Human IL6 enhances leptin action in mice
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Interleukin-6 is an inflammatory cytokine with pleiotropic effects upon nutrient homeostasis. Many reports show that circulating IL6 correlates with obesity and contributes to insulin resistance; however, IL6 can promote energy expenditure that improves glucose homeostasis.
We investigated nutrient homeostasis in C57BL/6J mice with sustained circulating human IL6 (hIL6) secreted predominantly from brain and lung (hIL6 tg mice).
The hIL6 tg mice displayed no features of systemic inflammation and were more insulin-sensitive than wild-type mice. On a high-fat diet, hIL6 tg mice were lean, had low leptin concentrations, consumed less food and expended more energy than wild-type mice. Like ob/ob mice, the ob/ob IL6 mice (generated by intercrossing ob/ob and hIL6 tg mice) were obese and glucose-intolerant. However, low-dose leptin injections increased physical activity and reduced both body weight and food intake in ob/ob IL6 mice, but was ineffective in ob/ob mice. Leptin increased hypothalamic signal transducer and activator of transcription-3 phosphorylation in ob/ob IL6 mice, whereas ob/ob mice barely responded.
Human IL6 enhanced central leptin action in mice, promoting nutrient homeostasis and preventing diet-induced obesity.
KeywordsDiabetes IL6 Insulin resistance Leptin Leptin sensitivity Obesity
AMP-activated protein kinase
Ciliary neurotrophic factor
Dual-energy X-ray absorptiometry
Glial fibrillary acidic protein
- hIL6 tg mice
Mice with sustained circulating human IL6 secreted predominantly from brain and lung
Leptin receptor, isoform b
Suppressor of cytokine signalling
Signal transducer and activator of transcription-3
Understanding how IL6 regulates central and peripheral nutrient homeostasis is complicated by contradictory and multi-systemic effects under various physiological states . IL6 is best known as a pro-inflammatory cytokine that regulates innate immunity and the acute-phase response. However, IL6 also has tissue-specific effects that can differ in humans and rodents, depending on context and timing of stimulation . IL6 promotes chronic inflammation, whereas it displays anti-inflammatory effects during acute inflammatory stimuli .
Obesity and its progression to diabetes are associated with chronic inflammation characterised by secretion of the proinflammatory cytokines resistin, TNFα and IL6 from adipocytes . Epidemiological data confirm that elevated circulating IL6 correlates with adiposity in humans . IL6 is generally thought to promote systemic insulin resistance, especially during obesity, because it is secreted from fat cells of insulin-resistant humans . However, in type 2 diabetes patients the plasma concentrations of IL6 and TNFα may best reflect the level of adiposity rather than insulin sensitivity during the euglycaemic–hyperinsulinaemic clamp . Yet TNFα might also be a principle cause of dysregulated insulin signalling, as it stimulates production of IL6, IL1 and C-reactive peptide . It is possible that IL6 opposes the action of TNFα upon insulin sensitivity, as physical exercise promotes secretion of IL6 from skeletal muscle, while improving insulin sensitivity and nutrient homeostasis [1, 8]. The question of how IL6 integrates multiple signalling cascades to coordinate nutrient homeostasis in mammals remains unanswered.
Cell-based experiments and in vivo studies in rodents show that IL6 promotes insulin resistance . In vivo, 90 min after IL6 injections plasma glucose and insulin concentrations increase . Infusion of IL6 for 3 h causes hepatic and muscle insulin resistance [11, 12]. In addition, hepatic insulin receptor signalling improves in ob/ob mice treated with neutralising antibodies against IL6 . Recently, electrotransfer of murine IL6 cDNA into skeletal muscle promoted liver inflammation and hyperinsulinaemia in mice .
