Hepatic leptin signalling and subdiaphragmatic vagal efferents are not required for leptin-induced increases of plasma IGF binding protein-2 (IGFBP-2) in ob/ob mice
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The fat-derived hormone leptin plays a crucial role in the maintenance of normal body weight and energy expenditure as well as in glucose homeostasis. Recently, it was reported that the liver-derived protein, insulin-like growth factor binding protein-2 (IGFBP-2), is responsible for at least some of the glucose-normalising effects of leptin. However, the exact mechanism by which leptin upregulates IGFBP-2 production is unknown. Since it is believed that circulating IGFBP-2 is predominantly derived from the liver and leptin has been shown to have both direct and indirect actions on the liver, we hypothesised that leptin signalling in hepatocytes or via brain–liver vagal efferents may mediate leptin control of IGFBP-2 production.
To address our hypothesis, we assessed leptin action on glucose homeostasis and plasma IGFBP-2 levels in both leptin-deficient ob/ob mice with a liver-specific loss of leptin signalling and ob/ob mice with a subdiaphragmatic vagotomy. We also examined whether restoring hepatic leptin signalling in leptin receptor-deficient db/db mice could increase plasma IGFBP-2 levels.
Continuous leptin administration increased plasma IGFBP-2 levels in a dose-dependent manner, in association with reduced plasma glucose and insulin levels. Interestingly, leptin was still able to increase plasma IGFBP-2 levels and improve glucose homeostasis in both ob/ob mouse models to the same extent as their littermate controls. Further, restoration of hepatic leptin signalling in db/db mice did not increase either hepatic or plasma IGFBP-2 levels.
Taken together, these data indicate that hepatic leptin signalling and subdiaphragmatic vagal inputs are not required for leptin upregulation of plasma IGFBP-2 nor blood glucose lowering in ob/ob mice.
KeywordsLeptin Liver IGFBP-2
Adenovirus expressing the gene for β-galactosidase
Adenovirus expressing the gene for Lepr-b
IGF binding protein-2
Leptin receptor, isoform b
Signal transducer and activator of transcription 3
IGFs and IGF binding proteins (IGFBPs) play an important role in regulating glucose metabolism [1, 2, 3]. IGF-1 specifically has been found to influence blood glucose concentrations directly by stimulating glucose uptake in target cells and indirectly by increasing the sensitivity of tissues to insulin . Effects of IGFs are modulated by IGFBPs through the ability of IGFBPs to sequester IGFs, but interestingly, several reports demonstrate that IGFBPs can activate cell surface receptors directly and stimulate cellular events independently of IGFs [1, 3, 4].
IGFBP-2 is the second most abundant plasma circulating IGFBP, but its physiological role remains poorly understood. Interestingly, mice overexpressing IGFBP-2 under the cytomegalovirus (CMV) promoter have mildly reduced postnatal body weight  and IGFBP-2 knockout mice have slightly increased body weight and body fat mass . Moreover, mice overexpressing IGFBP-2 under its native promoter are protected from high-fat-diet-induced obesity and glucose intolerance and display increased insulin sensitivity , while IGFBP-2 knockout mice on a chow diet showed no alterations in glucose tolerance . Further, in humans, low levels of IGFBP-2 are correlated with decreased insulin sensitivity and levels are also reduced in the presence of obesity . Overall, there appears to be an inverse association between IGFBP-2 and adiposity-related insulin resistance, but much is still unknown about the exact nature of this relationship.
Leptin has major effects on maintaining normal body weight and glucose metabolism and has been shown to increase hepatic transcription of Igfbp2 when administered either peripherally or centrally to lipodystrophic mice . Further, it was recently demonstrated that peripheral administration of leptin to leptin-deficient ob/ob mice potently upregulates both hepatic Igfbp2 mRNA and circulating IGFBP-2 levels . Moreover, Hedbacker et al.  showed that administration of an Igfbp2-expressing adenovirus to ob/ob mice had similar glucose lowering effects as leptin in various mouse models of insulin resistance and insulin-deficient diabetes and these effects were independent of body weight loss. Interestingly, in a small cohort of leptin-deficient people, it was observed that these patients had about 50% lower levels of plasma IGFBP-2 than controls and these levels were increased after 6 months of leptin treatment . These results indicate that leptin is involved in regulating circulating IGFBP-2 levels and that IGFBP-2 may play an intricate role in mediating the effects of leptin on metabolism. However, the exact site and mechanism by which leptin regulates IGFBP-2 production is still unclear.
