicate that hypothalamic insulin signalling is disrupted by orexin deficiency. Thus, orexin and insulin appear to exert cooperative actions on energy metabolism in the hypothalamus. Basal levels of Akt in the hypothalamus of wild-type mice were increased by intracerebroventricular injection of 4 μmol/l orexin at a rate of 0.25 μl/h, for a total of 14 days, using an osmotic minipump. The elevated levels of Akt phosphorylation in the hypothalamus of orexin knockout mice were not reversed, but, rather, enhanced by orexin administration (data not shown). It is possible that longer term or more finely tuned treatment with orexin, mimicking the physiological secretion pattern, might be necessary to rescue the abnormal hypothalamic Akt signalling in orexin deficiency.
Orexin neurons are known to discharge during active waking , and the excitability is inhibited by glucose [1, 4, 6]. In addition, changes in blood glucose levels negatively correlate with expression of the gene encoding prepro-orexin in the mouse hypothalamus . A recent study demonstrated that blood glucose is a crucial for the regulation of Akt phosphorylation levels in the cerebral cortex and hippocampus of mice, reporting that phosphorylation levels are lowered in the fasting state and elevated after glucose administration in non-diabetic mice, and that phosphorylation levels are elevated in streptozotocin-induced diabetic mice . We therefore anticipate that the basal level of Akt phosphorylation is extensively elevated in the brain, including the hypothalamus, under conditions where expression of the gene for prepro-orexin is reduced or lost. The glucosensing machinery of the orexin neurons may be at least partly involved in the conversion of hyperglycaemic signals into Akt phosphorylation through an as yet unknown mechanism.
A high-fat diet is known to increase the risk of insulin resistance. When fed a high-fat diet, obesity was more evident in female orexin knockout mice than wild-type female mice, and was associated with impaired fasting glucose, severe glucose intolerance and insulin resistance. In contrast, no such differences were observed between the orexin knockout male mice and the wild-type male mice. Although the biological basis of the difference between the sexes remains unclear, the present results indicate that alteration of Akt phosphorylation is not the key factor. We anticipate that leptin plays a pivotal role in this regard, because diet-induced obesity leads to leptin resistance that could further exacerbate insulin resistance [44, 45], and because brain uptake of leptin decreases in female and elderly humans . In fact, serum leptin levels were elevated exclusively in middle-aged orexin knockout female mice fed a normal chow diet, consistent with previous observation in female mice deficient in orexin neurons . Further study will be required to determine the influence of orexin deficiency in leptin signalling, especially in females.
In conclusion, the present results demonstrate that orexin is an essential factor for maintaining hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with advancing age. This may explain why the incidence of type 2 diabetes increases in orexin-deficient human narcolepsy without severe obesity. Furthermore, the expression of the gene for prepro-orexin is downregulated in diabetic ob/ob and db/db mice , and the downregulation is shown to be due to hyperglycaemia in these animals . These suggest that diabetic hyperglycaemia reduces the expression of the gene for prepro-orexin in the hypothalamus, and thereby further worsens the insulin resistance. Therefore, the orexin receptor that controls hypothalamic insulin signalling may be a new target to treat hyperglycaemia in patients with type 2 diabetes.
We thank S. Kobayashi, T. Uwano and H. Nishijo (University of Toyama) for their technical assistance. This study was supported in part by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to H. Tsuneki) and the Japan Diabetes Foundation.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.