Unlike in rodent studies, infusion of recombinant human IL6 (hIL6) to sustain physiological concentrations in healthy individuals or patients with diabetes increases lipolysis in the absence of adverse effects and enhances glucose infusion rates during a euglycaemic–hyperinsulinaemic clamp [15–17]. Moreover, adipose-derived hIL6 can have autocrine effects that increase leptin secretion and fat oxidation, and reduce expression and activity of lipoprotein lipase in human adipose tissues, a phenomenon that might attenuate progression of obesity and diabetes . Human IL6 also displays anti-inflammatory characteristics by inhibiting TNFα and IL1, and activating IL1 receptor antagonist and IL10 [19–21]. Moreover, in rodents IL6 has central effects similar to those of leptin in promotion of nutrient homeostasis and peripheral insulin sensitivity [1, 22]. Thus, the role of IL6 in the regulation of nutrient homeostasis is contradictory and incompletely resolved, possibly confounded by differences between human and murine cytokine action .
Leptin is secreted from adipose tissue in proportion to fat stores, informing the central nervous system of the peripheral energy supply. Dysregulated leptin action (ob/ob mice) increases food intake, while reducing energy expenditure. In addition, ob/ob mice display severe obesity and insulin resistance that progresses to diabetes . However, ordinary obesity in mice and humans is associated with elevated leptin concentrations, suggesting leptin resistance in the central nervous system as a principle cause [24, 25]. Interestingly, IL6 might be required for a normal leptin response, as adult Il6 −/− mice develop hyperphagia and obesity, which is difficult to prevent by peripheral leptin injections .
To establish the long-term systemic effect of hIL6 upon nutrient homeostasis in mice, we investigated glucose tolerance, energy expenditure and insulin action in transgenic C57BL/6J mice and ob/ob mice that secrete hIL6 constitutively into the circulation. Our results show that hIL6 promotes central leptin action in mice, together with its beneficial effects upon nutrient homeostasis.
Treatment of mice involved in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Children’s Hospital Boston. IL6 transgenic mice, which have been previously described , were generated by ten backcrosses for pure C57BL/6J background. Ob/ob mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Ob/ob IL6 mice were generated by mating ob +/− /IL6 tg mice with ob +/− mice. Animals were fed either regular chow diet with 9% of energy derived from fat or a high-fat diet (HFD) (Research Diets, New Brunswick, NJ, USA) with 45% of energy derived from fat.
Intraperitoneal glucose tolerance test was performed on mice fasted overnight for 16 h. Blood glucose levels were measured on random-fed or overnight-fasted animals in mouse-tail blood using a glucometer (Elite; Bayer, Leverkusen, Germany) and serum samples were collected for insulin measurements. Animals were then injected intraperitoneally with d-glucose (2 g/kg body weight) and blood glucose levels were measured . Blood insulin and leptin levels were determined using rat insulin and mouse leptin ELISA kits (Crystal Chem, Downers Grove, IL, USA). Lean and fat body mass were assessed by dual-energy X-ray absorptiometry (DEXA) (GE Lunar, Madison, WI, USA) .
Food intake, physical activity and energy expenditure
All measurements were performed over a 72 h period with a comprehensive laboratory animal monitoring system (Oxymax Windows 3.0.3; Columbus Instruments, Columbus, OH, USA). The data presented are average values obtained in these recordings.
Hypothalamic neuropeptide expression
Neuropeptide mRNA was analysed using quantitative real-time PCR with customised primers. Actin gene expression was used to normalise RNA content and the relative gene product amounts were reported as mean ± SEM of several animals.
Mice were fasted overnight (16 h) and then fed for 4 h. Tissues were removed under anaesthetic, homogenised and applied for direct immunoblotting (50 μg) with the indicated antibodies . Antibodies used in this study included: rabbit insulin receptor, IRS1 and IRS2 antibodies (Upstate Biotechnology, Billerica, MA, USA); antibodies against signal transducer and activator of transcription-3 (STAT3), phospho-specific STAT3 (Tyr307), phospho-Aktser473, Akt and β-actin (Cell Signaling Technology, Danvers, MA, USA); monoclonal antibody to suppressor of cytokine signalling (SOCS3) and phospho-tyrosine (Upstate Biotechnology). The intensity of signals was determined using a Kodak molecular imaging system.