The liver, which produces the long, signalling isoform of the leptin receptor [9, 10, 11], may be an important site for leptin action on increasing plasma IGFBP-2. It has been shown that increased hepatic Igfbp2 mRNA is associated with increased circulating IGFBP-2 [3, 12, 13], suggesting that the liver is a major source of plasma IGFBP-2. Further, subcutaneous administration of leptin to the periphery is able to increase both liver Igfbp2 mRNA [3, 8] and plasma IGFBP-2 levels . Thus, leptin may act directly on the liver to increase production and secretion of IGFBP-2. Intriguingly, however, central intracerebroventricular (ICV) doses of leptin that do not increase peripheral leptin levels are also able to increase hepatic Igfbp2 transcription . Consequently, we hypothesised that leptin may increase hepatic Igfbp2 expression by acting directly on leptin receptors in the liver or by acting on the brain and increasing liver expression of Igfbp2 via parasympathetic brain–liver vagal efferents. To test these hypotheses, we provided peripheral leptin treatment to ob/ob mice lacking hepatic leptin receptors and to ob/ob mice that had been subjected to a subdiaphragmatic vagotomy. In both cases, leptin was still able to reduce hyperglycaemia and hyperinsulinaemia while potently increasing circulating IGFBP-2 levels. Further, when we restored hepatic leptin signalling in db/db mice, which have a global loss of leptin signalling, neither hepatic nor plasma IGFBP-2 levels increased. Therefore, despite evidence suggesting an important role for the liver in mediating leptin-induced IGFBP-2 production, neither hepatic leptin signalling nor subdiaphragmatic vagal efferents are required for leptin to increase IGFBP-2 levels in the plasma of leptin-deficient ob/ob mice.
Male C57BL/6 mice (#000664), ob/ob mice (#000632), and db/db mice (#000697) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). For details on the generation of Leprflox/floxAlbcre ob/ob mice, see the electronic supplementary material (ESM). Mice were housed with a 12 h light–12 h dark cycle and had ad libitum access to water and a standard chow diet (#5015 Lab Diet, St Louis, MO, USA). db/db mice were treated intravenously with 1 × 109 plaque-forming units (pfu) of an adenovirus expressing the gene for either the long signalling isoform of the mouse leptin receptor (Ad-Lepr-b, kindly provided by M.G. Myers (University of Michigan) and C.J. Rhodes (University of Chicago) ) or β-galactosidase (Ad-β-gal) as a control. Subdiaphragmatic vagotomies were performed by the Jackson Laboratory. All procedures with animals were approved by the University of British Columbia Animal Care Committee and carried out in accordance with the Canadian Council on Animal Care guidelines.
PCR and RT-PCR analysis
Liver tissue was collected from mice and genomic DNA was extracted using DNeasy kits (Qiagen, Mississauga, Canada). RNA was isolated using either an RNeasy kit (Qiagen, Mississauga, ON, Canada) or TRI Reagent (Ambion, Streetsville, ON, Canada). Reverse transcription reactions were performed with a poly T primer using a Superscript First-Strand Synthesis kit (Invitrogen, Burlington, ON, Canada). See ESM for primer sequences.
Subdiaphragmatic vagotomies or sham surgeries were performed by the Jackson Laboratory in 3.5-week-old ob/ob mice using the ventral abdominal approach. See ESM for a brief description of the procedure.
Implantation of mini-osmotic pumps
Recombinant mouse leptin was from the National Hormone and Peptide Program (Torrance, CA, USA) and was reconstituted with sterile PBS. Mini-osmotic pumps (Alzet, Palo Alto, CA, USA) were loaded with recombinant mouse leptin or PBS (control) and pre-equilibrated according to manufacturer guidelines. The pumps were implanted subcutaneously in isoflurane-anaesthetised mice.
Plasma analyte analysis
Body weight, blood glucose, insulin, leptin and IGFBP-2 were measured following a 4 h fast unless specified otherwise. Blood glucose concentration was measured with a One Touch Ultra Glucometer (Life Scan, Milpitas, CA, USA) from the saphenous vein. Plasma insulin levels were measured using an Ultrasensitive Mouse Insulin ELISA (ALPCO, Salem, MA, USA), leptin levels were measured using a Mouse Leptin ELISA (Crystal Chem, Downers Grove, IL, USA) and circulating IGFBP-2 levels were measured using a Mouse/Rat IGFBP-2 ELISA (ALPCO).