Unless otherwise stated, mean values ± SEM were used to make comparisons between groups. Logistic regression or generalised linear regression (SPSS version 16; SPSS, Chicago, IL, USA) was used where indicated to establish significant difference (p < 0.05) when multiple categorical predictors were compared across the experiments. A generalised linear model was used to make comparisons across all samples, using the Bonferroni correction for multiple comparisons.
The effect of human IL6 on growth and diet-induced obesity in mice
The hIL6 tg mice maintained between 4 and 24 weeks of age on regular chow (9% of energy derived from fat) were slightly smaller than wild-type mice (Fig. 1c). By comparison, HFD (45% energy derived from fat) caused significant weight gain in wild-type mice, whereas HFD-fed hIL6 tg mice were slightly smaller than chow-fed wild-type mice (Fig. 1c, d). DEXA confirmed that the lean body mass of 24-week-old wild-type or hIL6 tg mice was not influenced by diet. By comparison, HFD increased adipose mass of wild-type mice twofold, but had no effect on hIL6 tg mice (Fig. 1e). Haematoxylin and eosin staining confirmed that adipocytes were 30% smaller (p < 0.05) in HFD-fed hIL6 tg mice than in wild-type mice (Fig. 1f, g). Thus, hIL6 tg mice were slightly smaller and had less visceral fat on a chow diet, whereas HFD-induced obesity was prevented.
Glucose tolerance and insulin sensitivity in hIL6 tg mice
Hepatic metabolism and signalling in hIL6 tg mice
As expected, liver SOCS3 levels were related to STAT3 phosphorylation. Compared with chow-fed wild-type mice, SOCS3 increased in chow-fed hIL6 tg mice, decreased in HFD-fed wild-type mice and increased tenfold in HFD-fed hIL6 tg mice (Fig. 3c, d). The postprandial IRS1 concentration decreased significantly in the liver of chow-fed and HFD-fed hIL6 tg mice, which correlated with increased SOCS3 concentration as previously described (Fig. 3c, e) . By comparison, hIL6 had no effect on liver IRS2 concentrations in chow-fed mice, whereas it prevented loss of IRS2 in HFD-fed hIL6 tg mice (Fig. 3d, f). Thus changes in IRS1 and IRS2 concentrations were poor predictors of the effect of hIL6 upon systemic glucose homeostasis and peripheral insulin sensitivity.
The insulin receptor mediates tyrosine phosphorylation of IRS1 and IRS2, which directly activates the phosphatidylinositol-3-kinase to Akt cascade in all cells. Insulin stimulated equally insulin receptor phosphorylation and AktSer473 phosphorylation in liver of chow-fed wild-type and hIL6 tg mice; however, insulin-stimulated insulin receptor phosphorylation in HFD-fed hIL6 tg mice was significantly stronger than in HFD-fed wild-type mice (Fig. 3g, h). Consistent with these results, insulin-stimulated AktSer473 phosphorylation in HFD-fed hIL6 tg mice was also increased compared with HFD-fed wild-type mice (Fig. 3g, i). This pattern of insulin receptor and Akt phosphorylation might explain in part the positive effect of hIL6 upon insulin sensitivity in hIL6 tg mice.
Regardless of diet, the hIL6 tg mice consumed more oxygen and expelled more CO2 than wild-type mice during the light and dark phases (Fig. 4c, d). They also displayed 20% more voluntary movement than wild-type controls, when controlling for age, time of day and diet (Fig. 4e). Thus, diet-induced obesity was probably avoided in hIL6 tg mice by a combination of reduced food intake and increased activity and energy expenditure, which was associated with increased leptin sensitivity.
Central regulation of feeding behaviour in hIL6 tg mice
We used semi-quantitative RT–PCR to determine whether hIL6 altered expression of Agrp and Pomc. As expected, feeding suppressed expression of the former and increased expression of the latter in chow-fed wild-type and hIL6 tg mice (Fig. 5e, f). By comparison, expression of Agrp and Pomc was reduced and insensitive to feeding in HFD-fed wild-type mice, whereas expression in HFD-fed hIL6 tg mice responded normally to feeding (Fig. 5e, f). Thus, hIL6 had multiple effects in the hypothalamus of mice on HFD; these effects were consistent with increased energy utilisation, reduced food intake and changes in peripheral glucose homeostasis.
The effect of human IL6 in ob/ob mice
Despite the fact that the adipose mass was equal in ob/ob and ob/ob IL6 mice at 12 weeks, the fed blood glucose and fasted insulin concentrations were reduced 50% in the latter (Fig. 6d, e). Despite this, ob/ob and ob/ob IL6 mice were diabetic, as their fasting blood glucose and glucose tolerance were dysregulated compared with wild-type and hIL6 tg mice (Fig. 6f). By 48 weeks of age, fed blood glucose was indistinguishable in wild-type, hIL6 tg and ob/ob IL6 mice, and significantly lower than in ob/ob mice (Fig. 6d). Thus, hIL6 promotes glucose homeostasis in old obese ob/ob IL6 mice.
To directly establish the effect of hIL6 upon leptin action, we injected leptin intraperitoneally into 12-week-old chow-fed mice. A typical daily dose of leptin (1 mg/kg body weight) decreased food intake and body weight of ob/ob and ob/ob IL6 mice (data not shown). To better distinguish the effect of leptin in these two groups, we injected a low dose of leptin (0.1 mg/kg) daily for 16 days. Compared with ob/ob mice, low-dose leptin significantly decreased body weight and increased locomotor activity of obese ob/ob IL6 mice; however, low-dose leptin had no effect in chow-fed wild-type or hIL6 tg mice, and was too low to significantly reduce food consumption by ob/ob mice (Δfood = −0.5±0.3 g, p > 0.05) (Fig. 6g–i). Low-dose leptin did, however, significantly reduce food consumption by ob/ob IL6 mice (Δfood = −1.0±0.2 g/day, p < 0.001) (Fig. 6i). To establish whether hIL6 increases central leptin signalling, we compared STAT3 phosphorylation in the hypothalamus of 1-year-old wild-type, hIL6 tg, ob/ob and ob/ob IL6 mice at 2 h after a single low-dose leptin injection. Compared with wild-type mice, basal and leptin-stimulated STAT3 phosphorylation was significantly increased in hIL6 tg mice, but significantly decreased in ob/ob mice (Fig. 6j). Remarkably, basal and leptin-stimulated STAT3 phosphorylation in ob/ob IL6 mice was indistinguishable from that in wild-type mice (Fig. 6j). These results support the hypothesis that circulating hIL6 augments central leptin signalling and action, revealing the principle mechanism by which hIL6 promotes nutrient homeostasis in hIL6 tg mice.
Despite evidence of pleotropic and contradictory actions of IL6 upon glucose tolerance in rodent models and human studies, our experiments show clearly that overexpression of hIL6 in brain and lung of hIL6 tg mice reduces daily food consumption and promotes energy expenditure. Consistent with the reduced adiposity, circulating insulin decreases and glucose tolerance improves, confirming that hIL6 promotes systemic insulin sensitivity, especially in animals on HFD. Moreover, circulating leptin and daily food consumption decreases, suggesting that hIL6 improves central leptin sensitivity or action.