Verification of vagotomy
Eight weeks post-surgery, the completeness of the subdiaphragmatic vagotomy was assessed using previously described methods . The test was based on the satiety effect of cholecystokinin (CCK), which is known to be mediated by the vagus nerve . For details of the procedure, see the ESM.
Data are represented as mean ± SEM and were analysed using a Student’s t test. Statistical significance was defined as p ≤ 0.05.
Low leptin levels are able to normalise hyperglycaemia and hyperinsulinaemia independent of weight loss
Leptin increases plasma IGFBP-2 levels in ob/ob mice in a dose-dependent manner
Leprflox/flox Albcre+ ob/ob mice have truncated hepatic leptin receptor transcripts
Hepatic leptin signalling is not required for leptin regulation of plasma IGFBP-2 levels
Effect of vagotomy on metabolic parameters in ob/ob mice
The most well-established effects of leptin are its effects on reducing body weight by decreasing food intake and increasing energy expenditure. However, even in one of the first studies showing the effects of leptin in ob/ob mice, it was seen that, at doses too low to affect body weight, leptin could still reverse hyperglycaemia . This observation has also been made recently by Hedbacker et al.  and in the present study (Fig. 1). Further, even in mouse models of type 1 diabetes, leptin can reverse hyperglycaemia independently of its effects on body weight and food intake [28, 29, 30]. Thus, one of the most potent effects of leptin in mice is actually in lowering glucose levels rather than in reducing body weight. Therefore, because of its glucose-lowering potential, it is of great interest to uncover the mechanism by which leptin potently reverses hyperglycaemia, even in the absence of weight loss.
It has been suggested that plasma IGFBP-2 may play a role in mediating the effects of leptin on glucose metabolism . Leptin treatment has been shown to significantly increase circulating IGFBP-2 levels in ob/ob mice and, remarkably, IGFBP-2 administration via adenovirus was able to ameliorate diabetes in leptin-deficient ob/ob mice . We saw that, even at the lowest dose of leptin (0.2 μg/day), which had effects on glucose homeostasis but not body weight, leptin was able to significantly increase IGFBP-2 levels compared with PBS-treated mice. The effect of leptin on circulating levels of IGFBP-2 was dose-dependent, and the highest dose of leptin treatment (5 μg/day) was able to increase plasma IGFBP-2 to levels found in normal, wild-type mice. These data further establish leptin as a potent regulator of plasma IGFBP-2 levels. However, the mechanism by which leptin increases plasma IGFBP-2 levels is still unknown.
The liver or the brain may be sites mediating the effects of leptin on increasing plasma IGFBP-2 levels [3, 8]. However, leptin was able to increase plasma IGFBP-2 levels in ob/ob mice lacking hepatic leptin signalling, indicating that hepatic leptin signalling is not required for leptin to increase levels of plasma IGFBP-2. Furthermore, when we re-expressed functional hepatic leptin receptors in hyperleptinaemic db/db mice, which have low plasma IGFBP-2 levels, neither hepatic nor plasma IGFBP-2 levels rose towards wild-type levels. Taken together, our data indicate that direct leptin action on the liver is neither required nor sufficient for mediating the effects of leptin on plasma IGFBP-2.
It is noteworthy that there were some time points where Leprflox/floxAlbcre+ob/ob mice treated with 5 μg/day leptin had significantly higher IGFBP-2 levels compared with controls, perhaps a reflection of the higher circulating leptin levels during leptin treatment. We speculate that the hepatic leptin receptor signalling domain may be involved in leptin clearance, resulting in the Leprflox/floxAlbcre+ob/ob mice having higher leptin levels during leptin treatment. Even though the kidney is reported to be the major site of leptin clearance [31, 32], it is possible that, at high levels of plasma leptin, such as those seen during continuous exogenous delivery of 5 μg/day leptin, the liver may be an important contributor to leptin clearance. Nonetheless, in both the Leprflox/floxAlbcre+ob/ob mice and the Leprflox/floxAlbcre−ob/ob controls treated with 5 μg/day leptin, leptin levels rose well above pre-treatment levels and, on the last day of leptin treatment, IGFBP-2 levels were nearly tenfold higher in both groups. In addition, Leprflox/floxAlbcre+mice, which had normal plasma leptin levels , also had normal plasma IGFBP-2 levels. Even hyperleptinaemic db/db mice with functional hepatic leptin receptors did not have altered IGFBP-2 levels compared with control db/db mice. Overall, our data show that, regardless of plasma leptin levels, direct leptin action on the liver is not required for leptin to increase plasma IGFBP-2.