Previous reports have shown that central leptin signalling requires IL6-mediated signals for a normal response. Thus Il6 −/− mice slowly develop obesity while circulating leptin increases, and obese Il6 −/− mice do not respond to intracranial leptin injections . By contrast, circulating leptin decreases significantly in hIL6 tg mice on chow or HFD. Since leptin signalling is required in the hypothalamus to suppress appetite and promote energy expenditure, hIL6 apparently augments leptin action: otherwise the hIL6 tg mice would consume more food and accumulate adipose mass . In our study, only ob/ob IL6 mice responded significantly to low-dose leptin injections with greater locomotor activity accompanied by decreased body weight and food consumption. Thus our results support the hypothesis that life-long hIL6 promotes central leptin signalling, which prevents diet-induced obesity in mice.
The signalling subunit gp130 of the IL6 receptor complex is similar structurally to the intracellular tail of the signalling-isoform of the leptin receptor, isoform b (LepRb) . Consistent with the shared regulation of STAT3 phosphorylation by leptin and IL6, Pomc and Agrp expression in our study was nearly normal in hIL6 tg mice on a HFD. However, the effect of hIL6 upon Pomc and Agrp regulation appears to occur through its effects upon leptin signalling, as ob/ob and ob/ob IL6 mice were equally hyperphagic.
STAT3 to SOCS3 signalling is stimulated by leptin in the hypothalamus and throughout the body by numerous factors including IL6, IFN-γ, IL10, CNTF (ciliary neurotrophic factor) and other gp130 signalling cytokines . However, the leptin response increased while SOCS3 production also increased in the hypothalamus of lean hIL6 tg mice, suggesting that SOCS3 does not inexorably block the leptin signal. Direct comparison of hypothalamic STAT3 phosphorylation in ob/ob and ob/ob IL6 mice shows that hIL6 weakly promoted STAT3 phosphorylation in the absence of leptin. Thus, hIL6 largely promotes the leptin-stimulated STAT3 to SOCS3 cascade, which maintains the normal relation between leptin and SOCS3.
We posit that IL6 receptor α-neurons are separate from LepRb-neurons, since hIL6 failed to normalise body weight or food intake in ob/ob IL6 mice. However, IL6 receptor α-neurons might converge upon a common efferent circuit, ordinarily regulated by LepRb neurons, to augment leptin signalling in ob/ob mice or wild-type mice on the HFD. A similar relation appears to exist between CNTF receptor neurons and LepRb neurons . LepRb and the CNTF receptor share structural homology and can activate similar signalling pathways in the hypothalamus. In the absence of CNTF receptor, CNTF can activate gp130 through a homodimer of IL6 receptor and leukaemia inhibitory factor receptor (LIFR) . CNTF can ameliorate obesity by circumventing diet-induced leptin resistance . It remains to be investigated whether CNTF mediates any of the central effects of IL6.
Cell-based experiments suggest that the IL6-stimulated STAT3 to SOCS3 cascade causes hepatic insulin resistance by inhibiting insulin receptor signalling and increasing IRS1 degradation . In parallel with increasing SOCS3 concentrations, IRS1 concentrations in the present study decreased in the postprandial liver of hIL6 tg mice; however, hIL6 prevented the near complete loss of insulin-stimulated insulin receptor autophosphorylation and the downstream phosphorylation of IRS2 and Aktser473 in animals on HFD, a finding consistent with improved systemic glucose tolerance. Recently, the negative effect of SOCS3 on insulin action has been questioned, since liver-specific Stat3 −/− mice with low SOCS3 concentrations were unable to suppress hepatic glucose production . The strongest effect of mIL6 upon liver metabolism might depend upon hypothalamic STAT3 signalling, which mediates the normalising effect of leptin on hepatic insulin action in rats on a HFD . Intracerebral ventricular insulin infusion has been shown to increase levels of mIL6 in the liver, which can increase hepatic STAT3 and through that suppress expression of gluconeogenic enzymes . Thus hepatic IL6 to STAT3 signalling triggered by brain insulin action could play an important role in nutrient homeostasis. However, in animals on a chow diet hIL6 might not be sufficient, because STAT3 phosphorylation did not increase in fasted hIL6 tg mice, while increasing equally in wild-type and hIL6 tg mice. Whereas the HFD inhibited hepatic STAT3 phosphorylation in our study, hIL6 strongly promoted basal and postprandial STAT3 phosphorylation. Thus a postprandial signal, perhaps initiated by insulin and/or leptin in the hypothalamus, appears to be essential for hepatic STAT3 phosphorylation.
The question of whether IL6 has positive or negative effects on metabolism is the subject of continuing controversy . The hypothesis that IL6 induces insulin resistance is challenged by findings that regular physical exercise increases insulin sensitivity while promoting production and release of IL6 from contracting skeletal muscle [40, 41]. IL6 can also increase peripheral insulin sensitivity and glucose tolerance by activating AMP-activated protein kinase (AMPK) in muscle [17, 42]. Here, however, hIL6 had no effect on AMPK phosphorylation or activity in hIL6 tg mice (data not shown). Further investigation regarding a potential of AMPK to mediate some of the effects of hIL6 is required.
The relation between IL6 and leptin in the central nervous system might play an important role on the effect of exercise upon nutrient homeostasis. Moderate exercise promotes peripheral insulin sensitivity and suppresses weight gain . Human IL6 secretion from skeletal muscle is dramatically increased during and after exercise . Our results are consistent with the hypothesis that the effect of exercise upon nutrient homeostasis and insulin sensitivity might be mediated through central effects of muscle-derived IL6 in promoting central leptin signalling.
Our results are consistent with the hypothesis that decreased fat mass in hIL6 tg mice, especially those on HFD, arises through increased energy expenditure. Thus oxygen consumption, CO2 production and physical activity were increased in the hIL6 tg mice. These data are consistent with previous reports that a single intracranial injection of IL6 increases oxygen consumption and energy expenditure by rats [22, 26].
Chronic cerebral expression of mIL6, using an IL6 transgene under the control of glial fibrillary acidic protein (GFAP) promoter, activates the hypothalamic–pituitary–adrenal axis, which increases corticosterone concentrations in stressed mice . However, in our experiments, plasma corticosterone concentrations were barely increased in unstressed hIL6 tg mice compared with control mice and increased equally in both mice during stress (data not shown). However, as in our hIL6 tg mice, the plasma leptin concentration was reduced in GFAP-IL6 transgenic mice . Since circulating IL6 was not elevated in those GFAP-IL6 mice, those results support the hypothesis that hIL6 promotes leptin action in the central nervous system.
Previous reports have shown that transgenic IL6 causes various pathologies of the immune system that can be fatal to mice [46, 47]. Human IL6 in C57BL/6J mice under the control of human immunoglobulin heavy-chain enhancer develop mesangial proliferative glomerulonephritis with massive IgG1 plasmacytosis . MTI/IL6 transgenic mice expressing murine IL6 constitutively in the liver developed progressive kidney damage and died between 12 and 20 weeks of age . Hepatic inflammation occurs in transgenic mouse secreting mIL6 from muscle . Despite the above, our hIL6 tg mice with circulating hIL6 secreted from brain and lung never displayed hepatic inflammation, acute inflammatory response or systemic inflammation. Perhaps the sites of IL6 secretion are critical for its systemic effect. In any case, hIL6 tg mice provide a unique system to investigate the role of hIL6 in central and peripheral nutrient homeostasis.
In summary, hIL6 protects mice from insulin resistance and obesity. Since this effect was not observed in ob/ob mice, hIL6 apparently augments central leptin action without substituting for leptin. Due to its immunoreactive nature, IL6 might never be a successful therapeutic treatment strategy; however, prolonged treatment with IL6 homologues with high accessibility to the central nervous system might show therapeutic promise in anti-obesity therapy.
This work was supported by American Diabetes Association grant 105RA143 and NIH ROI grant DK038712 to M. F. White. The authors thank A. Parlow, National Pituitary Program (NIDDK) for kindly providing us with the recombinant mouse leptin.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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