Central leptin signalling appears to play an important role in mediating the effect of leptin on energy homeostasis and glucose metabolism. Mice with a neuron-specific knockout of leptin receptors have increased adiposity and hyperinsulinaemia , whereas mice with disrupted peripheral leptin signalling but intact central leptin receptors have normal body weight and glucose metabolism . Furthermore, central leptin administration can modulate hepatic gene expression [8, 35, 36, 37]. Therefore, it is possible that leptin acts centrally to induce hepatic IGFBP-2 production and secretion via modulation of neural outputs to the liver. Interestingly, in leptin-deficient lipodystrophic mice, ICV delivery of leptin is able to upregulate Igfbp2 gene expression in the liver , which is suggested to be the main source of circulating IGFBP-2 [12, 13]. In addition, the central effects of leptin on modulating hepatic insulin sensitivity are reported to be mediated through the hepatic branch of the vagus nerve , which conveys parasympathetic innervation to the liver. To examine whether leptin acts on the brain to increase hepatic production of IGFBP-2 via brain–liver vagal communication, we treated vagotomised ob/ob mice with leptin. In spite of the initial metabolic differences in the vagotomised and sham-operated mice (Fig. 7), leptin treatment was able to increase circulating IGFBP-2 levels in both the vagotomised and sham-operated mice to an equal extent. Thus, even in the absence of subdiaphragmatic vagal innervations, leptin can still correct hyperglycaemia and hyperinsulinaemia in ob/ob mice, and brain–liver vagal efferents are not required for leptin to increase plasma IGFBP-2 levels.
While our data eliminate direct action of leptin on hepatocytes and brain–liver vagal efferents as the sole mediators of leptin-induced increases in plasma IGFBP-2, other possible mechanisms remain to be investigated. First, we did not eliminate the possibility that central leptin action can affect hepatic production and secretion of IGFBP-2 via sympathetic inputs to the liver, which remained intact in our mice. Further, while the liver is believed to be the major source of circulating IGFBP-2, other tissues may also contribute to plasma IGFBP-2 levels and these other tissues may play a bigger role when direct or indirect hepatic stimulation by leptin is absent. In fact, IGFBP-2 expression has been detected in several tissues, including kidney [38, 39], adrenal gland , brain [38, 41] and adipose [42, 43], all of which are known to secrete hormones and express the long, signalling isoform of the leptin receptor [9, 44, 45]. Visceral white adipose tissue in particular has recently been shown to express Igfbp2 mRNA in levels that correlate with plasma IGFBP-2 levels and, in the absence of leptin (ob/ob mice) or leptin signalling (db/db mice), Igfbp2 mRNA expression in visceral white adipose tissue is reduced . Furthermore, another possibility is that leptin may indirectly increase hepatic and plasma IGFBP-2 by modulating levels of other hormones that in turn act on the liver to increase IGFBP-2 levels. While we have eliminated the most obvious mechanisms by which leptin might regulate hepatic and plasma IGFBP-2, there are other possibilities that remain to be addressed. Considering the remarkable effects of leptin and IGFBP-2 on ameliorating diabetes, more research should be done to better understand the regulation of plasma IGFBP-2 levels by leptin.
We thank C. Donald (University of British Columbia) for her technical assistance with the animal studies and S.C. Chua (Albert Einstein College of Medicine) for his generous contribution of the Leprflox/flox mice. We would also like to thank M.G. Myers (University of Michigan) and C.J. Rhodes (University of Chicago) for providing the Ad-Lepr-b virus.
This work was funded by the Canadian Institutes of Health Research. FKH is supported by the Canadian Diabetes Association, HCD is supported by the Natural Sciences Research Council of Canada, and TJK is the recipient of a Senior Scholarship from the Michael Smith Foundation for Health Research.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors contributed to the conception and design or analysis and interpretation of data. Further, all authors contributed to drafting the article or revising it for important intellectual content. The approval of all authors was obtained before publication.
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